from mice immunized with pM-VEGF showed a significant enhanced cytotoxic activity against VEGF-secreting tumor cells, including EL-4 lymphoma, B16-F10 melanoma, and TC-1 carcinoma, as compared with those obtained from the mice immunized with the pMAE5△5 ''empty'' vector [33]. IFN-γ ELISPOT assay revealed a significant increase in the number of spots in spleen cells from mice immunized with pM-VEGF [33]. Vaccination with a mutated hVEGF121 gene inserted into the pMAE5△5 vector (pM-VEGFmut) produced similar *in vitro* and *in vivo* results, and remarkably reduced the number of spontaneous metastases in a murine model with Lewis lung carcinoma [33]. Serum VEGF levels decreased 8-fold in mice vaccinated with pM-VEGF or pM-VEGFmut as compared with those in pMAE5△5 treated mice [33]. A significant correlation was also found between the elevation of serum VEGF level and the increase of the tumor dimensions [33]. However, antibody responses against the GSThVEGF121 fusion protein or GST alone used as capture antigens in ELISA were undetect‐ able in animals vaccinated with pM-VEGF or pM-VEGFmut [33]. These findings indicate that human VEGF-harboring DNA vaccine can be employed for anti-angiogenic active immuno‐ therapy for cancers in mice and direct cell cytotoxicity contributes to the overall anti-tumor

Previous studies in rodent tumor models have indicated that immunization against xenogeneic growth factors is more likely to induce effective anti-tumor responses than immunization against the syngeneic growth factor [34]. In 2007, an investigation was conducted to assess the safety and anti-tumor and anti-angiogenic effects of a xenogeneic VEGF vaccine in pet dogs with spontaneous cancer. Nine dogs with soft tissue sarcoma were immunized with a recombinant human VEGF vaccine over a 16-week period [34]. The xenogeneic VEGF vaccine was well-tolerated by all dogs and resulted in induction of humoral responses against both human and canine VEGF in animals that remained in the study long enough to receive multiple immunizations [34]. Three of five multiply immunized dogs also experienced sustained decreases in circulating plasma VEGF concentrations and two dogs had a significant decrease in tumor microvessel density [34]. The overall tumor response (>50% decrease in tumor volume) rate was 30% for all treated dogs in the study. Thus, it was concluded that a xenogeneic VEGF vaccine may be a safe and effective alternative means of controlling tumor growth and

VEGF receptor-2 (VEGFR-2, also known as fetal liver kinse-1 (flk-1) in mouse and kinasecontaining domain receptor (KDR) in human) is the main receptor responsible for the VEGF-mediated angiogenic activity [6]. The impairment of vasculogenesis and death of embryo at day 8.5 were observed as the result of the targeted inactivation of flk-1 gene in mice [35]. Overexpression of KDR was found on activated endothelial cells of newly formed vessels [6]. It was discovered that the primary sequence of quail VEGFR-2 (qVEGFR-2) was 67% and 70% identical at the amino acid level with mouse and human homologues (flk-1 and KDR), respectively [36]. Immunotherapy with a vaccine based on quail homologous VEGFR-2 elicited protective and therapeutic anti-tumor immunity in both solid and hematopoietic tumor models in mice, such as LL/2 Lewis lung carcinoma, CT26 colon carcinoma, Meth A fibrosarcoma, MOPC-315 plasmacytoma, and EL-4 lymphoma [36]. Autoantibodies against flk-1 in the immunized mice were identified. Sera from qVEGFR-2-

effects observed in immunized mice [33].

370 Cancer Treatment - Conventional and Innovative Approaches

angiogenesis [34].

Fibroblast growth factor receptor-1 (FGFR-1) is expressed on endothelial cells and many types of tumors [39, 40]. The Xenopus homologue of FGFR-1 is 80% and 74% identical at the amino acid level with mouse FGFR-1 and human FGFR-1, respectively [41]. Therefore, FGFR-1 may be used as another ideal target for anti-angiogenesis therapy. Vaccination with Xenopus FGFR-1 (pxFR1) provoked protective and therapeutic effects in three murine tumor models, including Meth A fibrosarcoma cells, H22 hepatoma cells, and MA782/5S mammary carcinoma [41]. FGFR-1-specific autoantibodies were detected in sera of pxFR1-immunized mice by Western blot analysis, and the purified immunoglobulins effectively inhibited endothelial cell proliferation *in vitro* [41]. However, the immunoglobulins had no direct inhibitory effect on the proliferation of above three tumor cell lines [41]. Adoptive transfer of sera or purified immunoglobulin isolated from pxFR1-immunized mice into unimmunized mice provided effective protection against tumor growth, while adsorption of sera or immunoglobulin with FGFR-1-positive endothelial cells before adoptive transfer could abrogate its anti-tumor activity [41]. Autoantibodies deposited on the endothelial cells within tumor tissues and significantly suppressed intratumoral angiogenesis were found in pxFR1-immunized mice by histological examination [41]. Furthermore, this anti-tumor activity and production of FGFR-1 specific autoantibodies were abrogated by depletion of CD4+ T lymphocytes, again pointing to their essential helper function for antibody production [41].

dases, can selectively degrade components of the extracellular matrix [44]. *In vivo*, elevated stromal MMP-2 and MMP-9 activity is highly correlated with increased metastatic potential in most malignant tumors [45]. Increased activity of MMPs appears to permit the tumor to remodel its surrounding microenvironment, to grow in a permissive space, and to promote the development of supporting stroma, including angiogenesis [46]. Moreover, numerous pathological and clinical studies demonstrated that the MMPs were frequently overexpressed in various solid tumor cells and peritumoral stromal cells [46]. It was reported that the abrogation of MMP-2 alone resulted in the inhibition of the transition from the prevascular to the vascular stage during tumor development and then of tumor growth [47]. Furthermore, the suppression of tumor-induced angiogenesis and of invasion and metastasis of tumor cells could be observed in MMP-2-deficient mice [47]. These findings indicated that MMP-2 alone played an important role in angiogenesis and tumor growth. Sequence comparison analysis showed that the primary sequence of mouse MMP-2 at the amino acid level was 82% and 91% identical with chicken and human homologues, respectively [48]. It was reported that the plasmid DNA vaccination with chicken homologous MMP-2 (c-MMP-2)-based model antigen could induce both protective and therapeutic anti-tumor immunity in murine tumor models with LL/2 Lewis lung carcinoma, Meth A fibrosarcoma, and H22 hepatoma [48]. The elevation of MMP-2 in the sera of tumor-bearing mice was abrogated with the vaccination of c-MMP-2 [48]. The autoimmune response against MMP-2 may be provoked in a cross-reaction by the immunization with c-MMP-2, and the autoantibody targeting to MMP-2 was elevated and probably responsible for the anti-tumor activity [48]. Moreover, gelatinase activity of MMP-2, including both latent MMP-2 and active MMP-2, derived from the above mentioned three murine tumor models was apparently inhibited by the vaccination with c-MMP-2 [48]. However, the vaccination did not inhibit the gelatinase activity of MMP-9 [48]. These findings indicate that the activity of MMP-2 is impaired by immunization with c-MMP-2 in mice. Angiogenesis was apparently inhibited within tumors in immunized mice. The anti-tumor activity and production of auto-antibodies against MMP-2 were abrogated by depletion of

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In 2010, Yang et al. [49] used a cross-reactive serological expression cloning (SEREX) strategy (CR-SEREX) to identify novel xenogenic angiogenesis- and tumor-associated antigens in oocytes of *Xenopus laevis* and found that *Xenopus* receptor for hyaluronic-acid-mediated motility (xRHAMM) was the most frequently clone among 78 CR-SEREX positive clones, suggesting that xRHAMM has the strongest immunogenic potential for xenogenic immuno‐ therapy. It was demonstrated that expression of RHAMM is restricted to the testis, thymus, placenta, vascular endothelial cells, and various cancer cells, and RHAMM functions in vascular endothelial cell migration, angiogenesis, and in hyaluronic-acid-induced cell mobility [50]. In order to examine the anti-angiogenic effects, a DNA vaccine based on xRHAMM (pcDNA3.1-xRHAMM) was constructed [49]. Intramuscular vaccination of the cationic liposome encapsulated pcDNA3.1-xRHAMM DNA effectively induced a protective antitumor immunity against local tumor and lung metastasis in B16 melanoma mouse models. Angiogenesis was inhibited and cell apoptosis was increased within tumors. Anti-tumor

CD4+

*<IP_ADDRESS>. xRHAMM*

T lymphocytes [48].

#### *<IP_ADDRESS>. Integrins*

Integrins are heterodimeric transmembrane proteins consisting of α and β subunits with large extracellular domain and short cytoplasmic tail. They play very crucial roles in angiogenesis as the migration of endothelial cells is dependent on their adhesion to extracellular matrix proteins such as vitronectin [42]. αvβ3 is not generally found on blood vessels in normal tissues, but its expression is enhanced on newly developing blood vessels in human wound tissue, tumors, diabetic retinopathy, macular degeneration and rheumatoid arthritis, which implies that this integrin may play an important role in angiogenesis and development of neovascu‐ larization [42]. This distributive characteristic also makes αvβ3 an attractive target for tumor therapy [42]. A plasmid DNA encoding the ligand-binding domain of chicken integrin β3 was constructed to test this assumption. Immunization with chicken homologous integrin β3-based vaccine could elicit both protective and therapeutic anti-tumor immunity in murine tumor models with Meth A fibrosarcoma, H22 hepatoma, or MA782/5S mammary carcinoma [43]. Autoantibodies against integrin β3 in sera of the immunized mice were found by Western blot analysis and ELISA [43]. The purified immunoglobulins could effectively inhibit endothelial cell proliferation *in vitro*, and adoptive transfer of the purified immunoglobulins into nonimmunized mice could provide effective protection against tumor growth and markedly inhibit tumor angiogenesis [43]. The anti-tumor activity and the production of integrin β3 specific autoantibodies were CD4+ T lymphocyte-dependent [43].

#### *<IP_ADDRESS>. MMP*

Angiogenesis is an invasive process, requiring proteolysis of the extracellular matrix [44]. Inappropriate destruction of extracellular matrix components is involved in certain patholog‐ ical conditions, including arteriosclerosis, rheumatoid arthritis, and tumor aggression and metastasis [44]. The matrix metalloproteinases (MMPs), a family of extracellular endopepti‐ dases, can selectively degrade components of the extracellular matrix [44]. *In vivo*, elevated stromal MMP-2 and MMP-9 activity is highly correlated with increased metastatic potential in most malignant tumors [45]. Increased activity of MMPs appears to permit the tumor to remodel its surrounding microenvironment, to grow in a permissive space, and to promote the development of supporting stroma, including angiogenesis [46]. Moreover, numerous pathological and clinical studies demonstrated that the MMPs were frequently overexpressed in various solid tumor cells and peritumoral stromal cells [46]. It was reported that the abrogation of MMP-2 alone resulted in the inhibition of the transition from the prevascular to the vascular stage during tumor development and then of tumor growth [47]. Furthermore, the suppression of tumor-induced angiogenesis and of invasion and metastasis of tumor cells could be observed in MMP-2-deficient mice [47]. These findings indicated that MMP-2 alone played an important role in angiogenesis and tumor growth. Sequence comparison analysis showed that the primary sequence of mouse MMP-2 at the amino acid level was 82% and 91% identical with chicken and human homologues, respectively [48]. It was reported that the plasmid DNA vaccination with chicken homologous MMP-2 (c-MMP-2)-based model antigen could induce both protective and therapeutic anti-tumor immunity in murine tumor models with LL/2 Lewis lung carcinoma, Meth A fibrosarcoma, and H22 hepatoma [48]. The elevation of MMP-2 in the sera of tumor-bearing mice was abrogated with the vaccination of c-MMP-2 [48]. The autoimmune response against MMP-2 may be provoked in a cross-reaction by the immunization with c-MMP-2, and the autoantibody targeting to MMP-2 was elevated and probably responsible for the anti-tumor activity [48]. Moreover, gelatinase activity of MMP-2, including both latent MMP-2 and active MMP-2, derived from the above mentioned three murine tumor models was apparently inhibited by the vaccination with c-MMP-2 [48]. However, the vaccination did not inhibit the gelatinase activity of MMP-9 [48]. These findings indicate that the activity of MMP-2 is impaired by immunization with c-MMP-2 in mice. Angiogenesis was apparently inhibited within tumors in immunized mice. The anti-tumor activity and production of auto-antibodies against MMP-2 were abrogated by depletion of CD4+ T lymphocytes [48].

#### *<IP_ADDRESS>. xRHAMM*

acid level with mouse FGFR-1 and human FGFR-1, respectively [41]. Therefore, FGFR-1 may be used as another ideal target for anti-angiogenesis therapy. Vaccination with Xenopus FGFR-1 (pxFR1) provoked protective and therapeutic effects in three murine tumor models, including Meth A fibrosarcoma cells, H22 hepatoma cells, and MA782/5S mammary carcinoma [41]. FGFR-1-specific autoantibodies were detected in sera of pxFR1-immunized mice by Western blot analysis, and the purified immunoglobulins effectively inhibited endothelial cell proliferation *in vitro* [41]. However, the immunoglobulins had no direct inhibitory effect on the proliferation of above three tumor cell lines [41]. Adoptive transfer of sera or purified immunoglobulin isolated from pxFR1-immunized mice into unimmunized mice provided effective protection against tumor growth, while adsorption of sera or immunoglobulin with FGFR-1-positive endothelial cells before adoptive transfer could abrogate its anti-tumor activity [41]. Autoantibodies deposited on the endothelial cells within tumor tissues and significantly suppressed intratumoral angiogenesis were found in pxFR1-immunized mice by histological examination [41]. Furthermore, this anti-tumor activity and production of FGFR-1 specific autoantibodies were abrogated by depletion of CD4+ T lymphocytes, again pointing

Integrins are heterodimeric transmembrane proteins consisting of α and β subunits with large extracellular domain and short cytoplasmic tail. They play very crucial roles in angiogenesis as the migration of endothelial cells is dependent on their adhesion to extracellular matrix proteins such as vitronectin [42]. αvβ3 is not generally found on blood vessels in normal tissues, but its expression is enhanced on newly developing blood vessels in human wound tissue, tumors, diabetic retinopathy, macular degeneration and rheumatoid arthritis, which implies that this integrin may play an important role in angiogenesis and development of neovascu‐ larization [42]. This distributive characteristic also makes αvβ3 an attractive target for tumor therapy [42]. A plasmid DNA encoding the ligand-binding domain of chicken integrin β3 was constructed to test this assumption. Immunization with chicken homologous integrin β3-based vaccine could elicit both protective and therapeutic anti-tumor immunity in murine tumor models with Meth A fibrosarcoma, H22 hepatoma, or MA782/5S mammary carcinoma [43]. Autoantibodies against integrin β3 in sera of the immunized mice were found by Western blot analysis and ELISA [43]. The purified immunoglobulins could effectively inhibit endothelial cell proliferation *in vitro*, and adoptive transfer of the purified immunoglobulins into nonimmunized mice could provide effective protection against tumor growth and markedly inhibit tumor angiogenesis [43]. The anti-tumor activity and the production of integrin β3-

T lymphocyte-dependent [43].

