One important study of the basic science of IHs focused on the intrinsic vs. extrinsic origin theory, arguing that infantile hemangiomas arises from an intrinsic somatic mutation in an endothelial progenitor cell (Bischoff, 2002). By analyzing X-chromosome inactivation patterns using a polymorphism of the X-linked human androgen receptor gene (HUMARA), it was found that HemECs display a non-random pattern of X-chromosomal inactivation, demonstrating that each HemEC population was clonal and implicating a single progenitor cell as their origin. Additionally, non-endothelial cells isolated from hemangiomas did not display evidence of clonality, demonstrating that only endothelial cells within IHs are

pathogenesis or regulatory mechanisms that govern the progression of IHs.

and the authors of these reports did not report evidence of GLUT1 positivity.

between the array data and IH behavior.

**6.2 Intrinsic theory of hemangiomas**

clonal.

formation of IH.

Other studies have shown that the endothelial cells isolated from IHs behave abnormally when compared to isolated endothelial cells from foreskin/commercially available dermal microvascular endothelial cells. While HemECs display many of the same markers as normal endothelial cells such as CD31, vWF, and E-selectin (in response to LPS exposure), HemECs proliferate and migrate at a faster rate *in vitro* in response to treatment with exogenous VEGF-A when compared with human dermal microvascular endothelial cells (HDMECs) (Boye et al., 2001). A subsequent study showed that HemECs also possess properties similar to those of immature endothelial cells, cord blood endothelial progenitor cells (cbEPCs). When exposed to the angiogenesis inhibitor endostatin, cbEPCs paradoxically exhibited increased adhesion, proliferation and migration in response to a VEGF-A gradient, while HDMECs migrated more slowly in response to endostatin exposure. Eventually, the response of cbEPCs to the same experiments shifted to mirror that of mature endothelial cells as the cells were passaged in culture. HemECs exposed to endostatin also demonstrated behavior similar to that of cbEPCs, further demonstrating their abnormal behavior when compared with normal endothelial cells (Khan et al., 2006). Taken together, these data further support the theory that an anomaly intrinsic to the IH gives rise to the lesion.

Later investigations resulted in the isolation and characterization of a cell called the hemangioma stem cell (HemSC) (Khan et al., 2008). These HemSCs were demonstrated to be highly proliferative in culture, and capable of differentiating into endothelial, adipocyte, chondrocyte, osteocyte, and neuroglial lineages*.* Furthermore, when mixed with Matrigel and subsequently implanted as a subcutaneous Matrigel plug into the backs of nude mice, HemSCs formed GLUT1+ endothelial cell-lined functional blood vessels during the first few weeks. Four weeks after implantation, however, the Matrigel plugs were instead found to contain adipocytes. This was the first valid published animal model of IH, and its recapitulation of the natural history of IH gave further credence to the intrinsic theory of IH as well as evidence for the new theory of a stem cell as the cellular origin of IHs.

Some evidence also points to a possible neural crest cell origin for IHs. Waner and colleagues were the first to notice that segmental hemangiomas tend to occur at regions of embryological fusion (Waner et al., 2003). Several years later, Haggstrom and colleagues conducted a larger study, finding that certain segmental facial hemangiomas tend to respect embryological boundaries such as the maxillary and mandibular promineces, while others did not (Haggstrom et al., 2006b). Using their observations, they grouped the distribution of facial hemangiomas into four segments. Given the pattern of distribution of segmental facial hemangiomas, these groups also postulated that facial hemangiomas may have a neural crest cell origin. Expression of a marker for neural crest stem cells, p75, has also been demonstrated in proliferating IH (Itinteang et al., 2010).

#### **6.3 VEGF pathway**

Different pathways have been suggested as possibly being involved in the pathogenesis and development of IHs. The VEGF pathway, as one of the most important signaling molecules in normal and pathophysiological angiogenesis, has been the subject of much study. VEGFR-1 and VEGFR-2 are both expressed in IH. However, Jinnin et al demonstrated that VEGFR-2 signaling is constitutively active in cultured HemECs in the presence of VEGF-A, and that this signaling is a result of downregulated VEGFR-1 expression. VEGFR-1 is expressed at much lower levels as a result of aberrant increased interaction of a mutated

Infantile Hemangiomas: A Disease Model in the Study

and differentiation into HemEC phenotype.