Angiogenesis is an invasive process, requiring proteolysis of the extracellular matrix [44]. Inappropriate destruction of extracellular matrix components is involved in certain patholog‐ ical conditions, including arteriosclerosis, rheumatoid arthritis, and tumor aggression and metastasis [44]. The matrix metalloproteinases (MMPs), a family of extracellular endopepti‐

to their essential helper function for antibody production [41].

372 Cancer Treatment - Conventional and Innovative Approaches

*<IP_ADDRESS>. Integrins*

specific autoantibodies were CD4+

*<IP_ADDRESS>. MMP*

In 2010, Yang et al. [49] used a cross-reactive serological expression cloning (SEREX) strategy (CR-SEREX) to identify novel xenogenic angiogenesis- and tumor-associated antigens in oocytes of *Xenopus laevis* and found that *Xenopus* receptor for hyaluronic-acid-mediated motility (xRHAMM) was the most frequently clone among 78 CR-SEREX positive clones, suggesting that xRHAMM has the strongest immunogenic potential for xenogenic immuno‐ therapy. It was demonstrated that expression of RHAMM is restricted to the testis, thymus, placenta, vascular endothelial cells, and various cancer cells, and RHAMM functions in vascular endothelial cell migration, angiogenesis, and in hyaluronic-acid-induced cell mobility [50]. In order to examine the anti-angiogenic effects, a DNA vaccine based on xRHAMM (pcDNA3.1-xRHAMM) was constructed [49]. Intramuscular vaccination of the cationic liposome encapsulated pcDNA3.1-xRHAMM DNA effectively induced a protective antitumor immunity against local tumor and lung metastasis in B16 melanoma mouse models. Angiogenesis was inhibited and cell apoptosis was increased within tumors. Anti-tumor activity of xRHAMM was mediated by both the antigen-specific cellular and humoral responses against RHAMM, as confirmed by the depletion of immune cell subsets *in vivo*. Furthermore, the anti-angiogenic and anti-tumor effects induced by vaccination of pcDNA3.1 xRHAMM were significantly stronger than that induced by vaccination of the corresponding autologous counterpart pcDNA3.1-mRHAMM [49].

effect of angiostatin. These antibodies inhibit endothelial cell migration, block tumor cell- and bFGF-induced angiogenesis in the matrigel plug assay and prevent growth of transplanted tumors without impairing normal stromal or retina vessels [57]. Very recently, Arigoni et al. further showed that the pAmot-induced antibodies alter tumor vessel permeability and structure. These combined effects of vaccine-induced anti-Amot antibodies lead to inhibition of established clinically evident mammary tumors, massive tumor perivascular necrosis, and an effective tumor antigen presentation in a form of epitope spreading that induces an immune response against other oncoantigens overexpressed by tumor cells [61]. Greater tumor vessel permeability also markedly boosts the local accumulation of doxorubicin and enhances the anti-tumor effect of the drugs [61]. These data provide a rationale for the development of fresh anticancer treatments based on anti-Amot vaccination in conjunction with chemotherapy

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Taken together, it is obvious that vaccination with xenogeneic homologous molecules associ‐ ated with angiogenesis, such as pro-angiogenic factors, integrins, MMP, could induce antitumor immunity and thus might be a feasible strategy for cancer therapy with potential clinical

Given that vaccination with xenogeneic homologous molecules associated with tumor angiogenesis could effectively induce anti-tumor immunity, it can be assumed that vaccines based on non-xenogeneic homologous molecules, such as allogeneic homologues of some proangiogenic factors or other important molecules associated with angiogenesis, could also successfully induce specific and potent anti-tumor immunity. To date, several vaccines based on non-xenogeneic homologous molecules were used in anti-angiogenic active immunother‐

As has been discussed above, VEGF-mediated signaling pathway through VEGFR-2 is a ratelimiting step during tumor angiogenesis. Thus, VEGF/VEGFR-2 is still an ideal target in the non-xenogeneic homologous molecules-based anti-angiogenic strategy. Immunization of mice with VEGF receptor-2 (flk-1)-pulsed dendritic cells (DC) can break self-tolerance to VEGFR-2, induce CTL and antibody responses to VEGFR-2 [62]. Significant inhibition of tumor growth and metastasis was observed in both melanoma and Lewis lung carcinoma metastasis murine models [62]. Oral administration of mice with DNA vaccines encoding murine VEGFR-2 carried by attenuated *Salmonella typhimurium* could break the immune tolerance to VEGFR-2, induce CTL response to VEGFR-2, inhibit tumor cell-induced neoangiogenesis, and suppress the formation of spontaneous and experimental pulmonary metastases, with slight impact on wounds healing and no influence on hematopoiesis and pregnancy [63]. Immunization of mice with flk1-encoding mRNA-transfected DC could induce specific CTL response to VEGFR-2, partially inhibit the tumor cell-induced neoangiogenesis, and suppress tumor growth and metastasis in murine B16/F10.9 melanoma and MBT-2 bladder tumor models [64]. We studied

**2.2. Anti-angiogenic active immunotherapies based on non-xenogeneic homologous**

regimens.

applications.

**molecules**

apy for tumors.

*2.2.1. VEGFR-2*

### *<IP_ADDRESS>. DLL4*

Notch signaling has recently emerged as a critical regulator of developmental and tumor angiogenesis. Notch signaling in both endothelial and smooth muscle cells appears to provide critical regulatory information to these cells downstream of the initiating signal induced by VEGF [51, 52]. Studies in humans and mice have demonstrated that Notch ligand delta-like 4 (DLL4) is strongly expressed by the tumor vasculature and generally not by the tumor cells themselves. In various mouse models, strong DLL4 expression was observed in the majority of tumor vessels, contrasting with significantly lower vascular expression in adjacent normal tissues [51]. In humans, DLL4 expression was analyzed in tumors from kidney, bladder, colon, brain and breast [53, 54]. Robust DLL4 expression was observed specifically in the tumor vasculature in all of these tumor types, whereas DLL4 expression was low to undetectable in the vasculature of adjacent normal tissue. Furthermore, at least in the case of breast cancer, the degree of DLL4 expression correlated with outcome: tumors with high DLL4 in the vasculature progressed more rapidly [54]. These findings suggest that DLL4 is an attractive new thera‐ peutic anti-angiogenesis target. To generate the DLL4 plasmid vaccine, the cDNA encoding human DLL4 was cloned into the pVAX1 expression vector (DLL4 vaccine), which is specifically designed for the development of DNA vaccines and approved for use in humans. Immunization of Balb/c mice with DLL4 vaccine could bring about a break in tolerance against the self-antigen, DLL4. Readily detectable titers of serum antibodies against DLL4 were induced. Moreover, immunization with DLL4-encoding plasmid DNA severely retarded the growth of orthotopically implanted D2F2/E2 mammary carcinomas in mice by induction of a non-productive angiogenic response. In addition to the promising therapeutic effects, no evidence for a delayed wound healing response, or for toxicity associated with pharmacolog‐ ical blockade of DLL4 signaling, was observed in mice immunized with the DLL4 vaccine [55].

### *<IP_ADDRESS>. Angiomotin*

Angiomotin (Amot), one of angiostatin receptors [56], is a membrane-associated protein present on the endothelial cell surface of angiogenic tissues [57] characterized by conserved coiledcoil and carboxy termini-PDZ domains [58]. A shorter Amot isoform (p80) confers a hyper-migratory and invasive phenotype in transfected cells [59] and induces endothelial cell migration during angiogenesis [60]. The longer (p130) isoform localizes to tight junctions, regulates cell shape and appears to play a role in the later phase of angiogenesis [60]. It was demonstrated that increased Amot expression on tumor endothelia concomitant with the progression from pre-neoplastic lesions to full-fledged carcinoma, therefore, plasmid vaccine encoding human p80 Amot (pAmot) was constructed [57]. Immunization of mice with pAmot can overcome immune tolerance and induce a significant antibody response that mimic the effect of angiostatin. These antibodies inhibit endothelial cell migration, block tumor cell- and bFGF-induced angiogenesis in the matrigel plug assay and prevent growth of transplanted tumors without impairing normal stromal or retina vessels [57]. Very recently, Arigoni et al. further showed that the pAmot-induced antibodies alter tumor vessel permeability and structure. These combined effects of vaccine-induced anti-Amot antibodies lead to inhibition of established clinically evident mammary tumors, massive tumor perivascular necrosis, and an effective tumor antigen presentation in a form of epitope spreading that induces an immune response against other oncoantigens overexpressed by tumor cells [61]. Greater tumor vessel permeability also markedly boosts the local accumulation of doxorubicin and enhances the anti-tumor effect of the drugs [61]. These data provide a rationale for the development of fresh anticancer treatments based on anti-Amot vaccination in conjunction with chemotherapy regimens.

Taken together, it is obvious that vaccination with xenogeneic homologous molecules associ‐ ated with angiogenesis, such as pro-angiogenic factors, integrins, MMP, could induce antitumor immunity and thus might be a feasible strategy for cancer therapy with potential clinical applications.

#### **2.2. Anti-angiogenic active immunotherapies based on non-xenogeneic homologous molecules**

Given that vaccination with xenogeneic homologous molecules associated with tumor angiogenesis could effectively induce anti-tumor immunity, it can be assumed that vaccines based on non-xenogeneic homologous molecules, such as allogeneic homologues of some proangiogenic factors or other important molecules associated with angiogenesis, could also successfully induce specific and potent anti-tumor immunity. To date, several vaccines based on non-xenogeneic homologous molecules were used in anti-angiogenic active immunother‐ apy for tumors.

### *2.2.1. VEGFR-2*

activity of xRHAMM was mediated by both the antigen-specific cellular and humoral responses against RHAMM, as confirmed by the depletion of immune cell subsets *in vivo*. Furthermore, the anti-angiogenic and anti-tumor effects induced by vaccination of pcDNA3.1 xRHAMM were significantly stronger than that induced by vaccination of the corresponding

Notch signaling has recently emerged as a critical regulator of developmental and tumor angiogenesis. Notch signaling in both endothelial and smooth muscle cells appears to provide critical regulatory information to these cells downstream of the initiating signal induced by VEGF [51, 52]. Studies in humans and mice have demonstrated that Notch ligand delta-like 4 (DLL4) is strongly expressed by the tumor vasculature and generally not by the tumor cells themselves. In various mouse models, strong DLL4 expression was observed in the majority of tumor vessels, contrasting with significantly lower vascular expression in adjacent normal tissues [51]. In humans, DLL4 expression was analyzed in tumors from kidney, bladder, colon, brain and breast [53, 54]. Robust DLL4 expression was observed specifically in the tumor vasculature in all of these tumor types, whereas DLL4 expression was low to undetectable in the vasculature of adjacent normal tissue. Furthermore, at least in the case of breast cancer, the degree of DLL4 expression correlated with outcome: tumors with high DLL4 in the vasculature progressed more rapidly [54]. These findings suggest that DLL4 is an attractive new thera‐ peutic anti-angiogenesis target. To generate the DLL4 plasmid vaccine, the cDNA encoding human DLL4 was cloned into the pVAX1 expression vector (DLL4 vaccine), which is specifically designed for the development of DNA vaccines and approved for use in humans. Immunization of Balb/c mice with DLL4 vaccine could bring about a break in tolerance against the self-antigen, DLL4. Readily detectable titers of serum antibodies against DLL4 were induced. Moreover, immunization with DLL4-encoding plasmid DNA severely retarded the growth of orthotopically implanted D2F2/E2 mammary carcinomas in mice by induction of a non-productive angiogenic response. In addition to the promising therapeutic effects, no evidence for a delayed wound healing response, or for toxicity associated with pharmacolog‐ ical blockade of DLL4 signaling, was observed in mice immunized with the DLL4 vaccine [55].

Angiomotin (Amot), one of angiostatin receptors [56], is a membrane-associated protein present on the endothelial cell surface of angiogenic tissues [57] characterized by conserved coiledcoil and carboxy termini-PDZ domains [58]. A shorter Amot isoform (p80) confers a hyper-migratory and invasive phenotype in transfected cells [59] and induces endothelial cell migration during angiogenesis [60]. The longer (p130) isoform localizes to tight junctions, regulates cell shape and appears to play a role in the later phase of angiogenesis [60]. It was demonstrated that increased Amot expression on tumor endothelia concomitant with the progression from pre-neoplastic lesions to full-fledged carcinoma, therefore, plasmid vaccine encoding human p80 Amot (pAmot) was constructed [57]. Immunization of mice with pAmot can overcome immune tolerance and induce a significant antibody response that mimic the

autologous counterpart pcDNA3.1-mRHAMM [49].

374 Cancer Treatment - Conventional and Innovative Approaches

*<IP_ADDRESS>. DLL4*

*<IP_ADDRESS>. Angiomotin*

As has been discussed above, VEGF-mediated signaling pathway through VEGFR-2 is a ratelimiting step during tumor angiogenesis. Thus, VEGF/VEGFR-2 is still an ideal target in the non-xenogeneic homologous molecules-based anti-angiogenic strategy. Immunization of mice with VEGF receptor-2 (flk-1)-pulsed dendritic cells (DC) can break self-tolerance to VEGFR-2, induce CTL and antibody responses to VEGFR-2 [62]. Significant inhibition of tumor growth and metastasis was observed in both melanoma and Lewis lung carcinoma metastasis murine models [62]. Oral administration of mice with DNA vaccines encoding murine VEGFR-2 carried by attenuated *Salmonella typhimurium* could break the immune tolerance to VEGFR-2, induce CTL response to VEGFR-2, inhibit tumor cell-induced neoangiogenesis, and suppress the formation of spontaneous and experimental pulmonary metastases, with slight impact on wounds healing and no influence on hematopoiesis and pregnancy [63]. Immunization of mice with flk1-encoding mRNA-transfected DC could induce specific CTL response to VEGFR-2, partially inhibit the tumor cell-induced neoangiogenesis, and suppress tumor growth and metastasis in murine B16/F10.9 melanoma and MBT-2 bladder tumor models [64]. We studied the regulatory effects of IFN-γ on the differentiation and development of DC and found that IFN-γ is an autocrine mediator for DC maturation [65]. IFN-γ gene transfection could promote differentiation, development, and functional maturation of DC [66]. IFN-γ gene-modified DC had increased capacity to induce Th1 type immune response, and intratumoral injection of IFN-γ gene-modified DC in a murine model with pre-established B16 melanoma resulted in the potentiation of the anti-tumor effect of DC [66]. On the other hand, it was demonstrated that IFN-γ itself is also a negative regulator of neoangiogenesis [67]. In order to combine the anti-angiogenic immunotherapy with the cytokine immunotherapy, we constructed recombi‐ nant plasmid expressing murine VEGFR-2 extracellular domain (sVEGFR-2) and IFN-γ fusion protein, pcDNA3.1/sVEGFR-2-IFN-γ, and found that the fusion protein expressed by recombi‐ nant plasmid shared biological activities of both sVEGFR-2 and IFN-γ [68]. Immunization of mice with murine sVEGFR-2-IFN-γ fusion gene-transfected DC could significantly augment the CTL response to murine VEGFR-2 and pronouncedly inhibit tumor cell-induced angioge‐ nensis and tumor metastasis in comparison with murine sVEGFR2 gene-transfected DC [68].

In 2009, Seavey, et al developed *Listeria monocytogens* based VEGFR-2 vaccines that encode the peptide of VEGFR-2 extracellular domain fused to the first 441 residues of the microbial adjuvant listeriolysin O (*Lm*-LLO-Flk-E1 and *Lm*-LLO-Flk-E2) and the peptide of VEGFR-2 intracellular domain that also fused to LLO (*Lm*-LLO-Flk-I1), respectively [71, 72]. Immuni‐ zation of the mice with the *Listeria*-based Flk1 vaccines elicited potent antitumor CTL respons‐ es. Lm-LLO-Flk-1 was able to eradicate some established Her-2/neu+ breast tumors, reduce microvascular density in the remaining tumors, protect against tumor rechallenge and experimental metastases, and induce epitope spreading to various regions of the tumorassociated antigen Her-2/neu. Tumor eradication was found to be dependent on epitope spreading to Her-2/neu and was not solely due to the reduction of tumor vasculature [71]. In an autochthonous model for Her-2/neu+ breast cancer, theses vaccines could significantly delay tumor onset, while tumors that grew out overtime accumulated mutations in the Her-2/ neu molecule near or within CTL epitopes [72]. Moreover, vaccine efficacy did not affect normal wound healing nor have toxic side effects on pregnancy [71]. These data suggest that an anti-angiogenesis vaccine can overcome tolerance to the host vasculature driving epitope

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spreading to an endogenous tumor protein and drive active tumor regression.

the optimal dose for further clinical trials might be 2 mg/body or higher.

*2.2.2. bFGF*

Recently, a DNA vaccine (pSG.SS.Flk-1ECD.C3d3) encoding Flk-1 extracellular domain and the complement fragment C3d fusion protein was constructed [73]. Vaccination of mice with pSG.SS.Flk-1ECD.C3d3 could also elicit Flk-1 specific antibody response, leading to suppression of angiogenesis and tumor growth in bladder translational cell carcinoma mouse model, suggesting that C3d can be used as an adjuvant to enhance the immune response [73].

In 2010, Miyazawa, et al. [74] reported the results of phase I clinical trial combining of epitope peptide for VEGFR-2 (VEGFR2-169) with gemcitabine for patients with advanced pancreatic cancer. 18 HLA-A\*2402-positive patients with metastatic and unresectable pancreatic cancer were enrolled in the trial. Gemcitabine was administered at a dose of 1000 mg/m2 on days 1, 8, and 15 in a 28-day cycle. The VEGFR2-169 peptide was subcutaneously injected weekly in a dose-escalation manner (doses of 0.5, 1, and 2 mg/body, six patients/one cohort). Safety and immunological parameters were assessed. No severe adverse effect of grade 4 or higher was observed. Of the 18 patients who completed at least one course of the treatment, 15 (83%) developed immunological reactions at the injection sites. VEGFR2-169 specific CTLs were induced in 11 (61%) of the 18 patients. The disease control rate was 67%, and the median overall survival time was 8.7 months. This combination therapy for pancreatic cancer patients was tolerable at all doses. Peptide-specific CTL could be induced by the VEGFR2-169 peptide vaccine at a high rate, even in combination with gemcitabine. Therefore, they suggested that

Basic fibroblast growth factor (bFGF/FGF2) is an important proangiogenic factor, which is secreted by tumor cells and macrophages or released by extracelluar matrix, and functions in the autocrine or paracrine manner. FGF2 can upregulate the expression of several dominant pro-angiogenic factors, such as VEGF [75], and activator of plasminogen [76], and inhibit apoptosis of endothelial cells by bcl-2 pathway [77]. bFGF exerts its biological activities

In 2006, three CTL epitope candidates, designated as KDR1, KDR2 and KDR3, respectively, from VEGFR-2 with high binding affinity to the H-2Db molecule were predicted by two computer programs: Bimas and SYFPEITH [69]. Two of them, KDR2 and KDR3, were from the extracellular domain; KDR1 was from the intracellular part of the receptor [69]. Immuni‐ zation of mice with KDR2 or KDR3 peptide in combination with murine GM-CSF and agonist anti-mouse CD40 antibodies as adjuvant could break self-tolerance and induce specific immune responses in C57BL/6 mice [69]. Furthermore, immunization of mice with these two peptide epitopes elicited pronounced specific CTL responses to murine VEGFR-2, effectively inhibited VEGF-induced angiogenesis, and suppressed tumor growth in MC38 murine colon cancer model [69]. Similarly, the epitope peptides of human VEGFR-2 restricted by HLA-A\*0201 and HLA-A\*2402 were also identified by analyzing the binding affinities to the corresponding HLA molecules [70]. Antigen based on the epitope peptide with high binding affinity to human HLA-A\*0201 could successfully induce specific CTL response *in vitro* [70]. Furthermore, transgenic mice expressing HLA-A\*0201, A2/Kb, were generated, and the vascular endothelial cells in that mice could not only express human VEGFR-2 (KDR), but also express human MHC class Ι molecules [70]. After inoculation of A2/Kb with HLA-A\*0201 restricted VEGFR-2 epitope peptide, specific IFN-γ-expressed CTL was induced [70]. Immu‐ nization of tumor-bearing A2/Kb transgenic mice with VEGFR-2 epitope peptide could markedly inhibit tumor-induced angiogenesis, hereby inhibiting tumor growth in MC38 colon cancer and B16 melanoma models, and prolong survival of the tumor-bearing animals without fatal adverse effects [70]. To further study whether specific CTL response to KDR can be elicited in human or not, KDR epitope peptide vaccines were used to stimulate peripheral blood mononuclear cells derived from 6 cancer patients *in vitro*, and CTLs specific for the peptide epitope were successfully induced in all patients [70].

In comparison with the full-length protein, peptide vaccines like the aforementioned KDR epitope peptides can be easily synthesized in high purity and are less expensive. Moreover, immunization with such vaccines could avoid the potential dangers involving induction of an infection by recombinant viruses or exposure to a latently allergenic exogenous protein.

In 2009, Seavey, et al developed *Listeria monocytogens* based VEGFR-2 vaccines that encode the peptide of VEGFR-2 extracellular domain fused to the first 441 residues of the microbial adjuvant listeriolysin O (*Lm*-LLO-Flk-E1 and *Lm*-LLO-Flk-E2) and the peptide of VEGFR-2 intracellular domain that also fused to LLO (*Lm*-LLO-Flk-I1), respectively [71, 72]. Immuni‐ zation of the mice with the *Listeria*-based Flk1 vaccines elicited potent antitumor CTL respons‐ es. Lm-LLO-Flk-1 was able to eradicate some established Her-2/neu+ breast tumors, reduce microvascular density in the remaining tumors, protect against tumor rechallenge and experimental metastases, and induce epitope spreading to various regions of the tumorassociated antigen Her-2/neu. Tumor eradication was found to be dependent on epitope spreading to Her-2/neu and was not solely due to the reduction of tumor vasculature [71]. In an autochthonous model for Her-2/neu+ breast cancer, theses vaccines could significantly delay tumor onset, while tumors that grew out overtime accumulated mutations in the Her-2/ neu molecule near or within CTL epitopes [72]. Moreover, vaccine efficacy did not affect normal wound healing nor have toxic side effects on pregnancy [71]. These data suggest that an anti-angiogenesis vaccine can overcome tolerance to the host vasculature driving epitope spreading to an endogenous tumor protein and drive active tumor regression.

Recently, a DNA vaccine (pSG.SS.Flk-1ECD.C3d3) encoding Flk-1 extracellular domain and the complement fragment C3d fusion protein was constructed [73]. Vaccination of mice with pSG.SS.Flk-1ECD.C3d3 could also elicit Flk-1 specific antibody response, leading to suppression of angiogenesis and tumor growth in bladder translational cell carcinoma mouse model, suggesting that C3d can be used as an adjuvant to enhance the immune response [73].

In 2010, Miyazawa, et al. [74] reported the results of phase I clinical trial combining of epitope peptide for VEGFR-2 (VEGFR2-169) with gemcitabine for patients with advanced pancreatic cancer. 18 HLA-A\*2402-positive patients with metastatic and unresectable pancreatic cancer were enrolled in the trial. Gemcitabine was administered at a dose of 1000 mg/m2 on days 1, 8, and 15 in a 28-day cycle. The VEGFR2-169 peptide was subcutaneously injected weekly in a dose-escalation manner (doses of 0.5, 1, and 2 mg/body, six patients/one cohort). Safety and immunological parameters were assessed. No severe adverse effect of grade 4 or higher was observed. Of the 18 patients who completed at least one course of the treatment, 15 (83%) developed immunological reactions at the injection sites. VEGFR2-169 specific CTLs were induced in 11 (61%) of the 18 patients. The disease control rate was 67%, and the median overall survival time was 8.7 months. This combination therapy for pancreatic cancer patients was tolerable at all doses. Peptide-specific CTL could be induced by the VEGFR2-169 peptide vaccine at a high rate, even in combination with gemcitabine. Therefore, they suggested that the optimal dose for further clinical trials might be 2 mg/body or higher.

#### *2.2.2. bFGF*

the regulatory effects of IFN-γ on the differentiation and development of DC and found that IFN-γ is an autocrine mediator for DC maturation [65]. IFN-γ gene transfection could promote differentiation, development, and functional maturation of DC [66]. IFN-γ gene-modified DC had increased capacity to induce Th1 type immune response, and intratumoral injection of IFN-γ gene-modified DC in a murine model with pre-established B16 melanoma resulted in the potentiation of the anti-tumor effect of DC [66]. On the other hand, it was demonstrated that IFN-γ itself is also a negative regulator of neoangiogenesis [67]. In order to combine the anti-angiogenic immunotherapy with the cytokine immunotherapy, we constructed recombi‐ nant plasmid expressing murine VEGFR-2 extracellular domain (sVEGFR-2) and IFN-γ fusion protein, pcDNA3.1/sVEGFR-2-IFN-γ, and found that the fusion protein expressed by recombi‐ nant plasmid shared biological activities of both sVEGFR-2 and IFN-γ [68]. Immunization of mice with murine sVEGFR-2-IFN-γ fusion gene-transfected DC could significantly augment the CTL response to murine VEGFR-2 and pronouncedly inhibit tumor cell-induced angioge‐ nensis and tumor metastasis in comparison with murine sVEGFR2 gene-transfected DC [68].

In 2006, three CTL epitope candidates, designated as KDR1, KDR2 and KDR3, respectively,

computer programs: Bimas and SYFPEITH [69]. Two of them, KDR2 and KDR3, were from the extracellular domain; KDR1 was from the intracellular part of the receptor [69]. Immuni‐ zation of mice with KDR2 or KDR3 peptide in combination with murine GM-CSF and agonist anti-mouse CD40 antibodies as adjuvant could break self-tolerance and induce specific immune responses in C57BL/6 mice [69]. Furthermore, immunization of mice with these two peptide epitopes elicited pronounced specific CTL responses to murine VEGFR-2, effectively inhibited VEGF-induced angiogenesis, and suppressed tumor growth in MC38 murine colon cancer model [69]. Similarly, the epitope peptides of human VEGFR-2 restricted by HLA-A\*0201 and HLA-A\*2402 were also identified by analyzing the binding affinities to the corresponding HLA molecules [70]. Antigen based on the epitope peptide with high binding affinity to human HLA-A\*0201 could successfully induce specific CTL response *in vitro* [70]. Furthermore, transgenic mice expressing HLA-A\*0201, A2/Kb, were generated, and the vascular endothelial cells in that mice could not only express human VEGFR-2 (KDR), but also express human MHC class Ι molecules [70]. After inoculation of A2/Kb with HLA-A\*0201 restricted VEGFR-2 epitope peptide, specific IFN-γ-expressed CTL was induced [70]. Immu‐ nization of tumor-bearing A2/Kb transgenic mice with VEGFR-2 epitope peptide could markedly inhibit tumor-induced angiogenesis, hereby inhibiting tumor growth in MC38 colon cancer and B16 melanoma models, and prolong survival of the tumor-bearing animals without fatal adverse effects [70]. To further study whether specific CTL response to KDR can be elicited in human or not, KDR epitope peptide vaccines were used to stimulate peripheral blood mononuclear cells derived from 6 cancer patients *in vitro*, and CTLs specific for the peptide

In comparison with the full-length protein, peptide vaccines like the aforementioned KDR epitope peptides can be easily synthesized in high purity and are less expensive. Moreover, immunization with such vaccines could avoid the potential dangers involving induction of an infection by recombinant viruses or exposure to a latently allergenic exogenous protein.

molecule were predicted by two

from VEGFR-2 with high binding affinity to the H-2Db

376 Cancer Treatment - Conventional and Innovative Approaches

epitope were successfully induced in all patients [70].