**6.6 Renin-angiotensin system**

remain to be seen.

**6.7 Tumor endothelial cells**

**6.5 Notch signaling**

of Vascular Development, Aberrant Vasculogenesis and Angiogenesis 223

mRNA expression in HemECs down-regulated in response to serum relative to HDMECs. Thus Tie2 and its ligands Ang1 and Ang2 likely have an important role in the pathogenesis

Recent studies have also focused on the Notch signaling pathway, a highly conserved juxtacrine signaling pathway that is involved in cell fate determination during embryogenesis. Four Notch receptor genes (Notch1-4) were found to be expressed in IHs, as well as 2 Notch ligands, Jagged-1 and Delta-like-4 (Dll-4) (Wu & Kitajewski, 2009). Moreover, the Notch expression profile of proliferating hemangiomas is different than that of hemangiomas undergoing involution, with involuting hemangiomas demonstrating increased expression of Notch4 and Jagged-1 when compared with proliferating hemangiomas. HemSCs and HemECs also demonstrate completely different Notch expression profiles, with HemSCs expressing higher levels of Notch2 and Notch3, but lower levels of Notch1, Notch4, Jagged-1, and Dll-4 compared to HemECs (Wu et al., 2010). These data suggest a possible role for Notch signaling in the maintanence of HemSC pluripotency

Another area of focus on pathogenesis of IH is the renin-angiotensin system (Itinteang et al., 2011a). Proliferating hemangioma has been demonstrated to express both angiotensinconverting enzyme (ACE) and angiotensin receptor 2. Blast-like structures isolated from IH using an *in vitro* explant model expressed CD133, CD34, and VEGFR-2, as well as ACE. These blast-like structures increased in number following angiotenisin II treatment in a dose-response manner. The authors postulate that treatment of IH with propranolol exerts its effects at least partially through downregulation of renin activity of the kidney, leading to decreased conversion of angiotensinogen to angiotensin I and ultimately to decreased angiotensin II levels, thereby decreasing proliferation of IH. While interesting, whether or how the renin-angiotensin system contributes to the pathogenesis or development of IHs

It has long been known that as tumors develop and increase in size, they recruit blood vessels from their host in order to attempt to meet their increased metabolized demands by a process termed angiogenesis. The resultant vasculature is abnormal, and characterized by tortuous vessels, a disorganized sprouting, vessel leakiness, and loose associations between the vessels' endothelial cells and the basement membrane and pericytes that cover them. While an abnormal vasculature is a hallmark of solid tumors, it is only recently that there has been evidence suggesting that these blood vessels are composed of endothelial cells that are in and of themselves intrinsically abnormal as well. Endothelial cells isolated from human renal cell carcinoma have been reported to be more resistant to serum starvation and vincristineinduced apoptosis when compared to normal endothelial cells, exhibit enhanced Akt activation and decreased expression of the tumor suppressor gene PTEN, and do not undergo senescence (Bussolati et al., 2003). Murine Lewis lung carcinoma endothelial cells were characterized by a comparatively elongated cellular morphology, fewer cell-cell contacts, and increased levels of a host of endothelial cell-surface markers (Allport & Weissleder, 2003).

of hemangioma (Acevedo & Cheresh, 2008; Jinnin et al., 2008; Yu, et al., 2001).

anthrax toxin receptor-1 (ANTXR-1) with b1-integrin and VEGFR-2. The increase in this complex formation in HemECs appears to suppress NFAT-promoted transcription of VEGFR-1. As both VEGFR-1 and VEGFR-2 proteins bind to VEGF-A, and VEGFR-1 is believed to act as a negative regulator of VEGFR-2 signaling through sequestration of VEGF-A, a decrease in VEGFR-1 expression would lead to constitutive VEGFR-2 signaling. This constitutive VEGFR-2 signaling in the presence of VEGF-A helps explain the aberrant overgrowth of endothelial cells in IH.

Studies involving treatment options for IH, such as corticosteroids and propranolol, serve to further highlight the importance of the VEGF pathway in IH pathogenesis. Corticosteroids have been one of the first-line medical treatments for problematic hemangiomas. Greenberger and colleagues demonstrated that dexamethasone treatment of mice implanted with HemSCs in a Matrigel plug with led to a dose-dependent inhibition of microvessel density in those plugs. Pre-treating HemSCs with dexamethasone, or silencing VEGF-A expression in HemSCs using shRNA lentiviral particles, before implantation into mice also gave similar results. Furthermore, *in vitro* administration of dexamethasone to HemSCs resulted in a significant decrease in both mRNA transcript and protein expression levels of VEGF-A. The authors also showed that VEGF-A expression is present in IHs in the proliferating phase, but not in those in the involuting phase, strongly suggesting that early expression of VEGF-A by HemSCs has a crucial role in the pathogenesis of IH. (Greenberger et al., 2010b). In a subsequent study, the same authors demonstrated that treatment of HemSCs with dexamethasone suppresses the activity of nuclear factor kappa-light-chainenhancer of activated B cells (NF-B). They also showed that direct suppression of NF-B activity leads to decreased VEGF-A mRNA and protein expression, suggesting that NF-B regulates VEGF-A expression in HemSCs and providing a plausible mechanism explaining the clinical effect of corticosteroids on IH (Greenberger et al., 2010a).

Recently, propranolol has been demonstrated to be an effective drug for the treatment of IH. In 2008, Léauté-Labrèze and colleagues reported a dramatic response to propranolol treatment in a small cohort of eleven patients with IH (Léauté-Labrèze et al., 2008). However, the molecular mechanisms behind this effect are of considerable interest. Recent work by Lamy and colleagues in HUVECs has demonstrated that treatment with propranolol inhibited proliferation, chemotactic migration, and tube formation *in vitro*, as well as inhibition of VEGF-A-induced tyrosine phosphorylation of VEGFR-2 (Lamy et al., 2010). While this study demonstrates that propranolol can interfere with some of the important steps involved in angiogenesis, shortcomings of this study include lack of *in vivo* data and the fact that none of the experiments were conducted in HemECs and limits the application of the group's conclusions to IHs. More work is needed to elucidate the mechanism of action of propranolol on the accelerated involution of IH.

#### **6.4 TIE/Angiopoietin signaling**

The tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (TIE2)/angiopoietin signaling pathway is also another putative regulator of IH pathogenesis. Tie1, Tie2, and angiopoietin-2 (Ang2) are strongly expressed in cultured HemECs, with low expression of Ang1. Relative to human dermal microvascular endothelial cells (HDMECs), Tie2 mRNA and protein expression are increased in HemECs, and HemECs also demonstrate a concomitant increase in cellular responsiveness to Ang1 as measured by cellular proliferation and migration. Altered regulation of Ang2 has also been observed, with Ang2 mRNA expression in HemECs down-regulated in response to serum relative to HDMECs. Thus Tie2 and its ligands Ang1 and Ang2 likely have an important role in the pathogenesis of hemangioma (Acevedo & Cheresh, 2008; Jinnin et al., 2008; Yu, et al., 2001).