Basic fibroblast growth factor (bFGF/FGF2) is an important proangiogenic factor, which is secreted by tumor cells and macrophages or released by extracelluar matrix, and functions in the autocrine or paracrine manner. FGF2 can upregulate the expression of several dominant pro-angiogenic factors, such as VEGF [75], and activator of plasminogen [76], and inhibit apoptosis of endothelial cells by bcl-2 pathway [77]. bFGF exerts its biological activities through its binding to high affinity receptor, fibroblast growth factor receptor-1 (FGFR1). It was found that both peptide segments of synthetic human FGF2 heparin-binding structural domain and receptor-binding structural domain could inhibit the *in vitro* proliferation of human umbilical vein endothelial cells [39]. Immunization of mice with vaccine based on heparin-binding structural domain peptide could induce production of anti-FGF2 specific antibody, which could hamper the binding of FGF2 to heparin sulphate, and inhibit tumorinduced angiogenesis in a gelatin sponge model and tumor growth in a tumor metastatic model [39]. Surprisingly, despite an immune response toward FGF2, this modality of treatment did not affect wound healing as shown by the fact that the treatment did not alter the mean time of wound healing [78]. It also did not affect fertility, because the vaccinated females were not impaired in their ability to become pregnant, to support the growth and development of their embryos, and to deliver viable offspring when compared with control animals [78]. Furthermore, histological analyses did not reveal any alterations in organogenesis in these offsprings [78]. Therefore, the authors concluded that although vaccination against FGF2 induced a specific FGF2 antibody response and inhibited angiogenesis and tumor develop‐ ment in a pathological setting, it did not adversely alter normal physiological events dependent on FGF2.

*2.2.4. Legumain*

a prophylactic setting [85].

*2.2.5. Endoglin (CD105)*

, AroA–

(dam–

Tumor associated macrophages (TAMs) are well known to play a very important role in tumor angiogenesis and metastasis, as the abrogation of TAMs in tumor tissues effectively reduced several pro-tumor growth and angiogenesis factors, such as VEGF, TGF-β, TNF-α and MMP-9 [81]. Thus, the suppression of TAMs in the tumor-microenvironment provides a novel strategy to inhibit tumor growth and dissemination by remodeling the tumor's stroma. Legumain is an asparaginyl endopeptidase and a member of the C13 family of cystine proteases which was found to be highly upregulated in many murine and human tumor tissues and, furthermore, also overexpressed on TAMs in the murine tumor stroma, but absent or present at only very low levels in all normal tissues from which such tumors arose [81-84]. Recently, several oral minigene vaccines against murine MHC class I antigen epitopes of Legumain were constructed based on the binding predictions for these MHC class I molecules by the HLA peptide binding predictions program [85]. Expression vectors encoding these epitopes were designated as pLegu-H-2Dd and pLegu-H-2Kd respectively [85]. Oral administration of those vaccines by

Anti-Angiogenic Active Immunotherapy for Cancers: Dawn of a New Era?

significant suppression of angiogenesis in tumor tissues of D2F2 breast carcinoma in syngeneic BALB/c mice [85]. The possible mechanism of angiogenic inhibition involved the induction of a specific CTL response capable of killing Legumain positive cells, especially TAMs, which is likely to be responsible for anti-tumor angiogenesis [85]. Generally, the anti-angiogenic effect aided in the protection of BALB/c mice from lethal challenges with D2F2 breast tumor cells in

Endoglin, a 95 kDa cell surface protein expressed as a homodimer, functions as an accessory protein for kinase receptor complexes of the TGF-β superfamily and modulates TGF-β signaling [8]. Expression of CD105 is correlated with vascular density and poor prognosis [8]. Endoglin is over-expressed on proliferating endothelial cells in the breast tumor neovascula‐ ture and thus offers a target for anti-angiogenic therapy [8]. It was reported that an oral murine endoglin-encoding DNA vaccine carried by double attenuated Salmonella typhimurium

, AroA–

http://dx.doi.org/10.5772/55323

379

s patches, resulted in activation of

) resulted in

T cells

transforming them into attenuated Salmonella typhimurium (Dam–

) to a secondary lymphoid organ, i.e., Peyer'

antigen-presenting dendritic cells, induction of immune responses mediated by CD8+

against endoglin-positive target cells, and suppression of angiogenesis and dissemination of pulmonary metastases of D2F2 breast carcinoma cells presumably by eliminating proliferating endothelial cells in the tumor vasculature, thus providing an promising strategy to therapies for breast cancer [86]. More recently, Wood et al. [87] developed *Listeria*-based vaccines directed against CD105, Lm-LLO-CD105A and Lm-LLO-CD105B. The region of CD105 in Lm-LLO-CD105A vaccine contains at least three predicted H-2Kd epitopes, while the region of CD105 in Lm-LLO-CD105B contains at least two predicted H-2Kd epitopes. Immunization of the *Listeria*-based vaccines led to therapeutic responses against primary and metastatic tumors in the 4T1-Luc and NT-2 mouse models of breast cancer. In a mouse model for autochthonous Her-2/neu-driven breast cancer, Lm-LLO-CD105A vaccination prevented tumor incidence in 20% of mice by week 58 after birth while all control mice developed tumors by week 40. In

#### *2.2.3. EGFR*

Epidermal growth factor receptor (EGFR), a membrane surface sensor with tyrosine kinase activity, is widely distributed on the membrane of mammalian cells [79]. In the physiological condition, EGFR exerts, through binding to ligands (epidermal growth factor, EGF), its physiological activities in regulation of cell division, proliferation and differentiation [79]. Results from clinical studies show that high expression level of EGFR is frequently observed in non-small cell lung cancer, and has been implicated in aggressive biological behavior of tumor cells and poor prognosis of tumor patients [79]. Therefore, immunotherapy targeting EGFR should be another attractive approach to the treatment of EGFR-positive tumors. In murine tumor models with Lewis lung carcinoma and mammary cancer, immunization of mice with DC pulsed with recombinant ectodomain of mouse EGFR (DC-edMER) inhibited tumor angiogenesis, reduced tumor growth, and prolonged the survival of tumor-bearing mice [80]. Spleen cells isolated from DC-edMER-immunized mice showed a high frequency of EGFR-specific antibody-producing cells [80]. Anti-EGFR specific antibody was markedly elevated in sera of immunized mice and was shown to be effective against tumor growth by adoptive transfer [80]. Immunization with DC-edMER vaccine also elicited CTL responses [80]. Depletion of CD4+ T lymphocytes could completely abrogate the anti-tumor activity and generation of EGFR-specific antibody responses, whereas depletion of CD8+ T lymphocytes showed partial abrogation of the anti-tumor activity but antibody was still detected [80]. Furthermore, tumor-induced angiogenesis was suppressed in DC-edMER-immunized mice or mice treated with antibody adoptive transfer [80]. These findings indicate that vaccination with DC-edMER can induce both humoral and cellular anti-tumor immunity, and may suggest novel strategies for the treatment of EGFR-positive tumors through the induction of active immunity against EGFR [80].

### *2.2.4. Legumain*

through its binding to high affinity receptor, fibroblast growth factor receptor-1 (FGFR1). It was found that both peptide segments of synthetic human FGF2 heparin-binding structural domain and receptor-binding structural domain could inhibit the *in vitro* proliferation of human umbilical vein endothelial cells [39]. Immunization of mice with vaccine based on heparin-binding structural domain peptide could induce production of anti-FGF2 specific antibody, which could hamper the binding of FGF2 to heparin sulphate, and inhibit tumorinduced angiogenesis in a gelatin sponge model and tumor growth in a tumor metastatic model [39]. Surprisingly, despite an immune response toward FGF2, this modality of treatment did not affect wound healing as shown by the fact that the treatment did not alter the mean time of wound healing [78]. It also did not affect fertility, because the vaccinated females were not impaired in their ability to become pregnant, to support the growth and development of their embryos, and to deliver viable offspring when compared with control animals [78]. Furthermore, histological analyses did not reveal any alterations in organogenesis in these offsprings [78]. Therefore, the authors concluded that although vaccination against FGF2 induced a specific FGF2 antibody response and inhibited angiogenesis and tumor develop‐ ment in a pathological setting, it did not adversely alter normal physiological events dependent

378 Cancer Treatment - Conventional and Innovative Approaches

Epidermal growth factor receptor (EGFR), a membrane surface sensor with tyrosine kinase activity, is widely distributed on the membrane of mammalian cells [79]. In the physiological condition, EGFR exerts, through binding to ligands (epidermal growth factor, EGF), its physiological activities in regulation of cell division, proliferation and differentiation [79]. Results from clinical studies show that high expression level of EGFR is frequently observed in non-small cell lung cancer, and has been implicated in aggressive biological behavior of tumor cells and poor prognosis of tumor patients [79]. Therefore, immunotherapy targeting EGFR should be another attractive approach to the treatment of EGFR-positive tumors. In murine tumor models with Lewis lung carcinoma and mammary cancer, immunization of mice with DC pulsed with recombinant ectodomain of mouse EGFR (DC-edMER) inhibited tumor angiogenesis, reduced tumor growth, and prolonged the survival of tumor-bearing mice [80]. Spleen cells isolated from DC-edMER-immunized mice showed a high frequency of EGFR-specific antibody-producing cells [80]. Anti-EGFR specific antibody was markedly elevated in sera of immunized mice and was shown to be effective against tumor growth by adoptive transfer [80]. Immunization with DC-edMER vaccine also elicited CTL responses [80].

generation of EGFR-specific antibody responses, whereas depletion of CD8+

showed partial abrogation of the anti-tumor activity but antibody was still detected [80]. Furthermore, tumor-induced angiogenesis was suppressed in DC-edMER-immunized mice or mice treated with antibody adoptive transfer [80]. These findings indicate that vaccination with DC-edMER can induce both humoral and cellular anti-tumor immunity, and may suggest novel strategies for the treatment of EGFR-positive tumors through the induction of active

T lymphocytes could completely abrogate the anti-tumor activity and

T lymphocytes

on FGF2.

*2.2.3. EGFR*

Depletion of CD4+

immunity against EGFR [80].

Tumor associated macrophages (TAMs) are well known to play a very important role in tumor angiogenesis and metastasis, as the abrogation of TAMs in tumor tissues effectively reduced several pro-tumor growth and angiogenesis factors, such as VEGF, TGF-β, TNF-α and MMP-9 [81]. Thus, the suppression of TAMs in the tumor-microenvironment provides a novel strategy to inhibit tumor growth and dissemination by remodeling the tumor's stroma. Legumain is an asparaginyl endopeptidase and a member of the C13 family of cystine proteases which was found to be highly upregulated in many murine and human tumor tissues and, furthermore, also overexpressed on TAMs in the murine tumor stroma, but absent or present at only very low levels in all normal tissues from which such tumors arose [81-84]. Recently, several oral minigene vaccines against murine MHC class I antigen epitopes of Legumain were constructed based on the binding predictions for these MHC class I molecules by the HLA peptide binding predictions program [85]. Expression vectors encoding these epitopes were designated as pLegu-H-2Dd and pLegu-H-2Kd respectively [85]. Oral administration of those vaccines by transforming them into attenuated Salmonella typhimurium (Dam– , AroA– ) resulted in significant suppression of angiogenesis in tumor tissues of D2F2 breast carcinoma in syngeneic BALB/c mice [85]. The possible mechanism of angiogenic inhibition involved the induction of a specific CTL response capable of killing Legumain positive cells, especially TAMs, which is likely to be responsible for anti-tumor angiogenesis [85]. Generally, the anti-angiogenic effect aided in the protection of BALB/c mice from lethal challenges with D2F2 breast tumor cells in a prophylactic setting [85].

### *2.2.5. Endoglin (CD105)*

Endoglin, a 95 kDa cell surface protein expressed as a homodimer, functions as an accessory protein for kinase receptor complexes of the TGF-β superfamily and modulates TGF-β signaling [8]. Expression of CD105 is correlated with vascular density and poor prognosis [8]. Endoglin is over-expressed on proliferating endothelial cells in the breast tumor neovascula‐ ture and thus offers a target for anti-angiogenic therapy [8]. It was reported that an oral murine endoglin-encoding DNA vaccine carried by double attenuated Salmonella typhimurium (dam– , AroA– ) to a secondary lymphoid organ, i.e., Peyer' s patches, resulted in activation of antigen-presenting dendritic cells, induction of immune responses mediated by CD8+ T cells against endoglin-positive target cells, and suppression of angiogenesis and dissemination of pulmonary metastases of D2F2 breast carcinoma cells presumably by eliminating proliferating endothelial cells in the tumor vasculature, thus providing an promising strategy to therapies for breast cancer [86]. More recently, Wood et al. [87] developed *Listeria*-based vaccines directed against CD105, Lm-LLO-CD105A and Lm-LLO-CD105B. The region of CD105 in Lm-LLO-CD105A vaccine contains at least three predicted H-2Kd epitopes, while the region of CD105 in Lm-LLO-CD105B contains at least two predicted H-2Kd epitopes. Immunization of the *Listeria*-based vaccines led to therapeutic responses against primary and metastatic tumors in the 4T1-Luc and NT-2 mouse models of breast cancer. In a mouse model for autochthonous Her-2/neu-driven breast cancer, Lm-LLO-CD105A vaccination prevented tumor incidence in 20% of mice by week 58 after birth while all control mice developed tumors by week 40. In comparison with previous *Listeria*-based vaccines (Lm-LLO-HMWMAA-C [88] and Lm-LLO-FLK-I1 and Lm-LLO-FLK-E2 [71] ) targeting tumor vasculature, Lm-LLO-CD105A and Lm-LLO-CD105B demonstrated equivalent or superior efficacy against two transplantable mouse models of breast cancer. Mechanism analysis revealed that the anti-tumor therapeutic efficacy of *Listeria*-based CD105 vaccines was mediated by epitope spreading to endogenous tumor antigens and reduction in tumor vascularity [87]. These data suggest that CD105 therapeutic vaccines are highly effective in stimulating anti-angiogenesis and anti-tumor immune responses leading to therapeutic efficacy against primary and metastatic breast cancer.

inhibitors and angiogenic antibodies, anti-angiogenic active immunotherapy has its obvious merits. Provided that a break of immunological tolerance to positive regulators of angiogenesis is successfully induced, the long-lasting immune response to angiogenesis-related molecule will be present in the body, hereby providing long-lasting inhibitory effects on angiogenesis. Therefore, it is expected to be the more cost-effective strategy than angiogenic inhibitor or anti-

Anti-Angiogenic Active Immunotherapy for Cancers: Dawn of a New Era?

http://dx.doi.org/10.5772/55323

381

Here we divided anti-angiogenic active immunotherapy into two categories: therapies based on vaccination with xenogeneic homologous molecules and with non-xenogeneic homologous molecules related to angiogenesis. Presently, it is difficult to point out which one is better for clinical application because most of the outcomes reported to date were based on pre-clinical animal experiments. As VEGF-mediated signaling through its receptor VEGFR-2 is the key rate-limiting step in tumor angiogenesis, and plays the most impor‐ tant role in neovascularization, development, and progression of various tumors [6], as well as human VEGFR2-169 peptide vaccination could effectively break peripheral self toler‐ ance against VEGFR-2 in patients with metastatic and unresectable pancreatic cancer [74], anti-angiogenic active immunotherapy targeting VEGF or VEGFR-2 might be the most effective strategy among all these therapies. Moreover, considering the potential clinical application of anti-angiogenic immunotherapy based on the specific antibodies raised against a variety of angiogenesis-associated molecules in different tumor entities like glioma, renal cell cancer, and breast cancer, etc, a promising clinical application of antiangiogenic active immunotherapy alone or in combination with other anti-tumor strat‐ egies could be expected. However, there exist as well *caveats* and deficiencies in this strategy. Firstly, in the early phase of tumor growth when the tumor diameter is less than 2-3 mm, tumor cells simply depend on passive diffusion rather than blood perfusion to acquire enough oxygen and nutrition indispensable for growth. Therefore, anti-angiogen‐ ic therapy against tumor in this early stage might be ineffective when applied alone. Secondly, although current anti-angiogenic active immunotherapy is focused on specific targets, potential adverse effects might include impairment of wound healing and menstru‐ al cycle. Furthermore, this approach has also limited application perspectives in children with cancers. Therefore, along with recent developments in molecular biology and immunology, future studies will focus on multiple approaches, such as series analysis of gene expression to analyze the gene expression in normal endothelial cells and in prolifer‐ ative endothelial cells, phage display technology to search for new endothelial cell receptors, and proteomics to discover peptide segments or proteins regulating endothelial cell growth. These approaches are expected to discover more tumor-specific endothelial cell markers for the purpose of selecting specific targets for anti-angiogenic active immunother‐ apy. In addition, further studies are also required to optimize protocols how to construct vaccines to effectively break self-tolerance and to induce efficient immune response. With these issues being solved continuously, anti-angiogenic active immunotherapy for cancers

angiogenic antibody therapy where continuous use of the drugs is needed.

will become more applicable and effective.