#### **6.5 Notch signaling**

222 Tumor Angiogenesis

anthrax toxin receptor-1 (ANTXR-1) with b1-integrin and VEGFR-2. The increase in this complex formation in HemECs appears to suppress NFAT-promoted transcription of VEGFR-1. As both VEGFR-1 and VEGFR-2 proteins bind to VEGF-A, and VEGFR-1 is believed to act as a negative regulator of VEGFR-2 signaling through sequestration of VEGF-A, a decrease in VEGFR-1 expression would lead to constitutive VEGFR-2 signaling. This constitutive VEGFR-2 signaling in the presence of VEGF-A helps explain the aberrant

Studies involving treatment options for IH, such as corticosteroids and propranolol, serve to further highlight the importance of the VEGF pathway in IH pathogenesis. Corticosteroids have been one of the first-line medical treatments for problematic hemangiomas. Greenberger and colleagues demonstrated that dexamethasone treatment of mice implanted with HemSCs in a Matrigel plug with led to a dose-dependent inhibition of microvessel density in those plugs. Pre-treating HemSCs with dexamethasone, or silencing VEGF-A expression in HemSCs using shRNA lentiviral particles, before implantation into mice also gave similar results. Furthermore, *in vitro* administration of dexamethasone to HemSCs resulted in a significant decrease in both mRNA transcript and protein expression levels of VEGF-A. The authors also showed that VEGF-A expression is present in IHs in the proliferating phase, but not in those in the involuting phase, strongly suggesting that early expression of VEGF-A by HemSCs has a crucial role in the pathogenesis of IH. (Greenberger et al., 2010b). In a subsequent study, the same authors demonstrated that treatment of HemSCs with dexamethasone suppresses the activity of nuclear factor kappa-light-chainenhancer of activated B cells (NF-B). They also showed that direct suppression of NF-B activity leads to decreased VEGF-A mRNA and protein expression, suggesting that NF-B regulates VEGF-A expression in HemSCs and providing a plausible mechanism explaining

Recently, propranolol has been demonstrated to be an effective drug for the treatment of IH. In 2008, Léauté-Labrèze and colleagues reported a dramatic response to propranolol treatment in a small cohort of eleven patients with IH (Léauté-Labrèze et al., 2008). However, the molecular mechanisms behind this effect are of considerable interest. Recent work by Lamy and colleagues in HUVECs has demonstrated that treatment with propranolol inhibited proliferation, chemotactic migration, and tube formation *in vitro*, as well as inhibition of VEGF-A-induced tyrosine phosphorylation of VEGFR-2 (Lamy et al., 2010). While this study demonstrates that propranolol can interfere with some of the important steps involved in angiogenesis, shortcomings of this study include lack of *in vivo* data and the fact that none of the experiments were conducted in HemECs and limits the application of the group's conclusions to IHs. More work is needed to elucidate the

The tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (TIE2)/angiopoietin signaling pathway is also another putative regulator of IH pathogenesis. Tie1, Tie2, and angiopoietin-2 (Ang2) are strongly expressed in cultured HemECs, with low expression of Ang1. Relative to human dermal microvascular endothelial cells (HDMECs), Tie2 mRNA and protein expression are increased in HemECs, and HemECs also demonstrate a concomitant increase in cellular responsiveness to Ang1 as measured by cellular proliferation and migration. Altered regulation of Ang2 has also been observed, with Ang2

the clinical effect of corticosteroids on IH (Greenberger et al., 2010a).

mechanism of action of propranolol on the accelerated involution of IH.

**6.4 TIE/Angiopoietin signaling**

overgrowth of endothelial cells in IH.

Recent studies have also focused on the Notch signaling pathway, a highly conserved juxtacrine signaling pathway that is involved in cell fate determination during embryogenesis. Four Notch receptor genes (Notch1-4) were found to be expressed in IHs, as well as 2 Notch ligands, Jagged-1 and Delta-like-4 (Dll-4) (Wu & Kitajewski, 2009). Moreover, the Notch expression profile of proliferating hemangiomas is different than that of hemangiomas undergoing involution, with involuting hemangiomas demonstrating increased expression of Notch4 and Jagged-1 when compared with proliferating hemangiomas. HemSCs and HemECs also demonstrate completely different Notch expression profiles, with HemSCs expressing higher levels of Notch2 and Notch3, but lower levels of Notch1, Notch4, Jagged-1, and Dll-4 compared to HemECs (Wu et al., 2010). These data suggest a possible role for Notch signaling in the maintanence of HemSC pluripotency and differentiation into HemEC phenotype.

#### **6.6 Renin-angiotensin system**

Another area of focus on pathogenesis of IH is the renin-angiotensin system (Itinteang et al., 2011a). Proliferating hemangioma has been demonstrated to express both angiotensinconverting enzyme (ACE) and angiotensin receptor 2. Blast-like structures isolated from IH using an *in vitro* explant model expressed CD133, CD34, and VEGFR-2, as well as ACE. These blast-like structures increased in number following angiotenisin II treatment in a dose-response manner. The authors postulate that treatment of IH with propranolol exerts its effects at least partially through downregulation of renin activity of the kidney, leading to decreased conversion of angiotensinogen to angiotensin I and ultimately to decreased angiotensin II levels, thereby decreasing proliferation of IH. While interesting, whether or how the renin-angiotensin system contributes to the pathogenesis or development of IHs remain to be seen.

#### **6.7 Tumor endothelial cells**

It has long been known that as tumors develop and increase in size, they recruit blood vessels from their host in order to attempt to meet their increased metabolized demands by a process termed angiogenesis. The resultant vasculature is abnormal, and characterized by tortuous vessels, a disorganized sprouting, vessel leakiness, and loose associations between the vessels' endothelial cells and the basement membrane and pericytes that cover them. While an abnormal vasculature is a hallmark of solid tumors, it is only recently that there has been evidence suggesting that these blood vessels are composed of endothelial cells that are in and of themselves intrinsically abnormal as well. Endothelial cells isolated from human renal cell carcinoma have been reported to be more resistant to serum starvation and vincristineinduced apoptosis when compared to normal endothelial cells, exhibit enhanced Akt activation and decreased expression of the tumor suppressor gene PTEN, and do not undergo senescence (Bussolati et al., 2003). Murine Lewis lung carcinoma endothelial cells were characterized by a comparatively elongated cellular morphology, fewer cell-cell contacts, and increased levels of a host of endothelial cell-surface markers (Allport & Weissleder, 2003).

Infantile Hemangiomas: A Disease Model in the Study

to new insights into tumor angiogenesis.

January 2010, pp. 274-281.

pp. 527-530.

2002, pp. 220-204.

December 2005, pp. 19097-19102.

**8. References**

of Vascular Development, Aberrant Vasculogenesis and Angiogenesis 225

hematopoietic differentiation and should be investigated for their potential role in normal vasculogenesis. IHs also share many similarities with tumor endothelial cells, and further study and comparison with the unique characteristics of tumor endothelial cells could lead

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Hida and colleagues purified murine endothelial cells from human xenograft models of melanoma and liposarcoma and compared them with murine endothelial cells isolated from normal murine skin and adipose tissue. They found that endothelial cells isolated from tumors were cytogenetically different when compared to normal endothelial cells, displaying larger, heterogenous nuclei and aneuploidy that was exacerbated by passage in culture. Abnormal multiple centrosomes were also seen, and FISH analysis showed that the structural chromosomal aberrations were heterogenous, indicating that the mutations were not clonal (Hida et al., 2004). Possible mechanisms for this cytogenetic instability include the unique growth factor milieu in the tumor environment causing genetic instability; loss of tumor suppressor and checkpoint activity; uptake of human tumor oncogenes by fusion or phagocytosis of apoptotic bodies; and transdifferentiation of tumor cells into endothelial cells.

Xiong and colleages isolated endothelial cells from human hepatocellular carcinoma (HCC) and adjacent normal tissue, and reported that HCC endothelial cells demonstrate enhanced angiogenic activity including enhanced proliferation, resistance to apoptosis in the absence of serum, enhanced migration, and enhanced ability to form tubes when implanted into Matrigel in the absence of serum. These cells were also relatively resistant to chemotherapeutic drugs including adriamycin and 5-fluorouracil, and the antiangiogenic drug sorafenib. Tumor endothelial cells share many of the same characteristics of HemECs, and further research comparing the similarities and differences between the two cell types may give insight both into the origin of IHs and the unique properties of tumor endothelial cells (Xiong et al., 2009).