#### *2.2.6. Endothelial cell lysates-pulsed dendritic cells*

Dendritic cells (DCs) are the most potent professional antigen-presenting cells, they play crucial roles in the initiation of an immune response. DCs prepared from BALB/c mouse were pulsed with lysates of autologous or xenogeneic endothelium, and their anti-tumor effects were tested in two syngeneic models of colon cancer [89]. Immunization of endothelium lysates pulsed DCs could induce a break in self tolerance against endothelial cells and mount both the endothelium-specific CTL response and antibody response, leading to significant inhibition of tumor angiogenesis and the growth of subcutaneous tumors as well as pulmonary meta‐ stases in mice. Furthermore, the decrease in the mean vascular density of tumors correlates well with the extent of tumor inhibition [89]. Therefore, immunization of endothelium lysates pulsed DCs is also an effective modality of anti-angiogenic active immunotherapy for cancers, and should have important clinical implications for adjuvant cancer therapy.

### *2.2.7. Endothelial cell vaccine*

In 2008, Okaji Y, et al. [90] reported a pilot phase I clinical study in which glutaraldehydefixed human umbilical vein endothelial cells (HUVECs) were used as the vaccine. Six patients with recurrent malignant brain tumour and three patients with metastatic colorectal cancer were given intradermal injections of 5x107 HUVECs/dose, first month weekly, and then every 2 weeks (in total 230 vaccinations). ELISA and flow cytometry revealed immunoglobulin response against HUVECs' membrane antigens. ELISPOT and 51Cr-release cytotoxicity assay revealed a specific cellular immune response against HUVECs, which were lysed in an effectors:targets ratio-dependent manner.  Except for a DTH-like skin reaction at the injection site, no adverse effect of vaccination was observed. These results suggested that the endothelial vaccine can overcome peripheral tolerance of self-angiogenic antigens in clinical settings, and there‐ fore could be useful for adjuvant immunotherapy of cancer.

### **3. Concluding remarks**

Recent research achievements have disclosed inspiring pragmatic perspectives of antiangiogenic active immunotherapy for cancers. In comparison with application of angiogenic inhibitors and angiogenic antibodies, anti-angiogenic active immunotherapy has its obvious merits. Provided that a break of immunological tolerance to positive regulators of angiogenesis is successfully induced, the long-lasting immune response to angiogenesis-related molecule will be present in the body, hereby providing long-lasting inhibitory effects on angiogenesis. Therefore, it is expected to be the more cost-effective strategy than angiogenic inhibitor or antiangiogenic antibody therapy where continuous use of the drugs is needed.

comparison with previous *Listeria*-based vaccines (Lm-LLO-HMWMAA-C [88] and Lm-LLO-FLK-I1 and Lm-LLO-FLK-E2 [71] ) targeting tumor vasculature, Lm-LLO-CD105A and Lm-LLO-CD105B demonstrated equivalent or superior efficacy against two transplantable mouse models of breast cancer. Mechanism analysis revealed that the anti-tumor therapeutic efficacy of *Listeria*-based CD105 vaccines was mediated by epitope spreading to endogenous tumor antigens and reduction in tumor vascularity [87]. These data suggest that CD105 therapeutic vaccines are highly effective in stimulating anti-angiogenesis and anti-tumor immune responses leading to therapeutic efficacy against primary and metastatic breast cancer.

Dendritic cells (DCs) are the most potent professional antigen-presenting cells, they play crucial roles in the initiation of an immune response. DCs prepared from BALB/c mouse were pulsed with lysates of autologous or xenogeneic endothelium, and their anti-tumor effects were tested in two syngeneic models of colon cancer [89]. Immunization of endothelium lysates pulsed DCs could induce a break in self tolerance against endothelial cells and mount both the endothelium-specific CTL response and antibody response, leading to significant inhibition of tumor angiogenesis and the growth of subcutaneous tumors as well as pulmonary meta‐ stases in mice. Furthermore, the decrease in the mean vascular density of tumors correlates well with the extent of tumor inhibition [89]. Therefore, immunization of endothelium lysates pulsed DCs is also an effective modality of anti-angiogenic active immunotherapy for cancers,

In 2008, Okaji Y, et al. [90] reported a pilot phase I clinical study in which glutaraldehydefixed human umbilical vein endothelial cells (HUVECs) were used as the vaccine. Six patients with recurrent malignant brain tumour and three patients with metastatic colorectal cancer were given intradermal injections of 5x107 HUVECs/dose, first month weekly, and then every 2 weeks (in total 230 vaccinations). ELISA and flow cytometry revealed immunoglobulin response against HUVECs' membrane antigens. ELISPOT and 51Cr-release cytotoxicity assay revealed a specific cellular immune response against HUVECs, which were lysed in an effectors:targets ratio-dependent manner.  Except for a DTH-like skin reaction at the injection site, no adverse effect of vaccination was observed. These results suggested that the endothelial vaccine can overcome peripheral tolerance of self-angiogenic antigens in clinical settings, and there‐

Recent research achievements have disclosed inspiring pragmatic perspectives of antiangiogenic active immunotherapy for cancers. In comparison with application of angiogenic

and should have important clinical implications for adjuvant cancer therapy.

fore could be useful for adjuvant immunotherapy of cancer.

*2.2.6. Endothelial cell lysates-pulsed dendritic cells*

380 Cancer Treatment - Conventional and Innovative Approaches

*2.2.7. Endothelial cell vaccine*

**3. Concluding remarks**

Here we divided anti-angiogenic active immunotherapy into two categories: therapies based on vaccination with xenogeneic homologous molecules and with non-xenogeneic homologous molecules related to angiogenesis. Presently, it is difficult to point out which one is better for clinical application because most of the outcomes reported to date were based on pre-clinical animal experiments. As VEGF-mediated signaling through its receptor VEGFR-2 is the key rate-limiting step in tumor angiogenesis, and plays the most impor‐ tant role in neovascularization, development, and progression of various tumors [6], as well as human VEGFR2-169 peptide vaccination could effectively break peripheral self toler‐ ance against VEGFR-2 in patients with metastatic and unresectable pancreatic cancer [74], anti-angiogenic active immunotherapy targeting VEGF or VEGFR-2 might be the most effective strategy among all these therapies. Moreover, considering the potential clinical application of anti-angiogenic immunotherapy based on the specific antibodies raised against a variety of angiogenesis-associated molecules in different tumor entities like glioma, renal cell cancer, and breast cancer, etc, a promising clinical application of antiangiogenic active immunotherapy alone or in combination with other anti-tumor strat‐ egies could be expected. However, there exist as well *caveats* and deficiencies in this strategy. Firstly, in the early phase of tumor growth when the tumor diameter is less than 2-3 mm, tumor cells simply depend on passive diffusion rather than blood perfusion to acquire enough oxygen and nutrition indispensable for growth. Therefore, anti-angiogen‐ ic therapy against tumor in this early stage might be ineffective when applied alone. Secondly, although current anti-angiogenic active immunotherapy is focused on specific targets, potential adverse effects might include impairment of wound healing and menstru‐ al cycle. Furthermore, this approach has also limited application perspectives in children with cancers. Therefore, along with recent developments in molecular biology and immunology, future studies will focus on multiple approaches, such as series analysis of gene expression to analyze the gene expression in normal endothelial cells and in prolifer‐ ative endothelial cells, phage display technology to search for new endothelial cell receptors, and proteomics to discover peptide segments or proteins regulating endothelial cell growth. These approaches are expected to discover more tumor-specific endothelial cell markers for the purpose of selecting specific targets for anti-angiogenic active immunother‐ apy. In addition, further studies are also required to optimize protocols how to construct vaccines to effectively break self-tolerance and to induce efficient immune response. With these issues being solved continuously, anti-angiogenic active immunotherapy for cancers will become more applicable and effective.

### **Acknowledgements**

This work was supported by a grant from the Science and Technology Bureau of Hangzhou, Zhejiang Province, P.R. China (No. 20120633B30).

[11] Caldini R, Barletta E, Del Rosso M, et al. FGF2-mediated upregulation of urokinasetype plasminogen activator expression requires a MAP-kinase dependent activation

Anti-Angiogenic Active Immunotherapy for Cancers: Dawn of a New Era?

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383

[12] Ferrajoli A, Manshouri T, Estrov Z, et al. High levels of vascular endothelial growth factor receptor-2 correlate with shortened survival in chronic lymphocytic leukemia.

[13] Schneider BP, Sledge GW. Drug insight: VEGF as a therapeutic target for breast can‐

[14] Kanda S, Miyata Y, Kanetake H. Current status and perspective of antiangiogenic

[15] Rini BI, Rathmell WK. Biological aspects and binding strategies of vascular endothe‐ lial growth factor in renal cell carcinoma. Clin Cancer Res 2007; 13:741-746.

[16] Kerbel RS, Folkman J. Clinical translation of angiogenesis inhibitors. Nat Rev Cancer

[17] Shant K, Li CG. Targeting of vasculature in cancer and other angiogenic diseases.

[18] St Croix B, Rago C, Velculescu V, et al. Genes expressed in human tumor endotheli‐

[19] Reisfeld RA, Niethammer AG, Luo Y, et al. DNA vaccines designed to inhibit tumor growth by suppression of angiogenesis. Int Arch Allergy Immunol 2004; 133:295-304.

[20] Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, fluo‐ rouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004;

[21] Sandler A, Gray R, Perry MC, et al. Paclitaxel-carboplatin alone or with bevacizumab

[22] Miller K, Wang M, Gralow J, et al. Paclitaxel plus bevacizumab versus paclitaxel

[23] Willett CG, Duda DG, di Tomaso E, et al. Efficacy, safety, and biomarkers of neoadju‐ vant bevacizumab, radiation therapy, and fluorouracil in rectal cancer: a multidisci‐

[24] Holash J, Davis S, Papadopoulos N, et al. VEGF-Trap: a VEGF blocker with potent

[26] Sun Y, Wang J, Liu Y, et al. Results of phase III trial of rh-endostatin (YH-16) in ad‐ vanced non-small cell lung cancer (NSCLC) patients. J Clin Oncol 2005; 23:654S.

for non-small-cell lung cancer. N Engl J Med 2006; 355:2542-2550.

antitumor effects. Proc Natl Acad Sci USA 2002; 99:11393-11398.

[25] Kerbel RS. Tumor angiogenesis. N Engl J Med 2008; 358:2039-2049.

plinary phase II study. J Clin Oncol 2009; 27:3020-3026.

alone for metastatic breast cancer. N Engl J Med 2007; 357:2666-2676.

of poly (ADP-ribose) polymerase. J Cell Physiol 2005; 202: 125-134.

therapy for cancer: urinary cancer. Int J Clin Oncol 2006; 11:90-107.

Clin Cancer Res 2001; 7:795-799.

2002; 2:727-739.

350:2335-2342.

cer. Nat Clin Pract Oncol 2007; 4:181-189.

Trends Immunol 20001; 22:129-133.

um. Science 2000; 289:1197-1202.

### **Author details**

Jianping Pan\* and Lihuang Zhang

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Clinical Medicine, Zhejiang University City College School of Medicine, Hangzhou, P.R. China

### **References**

[11] Caldini R, Barletta E, Del Rosso M, et al. FGF2-mediated upregulation of urokinasetype plasminogen activator expression requires a MAP-kinase dependent activation of poly (ADP-ribose) polymerase. J Cell Physiol 2005; 202: 125-134.

**Acknowledgements**

**Author details**

Hangzhou, P.R. China

27: 549-557.

5:13-17.

2:795-803.

2003; 6:669-676.

Jianping Pan\*

**References**

Zhejiang Province, P.R. China (No. 20120633B30).

382 Cancer Treatment - Conventional and Innovative Approaches

and Lihuang Zhang

\*Address all correspondence to<EMAIL_ADDRESS>
spectives. Cancer Lett 2012; 320:130-137.

sels. Front Biosci 2011; 16:1413-1427.

This work was supported by a grant from the Science and Technology Bureau of Hangzhou,

Department of Clinical Medicine, Zhejiang University City College School of Medicine,

[1] Arbab AS. Activation of alternative pathways of angiogenesis and involvement of stem cells following anti-angiogenesis treatment in glioma. Histol Histopathol 2012;

[2] Shojaei F. Anti-angiogenesis therapy in cancer: current challenges and future per‐

[3] Hiratsuka S. Vasculogenensis, angiogenesis and special features of tumor blood ves‐

[4] Blagosklonny MV. Antiangiogenic therapy and tumor progression. Cancer Cell 2004;

[5] Ferrara N. VEGF and the quest for tumor angiogenesis factors. Nat Rev Cancer 2002;

[6] Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med

[7] Ferrara N, Kerbel RS. Angiogenesis as a therapeutic target. Nature 2005; 438:967-974.

[8] Hofmeister V, Schrama D, Becker JC, et al. Anti-cancer therapies targeting the tumor

[9] Shojaei F, Ferrara N. Antiangiogenesis to treat cancer and intraocular neovascular

[10] Veikkola T, Karkkainen M, Claesson-Welsh L, et al. Regulation of angiogenesis via

endothelial growth factor receptors. Cancer Rev 2000; 60:203-212.

stroma. Cancer Immunol Immunother 2008; 57: 1-17.

disorders. Lab Invest 2007; 87:227-230.

[27] McKeage MJ, Von Pawel J, Reck M, et al. Randomised phase II study of ASA404 combined with carboplatin and paclitaxel in previously untreated advanced nonsmall cell lung cancer. Br J Cancer 2008; 99:2006-2012.

[40] Valesky M, Spang AJ, Fisher GW, et al. Noninvasive dynamic fluorescence imaging of human melanomas reveals that targeted inhibition of bFGF or FGFR-1 in melano‐

Anti-Angiogenic Active Immunotherapy for Cancers: Dawn of a New Era?

http://dx.doi.org/10.5772/55323

385

[41] He QM, Wei YQ, Tian L, et al. Inhibition of tumor growth with a vaccine based on xenogeneic homologous fibroblast growth factor receptor-1 in mice. J Biol Chem

[42] Gutheil JC, Campbell TN, Pierce PR, et al. Targeted antiangiogenic therapy for cancer using Vitaxin: a humanized monoclonal antibody to the Integrin αvβ3. Clin Cancer

[43] Lou YY, Wei QY, Yang L, et al. Immunogene therapy of tumors with vaccine based on the ligand binding domain of chick homologous integrin beta3. Immunol Invest

[44] William G, Stetler S. Matrix metalloproteinases in angiogenesis: a moving target for

[45] Liotta LA, Steeg PS, Stetler-Stevenson WG. Cancer metastasis and angiogenesis: an

[46] Albini A, Melchiori A, Santi L, et al. Tumor cell invasion inhibited by TIMP-2. J Natl

[47] Itoh T, Tanioka M, Yoshida H, et al. Reduced angiogenesis and tumor progression in

[48] Su JM, Wei YQ, Tian L, et al. Active immunotherapy of cancer with vaccine on the basis of chicken homologous matrix metalloproteinase-2. Cancer Res 2003;

[49] Yang HS, Zhang DM, Deng HX, et al.Antitumor and anti-angiogenesis immunity in‐ duced by CR-SEREX-identified Xenopus RHAMM.Cancer Sci 2010; 101:862-868.

[50] Hardwick C, Hoare K, Owens R, et al. Molecular cloning of a novel hyaluronan re‐

[51] Kuhnert F, Kirshner JR, Thurston G. Dll4-Notch signaling as a therapeutic target in

[52] Gu JW, Rizzo P, Pannuti A, et al. Notch signals in the endothelium and cancer "stem-

[53] Ridgway J, Zhang G, Wu Y, et al. Inhibition of Dll4 signalling inhibits tumour

[54] Jubb AM, Soilleux EJ, Turley H, et al. Expression of vascular notch ligand delta-like 4 and inflammatory markers in breast cancer. Am J Pathol 2010; 176:2019-2028.

ceptor that mediates tumor cell motility. J Cell Biol 1992; 117:1343-1350.

like" cells: opportunities for cancer therapy. Vascular Cell 2012; 4: 7.

growth by deregulating angiogenesis. Nature 2006; 444:1083-1087.

therapeutic intervention. J Clin Invest 1999; 103:1237-1241.

gelatinase A-deficient mice. Cancer Res 1998; 58:1048-1051.

imbalance of positive and negative regulation. Cell 1991; 64:327-336.

ma cells blocks tumor growth by apoptosis. Mol Med 2002; 8: 103-112.

2003; 278:21831-21836.

Res 2000; 6:3056-3061.

Cancer Inst 1991; 83: 775-779.

tumor angiogenesis.Vasc Cell 2011; 3:20.

2002; 31:51-69.

63:600-607.

[40] Valesky M, Spang AJ, Fisher GW, et al. Noninvasive dynamic fluorescence imaging of human melanomas reveals that targeted inhibition of bFGF or FGFR-1 in melano‐ ma cells blocks tumor growth by apoptosis. Mol Med 2002; 8: 103-112.

[27] McKeage MJ, Von Pawel J, Reck M, et al. Randomised phase II study of ASA404 combined with carboplatin and paclitaxel in previously untreated advanced non-

[28] Kornberg TB, Krasnow MA. The Drosophila genome sequence: implications for biol‐

[29] Wei YQ, Wang QR, Zhao X, et al. Immunotherapy of tumors with xenogeneic endo‐

[30] Asakage M, Tsuno NH, Kitayama J, et al. Early-outgrowth of endothelial progenitor cells can function as antigen-presenting cells. Cancer Immunol Immunother 2006; 55:

[31] Kerbel RS. Tumor angiogenesis: past, present and the near future. Carcinogenesis

[32] Wei YQ, Huang MJ, Yang L, et al. Immunogene therapy of tumors with vaccine based on xenopus homologous vascular endothelial growth factors as a model anti‐

[33] Bequet-Romero M, Ayala M, Acevedo BE, et al. Prophylactic naked DNA vaccination with the human vascular endothelial growth factor induces an anti-tumor response

[34] Kamstock D, Elmslie R, Thamm D, et al. Evaluation of a xenogeneic VEGF vaccine in dogs with soft tissue sarcoma. Cancer Immunol Immunother 2007; 56: 1299-1309. [35] Shalaby F, Rossant J, Yamaguchi TP, et al. Failure of blood-island formation and vas‐

[36] Liu JY, Wei YQ, Yang L, et al. Immunotherapy of tumors with vaccine based on quail homologous vascular endothelial growth factor receptor-2. Blood 2003;

[37] Wei Y, Sun Y, Song C, et al. Enhancement of DNA vaccine efficacy by targeting the xenogeneic human chorionic gonadotropin, survivin and vascular endothelial growth factor receptor 2 combined tumor antigen to the major histocompatibility

[38] Li ZJ, Zhu H, Ma BY, et al. Inhibitory effect of Bifidobacterium infantis-mediated sKDR prokaryotic expression system on angiogenesis and growth of Lewis lung can‐

[39] Plum SM, Holaday JW, Ruiz A, et al. Administration of a liposomal FGF-2 peptide vaccine leads to abrogation of FGF-2-mediated angiogenesis and tumor develop‐

small cell lung cancer. Br J Cancer 2008; 99:2006-2012.

ogy and medicine. Science 2000; 287: 2218-2220.

384 Cancer Treatment - Conventional and Innovative Approaches

708-716.

2000; 21:505-515.

102:1815-1823.

thelial cells as a vaccine. Nat Med 2000; 6:1160-1166.

gen. Pro Natl Acad Sci USA 2001; 98:11545-11550.

in C57Bl/6 mice. Angiogenesis 2007; 10:23-34.

culogenesis in Flk-1-deficient mice. Nature 1995; 376:62-66.

complex class II pathway. J Gene Med 2012; 14: 353-362.

cer in mice. BMC Cancer 2012; 12:155.

ment. Vaccine 2000; 19:1294-1303.

[55] Haller BK, Bråve A, Wallgard E, et al. Therapeutic efficacy of a DNA vaccine target‐ ing the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma. Onco‐ gene 2010; 29:4276-4286.

[68] Pan J, Heiser A, Marget M, et al. Enhanced antimetastatic effect of fetal liver kinase 1 extracellular domain and interferon-gamma fusion gene-modified dendritic cell vac‐

from mouse VEGFR2 that can inhibit angiogenesis and tumor growth. J Immunother

[70] Wada S, Tsunoda T, Baba T, et al. Rationale for antiangiogenic cancer therapy with vaccination using epitope peptides derived from human vascular endothelial growth

[71] Seavey MM, Maciag PC, Al-Rawi N, et al. An anti-vascular endothelial growth factor receptor 2/fetal liver kinase-1 Listeria monocytogenes anti-angiogenesis cancer vac‐ cine for the treatment of primary and metastatic Her-2/neu+ breast tumors in a

[72] Seavey MM, Paterson Y. Anti-Angiogenesis immunotherapy induces epitope spread‐ ing to Her-2/neu resulting in breast tumor immunoediting. Breast Cancer 2009;

[73] Liang PH, Zhang KQ, Xu GL, et al. Construction of a DNA vaccine encoding Flk-1 extracellular domain and C3d fusion gene and investigation of its suppressing effect

[74] Miyazawa M, Ohsawa R, Tsunoda T, et al. Phase I clinical trial using peptide vaccine for human vascular endothelial growth factor receptor 2 in combination with gemci‐ tabine for patients with advanced pancreatic cancer. Cancer Sci 2008; 101:433-439. [75] Tokuda H, Kozawa O, Uematsu T, et al. Basic fibroblast growth factor stimulates vas‐ cular endothelial growth factor release in osteoblasts: divergent regulation by p44/p42 mitogen-activated protein kinase and p38 mitogen-activated protein kinase.

[76] Dias S, Hattori K, Zhu Z, et al. Autocrine stimulation of VEGFR-2 activates human

[77] Karsan A, Yee E, Poirier GG, et al. Fibroblast growth factor-2 inhibits endothelial cell apoptosis by Bcl-2-dependent and independent mechanisms. Am J Pathol 1997;

[78] Plum SM, Vu HA, Mercer B, et al. Generation of a specific immunological response to FGF-2 does not affect wound healing or reproduction. Immunopharmacol Immu‐

[79] Harari PM. Epidermal growth factor receptor inhibition strategies in oncology. En‐

leukemic cell growth and migration. J Clin Invest 2000; 106: 511-521.

on tumor growth. Cancer Immunol Immunother 2010; 59:93-101.

Anti-Angiogenic Active Immunotherapy for Cancers: Dawn of a New Era?

T-cell epitopes

387

http://dx.doi.org/10.5772/55323

cine. Gene Ther 2005; 12:742-750.

factor 2. Cancer Res 2005; 65:4939-4946.

J Bone Miner Res 2000; 15: 2371-2379.

2006; 29:32-40.

1:19-30.

151:1775-1784.

notoxicol 2004; 26:29-41.

docr Relat Cancer 2004; 11: 689-708.

[69] Dong Y, Qian J, Ramy I, et al. Identification of H-2Db

mouse model. J Immunol 2009; 182(9):5537-5546.

[68] Pan J, Heiser A, Marget M, et al. Enhanced antimetastatic effect of fetal liver kinase 1 extracellular domain and interferon-gamma fusion gene-modified dendritic cell vac‐ cine. Gene Ther 2005; 12:742-750.

[55] Haller BK, Bråve A, Wallgard E, et al. Therapeutic efficacy of a DNA vaccine target‐ ing the endothelial tip cell antigen delta-like ligand 4 in mammary carcinoma. Onco‐

[56] Troyanovsky B, Levchenko T, Månsson G, et al. Angiomotin: an angiostatin binding protein that regulates endothelial cell migration and tube formation. J Cell Biol. 2001;

[57] Holmgren L, Ambrosino E, Birot O, et al. A DNA vaccine targeting angiomotin in‐ hibits angiogenesis and suppresses tumor growth. Proc Natl Acad Sci USA 2006; 103:

[58] Bratt A, Wilson WJ, Troyanovsky B, et al. Angiomotin belongs to a novel protein family with conserved coiled-coil and PDZ binding domains. Gene 2002; 298:69-77.

[59] Levchenko T, Bratt A, Arbiser JL, et al. Angiomotin expression promotes heman‐

[60] Ernkvist M, Birot O, Sinha I, et al. Differential roles of p80- and p130-angiomotin in the switch between migration and stabilization of endothelial cells. Biochim Biophys

[61] Arigoni M, Barutello G, Lanzardo S, et al. A vaccine targeting angiomotin induces an antibody response which alters tumor vessel permeability and hampers the growth

[62] Li Y, Wang MN, Li H, et al. Active immunization against the vascular endothelial growth factor receptor flk1 inhibits tumor angiogenesis and metastasis. J Exp Med

[63] Nithammer AG, Xiang R, Becker JC, et al. A DNA vaccine against VEGF receptor 2 prevents effective angiogenesis and inhibits tumor growth. Nat Med 2002;

[64] Nair S, Boczkowski D, Moeller B, et al. Synergy between tumor immunotherapy and

[65] Pan J, Zhang M, Wang J, et al. Interferon-gamma is an autocrine mediator for den‐

[66] Pan J, Zhang M, Wang J, et al. Intratumoral injection of interferon-gamma genemodified dendritic cells elicits potent antitumor effects: effective induction of tumor-

[67] Qin Z, Blankenstein T. CD4+ T cell-mediated tumor rejection involves inhibition of angiogenesis that is dependent on IFN gamma receptor expression by nonhemato‐

specific CD8+ CTL response. J Cancer Res Clin Oncol 2005; 131:468-478.

gioendothelioma invasion. Oncogene 2004; 23:1469-1473.

of established tumors. Angiogenesis 2012; 15:305-316.

antiangiogenic therapy. Blood 2003; 102: 964-971.

poietic cells. Immunity 2000; 12:677-686.

dritic cell maturation. Immunol Lett 2004; 94:141-151.

gene 2010; 29:4276-4286.

386 Cancer Treatment - Conventional and Innovative Approaches

Acta 2008; 1783: 429-437.

2002; 195: 1575-1584.

8:1369-1375.

152: 1247-1254.

9208-9213.

[80] Hu B, Wei YQ, Tian L, et al. Active antitumor immunity elicited by vaccine based on recombinant form of epidermal growth factor receptor. J Immunother 2005; 28:236-244.

**Section 4**

**Multidisciplinarity in Cancer Therapy: Nutrition**

**and Beyond**

**Multidisciplinarity in Cancer Therapy: Nutrition and Beyond**

[80] Hu B, Wei YQ, Tian L, et al. Active antitumor immunity elicited by vaccine based on recombinant form of epidermal growth factor receptor. J Immunother 2005;

[81] Luo Y, Zhou H, Krueger J, et al. Targeting tumor-associated macrophages as a novel

[82] Liu C, Sun C, Huang H, et al. Overexpression of legumain in tumors is significant for invasion / metastasis and a candidate enzymatic target for prodrug therapy. Cancer

[83] Murthy RV, Arbman G, Gao J, et al. Legumain expression in relation to clinicopatho‐ logic and biological variables in colorectal cancer. Clin Cancer Res 2005; 11:2293-2299.

[84] Oosterling SJ, van der Bij GJ, Meijer GA, et al. Macrophages direct tumor histology

[85] Lewen S, Zhou H, Hu HD, et al. A Legumain-based minigene vaccine targets the tu‐ mor stroma and suppresses breast cancer growth and angiogenesis. Cancer Immunol

[86] Lee SH, Mizutani N, Mizutani M, et al. Endoglin (CD105) is a target for an oral DNA vaccine against breast cancer. Cancer Immunol Immunother 2006; 55:1565-1574. [87] Wood LM, Pan ZK, Guirnalda P, et al. Targeting tumor vasculature with novel Liste‐ ria-based vaccines directed against CD105. Cancer Immunol Immunother 2011;

[88] Maciag PC, Seavey MM, Pan ZK, et al. Cancer immunotherapy targeting the high molecular weight melanoma-associated antigen protein results in a broad antitumor response and reduction of pericytes in the tumor vasculature. Cancer Res 2008; 68:

[89] Yoneyama S, Okaji Y, Tsuno NH, et al. A study of dendritic and endothelial cell in‐ teractions in colon cancer in a cell line and small mammal model. Eur J Surg Oncol

[90] Okaji Y, Tsuno NH, Tanaka M, et al. Pilot study of anti-angiogenic vaccine using fixed whole endothelium in patients with progressive malignancy after failure of

conventional therapy. Eur J Cancer 2008; 44:383-390.

and clinical outcome in a colon cancer model. J Pathol 2005; 207:147-155.

strategy against breast cancer. J Clin Invest 2006; 116:2132-2141.

28:236-244.

60:931-942.

8066-8075.

2007; 33:1191-1198.

Res 2003; 63:2957-2964.

388 Cancer Treatment - Conventional and Innovative Approaches

Immunother 2008; 57:507-515.

**Chapter 17**

**Nutrigenomics and Cancer Prevention**

Cancer is fundamentally a genetic disease. At the beginning of the process is an alteration in the DNA of a single cell. This change in DNA can be caused by several factors, chemical, physical or biological phenomena. The stage of promotion is the second stage of carcinogenesis. The genetically altered cells, ie, "initiated," suffer the effects of carcinogens classified as oncopromo‐ tores. The initiated cell is transformed into a malignant cell, a slow and gradual process. For this transformation to occur, you need a long and continuous contact with the carcinogen promot‐ er. The stage of progression is the third and final stage and is characterized by uncontrolled proliferationof cells andirreversiblychanged.Atthis stage canceris alreadyinstalled,progress‐ ing to the emergence of the first clinical manifestations of the disease [1]. In this sense, the diet plays a key role in various stages of cancer development. The process of carcinogenesis may be affected by nutritional factors through mechanisms that promote or inhibit its development. Some foods can contain not only carcinogens, but also other substances that act to reduce the damage to the cell's genetic material caused by environmental mutagens. The observation of cancer in an individual does not identify the causative agent(s). However, epidemiological data on populations do indicate that a large fraction of human cancers are associated with lifestyle/ diet. Such studies may also help identify the etiologic agents but unless there are good doseresponse data for humans and/or animal models, the probability of identifying the agent is not high. Cancers may result from endogenous reactions, such as oxidations or from exogenous agents, such as tobacco smoke (lung cancer), sunlight exposure (skin cancer), aflatoxin (liver

cancer), and relatively high doses of ionizing radiations (many types of cancers) [2].

The importance of nutrition in health is not a new idea. More than two thousand years ago, Hippocrates, the father of Western medicine, wrote: "Let food be thy medicine and medicine be thy food." What has changed since the time of Hippocrates is our understanding of the details of how nutrition affects our health. Researchers are getting more knowledge as to what foods or bioactive food compounds and how they can interact with our bodies promoting

and reproduction in any medium, provided the original work is properly cited.

© 2013 Nepomuceno; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Additional information is available at the end of the chapter

Júlio César Nepomuceno

http://dx.doi.org/10.5772/55429

**1. Introduction**

### **Chapter 17**

### **Nutrigenomics and Cancer Prevention**

### Júlio César Nepomuceno

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55429

### **1. Introduction**

Cancer is fundamentally a genetic disease. At the beginning of the process is an alteration in the DNA of a single cell. This change in DNA can be caused by several factors, chemical, physical or biological phenomena. The stage of promotion is the second stage of carcinogenesis. The genetically altered cells, ie, "initiated," suffer the effects of carcinogens classified as oncopromo‐ tores. The initiated cell is transformed into a malignant cell, a slow and gradual process. For this transformation to occur, you need a long and continuous contact with the carcinogen promot‐ er. The stage of progression is the third and final stage and is characterized by uncontrolled proliferationof cells andirreversiblychanged.Atthis stage canceris alreadyinstalled,progress‐ ing to the emergence of the first clinical manifestations of the disease [1]. In this sense, the diet plays a key role in various stages of cancer development. The process of carcinogenesis may be affected by nutritional factors through mechanisms that promote or inhibit its development. Some foods can contain not only carcinogens, but also other substances that act to reduce the damage to the cell's genetic material caused by environmental mutagens. The observation of cancer in an individual does not identify the causative agent(s). However, epidemiological data on populations do indicate that a large fraction of human cancers are associated with lifestyle/ diet. Such studies may also help identify the etiologic agents but unless there are good doseresponse data for humans and/or animal models, the probability of identifying the agent is not high. Cancers may result from endogenous reactions, such as oxidations or from exogenous agents, such as tobacco smoke (lung cancer), sunlight exposure (skin cancer), aflatoxin (liver cancer), and relatively high doses of ionizing radiations (many types of cancers) [2].

The importance of nutrition in health is not a new idea. More than two thousand years ago, Hippocrates, the father of Western medicine, wrote: "Let food be thy medicine and medicine be thy food." What has changed since the time of Hippocrates is our understanding of the details of how nutrition affects our health. Researchers are getting more knowledge as to what foods or bioactive food compounds and how they can interact with our bodies promoting

© 2013 Nepomuceno; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

health. The Human Genome Project was one of the key factors that enable the study of genefood interactions and promotion of health. Discoveries in genetics make it possible to under‐ stand the effects of nutrients in processes at the molecular level in the body and also the variable effects of dietary components on each individual. Research has shown that the nutrients affect gene expression and formation of several proteins that are important in the formation and maintenance of tissues. So, faced with this interaction genomics and nutrition, emerges Nutrigenomics aiming to understand the functions of all genes and their interactions with food, in order to promote health and reduce the risk of developing diseases [3].

nucleotide, resulting in two methylated cytosine residues sitting diagonally to each other on opposing DNA strands [6]. DNA methylation, which modifies a cytosine base at the CpG dinucleotide residues with methyl groups, is catalyzed by DNA methyltransferases (Dnmt) and regulates gene expression patterns by altering chromatin structures. Currently, 5 different Dnmt are known: Dnmt1, Dnmt2, Dnmt 3a, Dnmt3b and DnmtL [7]. The Polycomb group protein EZH2 directly controls DNA methylation (Figure 1). EZH2 serves as a recruitment platform for DNA methyltransferases, thus highlighting a previously unrecognized direct

Nutrigenomics and Cancer Prevention http://dx.doi.org/10.5772/55429 393

**Figure 1.** Polycomb Group (PcG) protein EZH2 serves as a recruitment platform for DNA methyltransferases (http://

DNA methylation is essential for cell differentiation and embryonic development. Moreover, in some cases, methylation has observed to play a role in mediating gene expression. In mammals, methylation is found sparsely but globally, distributed in definite CpG sequences throughout the entire genome, with the exception of CpG islands, or certain stretches (ap‐ proximately 1 kilobase in length) where high CpG contents are found. The methylation of these sequences can lead to inappropriate gene silencing, such as the silencing of tumor suppressor genes in cancer cells [6]. A large amount of research on DNA methylation and disease has focused on cancer and tumor suppressor genes. Tumor suppressor genes are often silenced in cancer cells due to hypermethylation. In contrast, the genomes of cancer cells have been shown to be hypomethylated overall when compared to normal cells, with the exception of hyper‐ methylation events at genes involved in cell cycle regulation, tumor cell invasion, DNA repair, and other events in which silencing propagates metastasis. In fact, in certain cancers, such as that of the colon, hypermethylation is detectable early and might serve as a biomarker for the

In the nutritional field, epigenetics is exceptionally important, because nutrients and bioactive food components can modify epigenetic phenomena and alter the expression of genes at the transcriptional level. Nutrients can reverse or change epigenetic phenomena such as DNA

connection between two key epigenetic repression systems [8].

www.ulb.ac.be/medecine/fukslab/research.htm).

disease [6] (See Figure 2).

Nutrigenomics studies the modulating effect of the chemical compounds in foods and on the stability of DNA synthesis and gene expression. The nutrients are able to affect the genome and its expression through the synthesis of nucleotides, prevention and repair of DNA damage, or through epigenetic mechanisms including methylation of histones, proteins responsible for chromatin structure that play an important role in regulating gene expression. Those meth‐ odological approaches are based on nutrition, molecular biology, and genomics. Integration of these disciplines is leading to identification and understanding of individual and population differences and similarities in gene expression, or phenotype, in response to diet. We can consider nutrigenomics as a multidisciplinary science that applies the genomic techniques besides the biochemical and epidemiological aspects, with the aim to understand the etiologic aspects of chronic diseases such as cardiovascular diseases, diabetes, obesity and cancer [4].

An understanding of scientific information about the composition and functions of genomes, has created unprecedented opportunities for increasing our understanding of how nutrients modulate gene and protein expression and ultimately influence cellular and organismal metabolism. On that basis, the purpose of this chapter is to make a broad review study to evaluate the modulation between compounds found in nutrients and their interactions with on the genomic stability and control of gene expression.

### **2. Nutrition and epigenetics**

All the cells in the body have identical genomes. However, each cell has one of many "epige‐ nomes", unique sets of epigenetic instructions for establishing and maintaining lineagespecific expression profiles. The genome is programmed to express appropriate sets of genes, in particular tissues, at specific time points during the individual's life. Epigenetic events create a memory of cell identity, maintaining genomic functions such as the maintenance of cell identity after differentiation, the propagation of essential features of chromosomal architecture and dosage compensation [5]. Epigenetic mechanisms are capable of modulating gene expression through changes in the chromosomes structure. Chromosomes are formed from the condensation of the chromatin, which is formed by a complex of DNA, and unique proteins called histone. Examples of epigenetic mechanisms may be mentioned as DNA methylation and histone acetylation [3].

DNA methylation occurs at the cytosine bases of eukaryotic DNA, which are converted to 5 methylcytosine. The altered cytosine residues are usually immediately adjacent to a guanine nucleotide, resulting in two methylated cytosine residues sitting diagonally to each other on opposing DNA strands [6]. DNA methylation, which modifies a cytosine base at the CpG dinucleotide residues with methyl groups, is catalyzed by DNA methyltransferases (Dnmt) and regulates gene expression patterns by altering chromatin structures. Currently, 5 different Dnmt are known: Dnmt1, Dnmt2, Dnmt 3a, Dnmt3b and DnmtL [7]. The Polycomb group protein EZH2 directly controls DNA methylation (Figure 1). EZH2 serves as a recruitment platform for DNA methyltransferases, thus highlighting a previously unrecognized direct connection between two key epigenetic repression systems [8].

health. The Human Genome Project was one of the key factors that enable the study of genefood interactions and promotion of health. Discoveries in genetics make it possible to under‐ stand the effects of nutrients in processes at the molecular level in the body and also the variable effects of dietary components on each individual. Research has shown that the nutrients affect gene expression and formation of several proteins that are important in the formation and maintenance of tissues. So, faced with this interaction genomics and nutrition, emerges Nutrigenomics aiming to understand the functions of all genes and their interactions with

Nutrigenomics studies the modulating effect of the chemical compounds in foods and on the stability of DNA synthesis and gene expression. The nutrients are able to affect the genome and its expression through the synthesis of nucleotides, prevention and repair of DNA damage, or through epigenetic mechanisms including methylation of histones, proteins responsible for chromatin structure that play an important role in regulating gene expression. Those meth‐ odological approaches are based on nutrition, molecular biology, and genomics. Integration of these disciplines is leading to identification and understanding of individual and population differences and similarities in gene expression, or phenotype, in response to diet. We can consider nutrigenomics as a multidisciplinary science that applies the genomic techniques besides the biochemical and epidemiological aspects, with the aim to understand the etiologic aspects of chronic diseases such as cardiovascular diseases, diabetes, obesity and cancer [4]. An understanding of scientific information about the composition and functions of genomes, has created unprecedented opportunities for increasing our understanding of how nutrients modulate gene and protein expression and ultimately influence cellular and organismal metabolism. On that basis, the purpose of this chapter is to make a broad review study to evaluate the modulation between compounds found in nutrients and their interactions with

All the cells in the body have identical genomes. However, each cell has one of many "epige‐ nomes", unique sets of epigenetic instructions for establishing and maintaining lineagespecific expression profiles. The genome is programmed to express appropriate sets of genes, in particular tissues, at specific time points during the individual's life. Epigenetic events create a memory of cell identity, maintaining genomic functions such as the maintenance of cell identity after differentiation, the propagation of essential features of chromosomal architecture and dosage compensation [5]. Epigenetic mechanisms are capable of modulating gene expression through changes in the chromosomes structure. Chromosomes are formed from the condensation of the chromatin, which is formed by a complex of DNA, and unique proteins called histone. Examples of epigenetic mechanisms may be mentioned as DNA methylation

DNA methylation occurs at the cytosine bases of eukaryotic DNA, which are converted to 5 methylcytosine. The altered cytosine residues are usually immediately adjacent to a guanine

food, in order to promote health and reduce the risk of developing diseases [3].

on the genomic stability and control of gene expression.

**2. Nutrition and epigenetics**

392 Cancer Treatment - Conventional and Innovative Approaches

and histone acetylation [3].

**Figure 1.** Polycomb Group (PcG) protein EZH2 serves as a recruitment platform for DNA methyltransferases (http:// www.ulb.ac.be/medecine/fukslab/research.htm).

DNA methylation is essential for cell differentiation and embryonic development. Moreover, in some cases, methylation has observed to play a role in mediating gene expression. In mammals, methylation is found sparsely but globally, distributed in definite CpG sequences throughout the entire genome, with the exception of CpG islands, or certain stretches (ap‐ proximately 1 kilobase in length) where high CpG contents are found. The methylation of these sequences can lead to inappropriate gene silencing, such as the silencing of tumor suppressor genes in cancer cells [6]. A large amount of research on DNA methylation and disease has focused on cancer and tumor suppressor genes. Tumor suppressor genes are often silenced in cancer cells due to hypermethylation. In contrast, the genomes of cancer cells have been shown to be hypomethylated overall when compared to normal cells, with the exception of hyper‐ methylation events at genes involved in cell cycle regulation, tumor cell invasion, DNA repair, and other events in which silencing propagates metastasis. In fact, in certain cancers, such as that of the colon, hypermethylation is detectable early and might serve as a biomarker for the disease [6] (See Figure 2).

In the nutritional field, epigenetics is exceptionally important, because nutrients and bioactive food components can modify epigenetic phenomena and alter the expression of genes at the transcriptional level. Nutrients can reverse or change epigenetic phenomena such as DNA

methylation in *Apis* is used for storing epigenetic information, that the use of that information can be differentially altered by nutritional input, and that the flexibility of epigenetic modifi‐ cations underpins, profound shifts in developmental fates, with massive implications for

During our lifetime, nutrients can modify physiologic and pathologic processes through epigenetic mechanisms that are critical for gene expression (summarized in Table 1). Modu‐ lation of these processes through diet or specific nutrients may prevent diseases and maintain health. However, it is very hard to delineate the precise effect of nutrients or bioactive food components on each epigenetic modulation and their associations with physiologic and pathologic processes in our body, because the nutrients also interact with genes, other nutrients, and other lifestyle factors. Furthermore, each epigenetic phenomenon also interacts

**Nutrient or diet Epigenetic mechanism**

modifications

Nutrigenomics and Cancer Prevention http://dx.doi.org/10.5772/55429 395

methylation

methylation

Choline DNA methylation Protein restriction DNA methylation, histone

Alcohol DNA methylation

Calorie restriction Histone acetylation

Genistein DNA methylation, microRNA (-)-Epigallocatechin-3-gallate DNA methylation, PRC

Embryonic Folate DNA methylation, imprinting

Stem cell Butyrate Histone acetylation, DNA

Aging Folate DNA methylation

Immune function Folate DNA methylation

Retinoic acid PRC

Cancer Methyl-deficient diet Histone modification, microRNA

Obesity, insulin High-fat diet DNA methylation, microRNA

Inflammation Resveratrol Histone acetylation

**Table 1.** Epigenetic roles of nutrition in physiologic and pathologic processes

Neurocognition Choline DNA methylation, histone

Curcumin microRNA

Methyl-deficient diet microRNA

Methyl-deficient diet DNA methylation Curcumin Histone acetylation

AdoMet Histone methylation

reproductive and behavioral status.

development

resistance

Data in reference [7].

with the others, adding to the complexity of the system [7].

**Figure 2.** DNA methylation and cancer. This diagram shows a representative region of genomic DNA in a normal cell. The region contains repeat-rich, hypermethylated pericentromeric heterochromatin and an actively transcribed tumor suppressor gene (TSG) associated with a hypomethylated CpG island (indicated in red). In tumor cells, repeat-rich het‐ erochromatin becomes hypomethylated, and this contributes to genomic instability (a hallmark of tumor cells) through increased mitotic recombination events. *De novo* methylation of CpG islands also occurs in cancer cells, and it can result in the transcriptional silencing of growth-regulatory genes. These changes in methylation are early events in tumorigenesis. (See reference [9].)

methylation and histone modifications, thereby modifying the expression of critical genes associated with physiologic and pathologic processes, including embryonic development, aging, and carcinogenesis [7].

The most interesting study linking diet and epigenetics was made by Kucharski et al. [10], about nutritional control of reproductive status in honeybees via DNA methylation. Fertile queens and sterile workers are alternative forms of the adult female honeybee that develop from genetically identical larvae following differential feeding with royal jelly. Royal jelly is a complex, protein-rich substance secreted from glands on the heads of worker bees.  The authors observed that larvae fed with royal jelly developed functional ovaries and a larger abdomen for egg laying, while worker bees remain sterile. She'll also develop the necessary behaviors to act as queen, such as killing rival queens, making communication sounds known as "piping," and going on "mating flights." The queen is fed royal honey exclusively for the rest of her life. They showed that royal jelly silences a key gene (Dnmt3), which codes for an enzyme involved in genome-wide gene silencing. When Dnmt3 is active in bee larvae, the queen genes are epigenetically silenced and the larvae develop into the default "worker" variety. But when royal jelly turns Dnmt3 off, certain genes jump into action that turn the lucky larvae into queens. The authors suggested that DNA methylation in *Apis* is used for storing epigenetic information, that the use of that information can be differentially altered by nutritional input, and that the flexibility of epigenetic modifi‐ cations underpins, profound shifts in developmental fates, with massive implications for reproductive and behavioral status.

During our lifetime, nutrients can modify physiologic and pathologic processes through epigenetic mechanisms that are critical for gene expression (summarized in Table 1). Modu‐ lation of these processes through diet or specific nutrients may prevent diseases and maintain health. However, it is very hard to delineate the precise effect of nutrients or bioactive food components on each epigenetic modulation and their associations with physiologic and pathologic processes in our body, because the nutrients also interact with genes, other nutrients, and other lifestyle factors. Furthermore, each epigenetic phenomenon also interacts with the others, adding to the complexity of the system [7].

**Table 1.** Epigenetic roles of nutrition in physiologic and pathologic processes

methylation and histone modifications, thereby modifying the expression of critical genes associated with physiologic and pathologic processes, including embryonic development,

**Figure 2.** DNA methylation and cancer. This diagram shows a representative region of genomic DNA in a normal cell. The region contains repeat-rich, hypermethylated pericentromeric heterochromatin and an actively transcribed tumor suppressor gene (TSG) associated with a hypomethylated CpG island (indicated in red). In tumor cells, repeat-rich het‐ erochromatin becomes hypomethylated, and this contributes to genomic instability (a hallmark of tumor cells) through increased mitotic recombination events. *De novo* methylation of CpG islands also occurs in cancer cells, and it can result in the transcriptional silencing of growth-regulatory genes. These changes in methylation are early events in

The most interesting study linking diet and epigenetics was made by Kucharski et al. [10], about nutritional control of reproductive status in honeybees via DNA methylation. Fertile queens and sterile workers are alternative forms of the adult female honeybee that develop from genetically identical larvae following differential feeding with royal jelly. Royal jelly is a complex, protein-rich substance secreted from glands on the heads of worker bees.  The authors observed that larvae fed with royal jelly developed functional ovaries and a larger abdomen for egg laying, while worker bees remain sterile. She'll also develop the necessary behaviors to act as queen, such as killing rival queens, making communication sounds known as "piping," and going on "mating flights." The queen is fed royal honey exclusively for the rest of her life. They showed that royal jelly silences a key gene (Dnmt3), which codes for an enzyme involved in genome-wide gene silencing. When Dnmt3 is active in bee larvae, the queen genes are epigenetically silenced and the larvae develop into the default "worker" variety. But when royal jelly turns Dnmt3 off, certain genes jump into action that turn the lucky larvae into queens. The authors suggested that DNA

aging, and carcinogenesis [7].

tumorigenesis. (See reference [9].)

394 Cancer Treatment - Conventional and Innovative Approaches

### **2.1. Diet and genomic stability**

Eukaryotic DNA replication starts at multiple sites throughout the genome and is necessarily coordinated with transcription, sister chromatid cohesion, nucleosome assembly and cell cycle progression. In addition to the complexity of the replication reaction it, during replication cells need to deal with DNA damage and stalled forks, originated inevitably by the action of exogenous and endogenous agents. The success of this process is crucial to preserve genome stability, and the inability to deal with DNA lesions during replication or to protect or restart stalled forks leads to DNA breaks, chromosomal rearrangements, and mutations that can cause the loss of cell viability, but in addition errors in DNA replication result in a large number of human syndromes, including premature aging, various cancer predispositions and genetic abnormalities. To solve or reduce these problems, cells use repair and detoxification pathways as well as surveillance mechanisms, called checkpoints, which serve to detect the problem and coordinate repair with chromosome segregation and progression through the cell cycle (see Figure 3. www.genomic-instability.org/).

Maintaining genomic stability in the face of replication and recombination requires a huge variety of different damage response proteins. A cell's ability to decide when and where to deploy this DNA repair kit is critical to prevent tumor development [11].

There is evidence that inappropriate nutrient supply can cause sizeable levels of genome mutation and alter expression of genes required for genome maintenance. Deficiencies in several micronutrients have been shown to cause DNA damage and are thought to be associated with a number of serious human diseases: folic acid, niacin, vitamin B6 and B12 deficiency may increase the risk of colon cancer, heart disease and neurological dysfunction due to chromosome breaks and disabled DNA repair [12]. On the other hand, as seen in reference [13], the authors believe that caloric restriction (CR) is an 'intervention' that alters the activation of specific 'stress response genes', key enzymes in DNA repair pathways, which then results in upregulation of 'DNA repair' capacity. Enhanced DNA repair reduces the levels of DNA damage, consequently reducing mutation frequency, which would result in mainte‐ nance of genomic stability.

**3. Diet and cancer prevention**

ty.org/).

Current cancer models comprise those that are inherited through the germline and represent only ∼5% of total cases of human cancers. These tumors originate because of mutational events. The remaining ∼95% originate as sporadic events and evolve as a result of exposure to the environment, which includes exposure to both environmental contaminants and dietary agents. The multistage model of carcinogenesis identifies various phases, initiation, promo‐ tion, and progression, appears to be influenced by tissue microenvironment and organization. Significant opportunities in nutrition and cancer prevention exist in the early stages of initiation and promotion prior to clonal expansion of heterogeneous populations. Nutrige‐ nomics represents a strategy that can be applied to the study and prevention of many diseases including cancer. DNA methylation and histone modifications are epigenetic events that mediate heritable changes in gene expression and chromatin organization in the absence of changes in the DNA sequence. The age-increased susceptibility to cancer may derive from accumulation of epigenetic changes and represents a potential target for therapies with bioactive compounds. Factors that mediate the response to dietary factors include nuclear receptors and transcription factors, which function as sensors to dietary components and determine changes in the profile of transcripts [15]. Milner and Romagnolo [15] affirm that the opportunity of targeting nutrients–gene interactions to influence the cancer process is modu‐ lated by genetic variations in human populations, epigenetic modifications that selectively and permanently alter gene expression, by complex interactions/associations among dietary components, and heterogeneity of cells within a certain tumor. Therefore, integration of information about gene polymorphisms, identification of gene targets that regulate cell and

**Figure 3.** A general view of DNA insults and consequences on cell cycle and DNA repair (www.genomic-instabili‐

Nutrigenomics and Cancer Prevention http://dx.doi.org/10.5772/55429 397

Recommended dietary allowances (RDAs) of micronutrients have been traditionally defined as those levels necessary to prevent symptoms of deficiency diseases. There is increasing evidence that higher levels of many such micronutrients may be necessary for various DNA maintenance reactions, and that the current RDAs for some micronutrients may be inadequate to protect against genomic instability. Dietary imbalance may increase gene mutation and chromosome aberrations in human populations, similar to exposure to radiation, mutagens and carcinogens. Diet may well be a key factor in determining genomic stability since it impacts on all relevant pathways, i.e. exposure to dietary carcinogens, activation/detoxification of carcinogens, DNA repair, DNA synthesis and apoptosis, as mentioned previously. Many micronutrient minerals and vitamins act as substrates and/or co-factors in key DNA mainte‐ nance reactions, and the exact concentration of these in the cell may be critical. Sub-optimal levels of key micronutrients required for DNA maintenance will reduce genomic stability, producing similar effects to inherited genetic disorders or exposure to carcinogens [14].

**Figure 3.** A general view of DNA insults and consequences on cell cycle and DNA repair (www.genomic-instabili‐ ty.org/).

### **3. Diet and cancer prevention**

**2.1. Diet and genomic stability**

396 Cancer Treatment - Conventional and Innovative Approaches

Figure 3. www.genomic-instability.org/).

nance of genomic stability.

Eukaryotic DNA replication starts at multiple sites throughout the genome and is necessarily coordinated with transcription, sister chromatid cohesion, nucleosome assembly and cell cycle progression. In addition to the complexity of the replication reaction it, during replication cells need to deal with DNA damage and stalled forks, originated inevitably by the action of exogenous and endogenous agents. The success of this process is crucial to preserve genome stability, and the inability to deal with DNA lesions during replication or to protect or restart stalled forks leads to DNA breaks, chromosomal rearrangements, and mutations that can cause the loss of cell viability, but in addition errors in DNA replication result in a large number of human syndromes, including premature aging, various cancer predispositions and genetic abnormalities. To solve or reduce these problems, cells use repair and detoxification pathways as well as surveillance mechanisms, called checkpoints, which serve to detect the problem and coordinate repair with chromosome segregation and progression through the cell cycle (see

Maintaining genomic stability in the face of replication and recombination requires a huge variety of different damage response proteins. A cell's ability to decide when and where to

There is evidence that inappropriate nutrient supply can cause sizeable levels of genome mutation and alter expression of genes required for genome maintenance. Deficiencies in several micronutrients have been shown to cause DNA damage and are thought to be associated with a number of serious human diseases: folic acid, niacin, vitamin B6 and B12 deficiency may increase the risk of colon cancer, heart disease and neurological dysfunction due to chromosome breaks and disabled DNA repair [12]. On the other hand, as seen in reference [13], the authors believe that caloric restriction (CR) is an 'intervention' that alters the activation of specific 'stress response genes', key enzymes in DNA repair pathways, which then results in upregulation of 'DNA repair' capacity. Enhanced DNA repair reduces the levels of DNA damage, consequently reducing mutation frequency, which would result in mainte‐

Recommended dietary allowances (RDAs) of micronutrients have been traditionally defined as those levels necessary to prevent symptoms of deficiency diseases. There is increasing evidence that higher levels of many such micronutrients may be necessary for various DNA maintenance reactions, and that the current RDAs for some micronutrients may be inadequate to protect against genomic instability. Dietary imbalance may increase gene mutation and chromosome aberrations in human populations, similar to exposure to radiation, mutagens and carcinogens. Diet may well be a key factor in determining genomic stability since it impacts on all relevant pathways, i.e. exposure to dietary carcinogens, activation/detoxification of carcinogens, DNA repair, DNA synthesis and apoptosis, as mentioned previously. Many micronutrient minerals and vitamins act as substrates and/or co-factors in key DNA mainte‐ nance reactions, and the exact concentration of these in the cell may be critical. Sub-optimal levels of key micronutrients required for DNA maintenance will reduce genomic stability, producing similar effects to inherited genetic disorders or exposure to carcinogens [14].

deploy this DNA repair kit is critical to prevent tumor development [11].