Interestingly, Dudley and colleagues have recently reported that tumor endothelial cells isolated from a transgenic mouse model in which the mice develop prostate adenocarcinoma at puberty demonstrate some of the features of mesenchymal stem cells. When cultured *in vitro* and stimulated with osteogenic medium, clonal populations of these prostate tumor endothelial cells demonstrated alkaline phosphatase activity, expressed bone-specific markers such as osteopontin and osteocalcin, and underwent mineralization. These same cells formed cartilage-like tissues when cultured in chondrogenic medium, with concomitant upregulation of cartilage-specific genes. However, these tumor endothelial cells could not be differentiated to form adipocytes, distinguishing them from normal mesenchymal stem cells and HemSCs (Dudley et al., 2008).

#### **7. Conclusion**

IHs remain poorly understood but may have serious clinical consequences. While many details of their pathogenesis and regulatory mechanisms are still not known, they most likely originate from a hemangioma stem cell (HemSC) that abnormally differentiate into hemangioma endothelial cells (HemEC). These HemECs are different from mature endothelial cells in that they retain features of immature endothelial progenitor cells, suggesting an abnormality in stem cell differentiation. In addition, many different signaling mechanisms have been studied as potential candidates for regulating the transition of IH from the proliferating to involuting phase, and from HemSC to HemEC.

Understanding the mechanisms that regulate IHs will allow directed therapy for affected infants. Moreover, IHs also serve as a model of aberrant vasculogenesis and allow for greater understanding of vascular development, including the role of the VEGF, Notch, and renin-angiotensin signaling pathways. These mechanisms may give insight into hematopoietic differentiation and should be investigated for their potential role in normal vasculogenesis. IHs also share many similarities with tumor endothelial cells, and further study and comparison with the unique characteristics of tumor endothelial cells could lead to new insights into tumor angiogenesis.

#### **8. References**

224 Tumor Angiogenesis

Hida and colleagues purified murine endothelial cells from human xenograft models of melanoma and liposarcoma and compared them with murine endothelial cells isolated from normal murine skin and adipose tissue. They found that endothelial cells isolated from tumors were cytogenetically different when compared to normal endothelial cells, displaying larger, heterogenous nuclei and aneuploidy that was exacerbated by passage in culture. Abnormal multiple centrosomes were also seen, and FISH analysis showed that the structural chromosomal aberrations were heterogenous, indicating that the mutations were not clonal (Hida et al., 2004). Possible mechanisms for this cytogenetic instability include the unique growth factor milieu in the tumor environment causing genetic instability; loss of tumor suppressor and checkpoint activity; uptake of human tumor oncogenes by fusion or phagocytosis of apoptotic bodies; and transdifferentiation of tumor cells into endothelial

Xiong and colleages isolated endothelial cells from human hepatocellular carcinoma (HCC) and adjacent normal tissue, and reported that HCC endothelial cells demonstrate enhanced angiogenic activity including enhanced proliferation, resistance to apoptosis in the absence of serum, enhanced migration, and enhanced ability to form tubes when implanted into Matrigel in the absence of serum. These cells were also relatively resistant to chemotherapeutic drugs including adriamycin and 5-fluorouracil, and the antiangiogenic drug sorafenib. Tumor endothelial cells share many of the same characteristics of HemECs, and further research comparing the similarities and differences between the two cell types may give insight both into the origin of IHs and the unique properties of tumor endothelial

Interestingly, Dudley and colleagues have recently reported that tumor endothelial cells isolated from a transgenic mouse model in which the mice develop prostate adenocarcinoma at puberty demonstrate some of the features of mesenchymal stem cells. When cultured *in vitro* and stimulated with osteogenic medium, clonal populations of these prostate tumor endothelial cells demonstrated alkaline phosphatase activity, expressed bone-specific markers such as osteopontin and osteocalcin, and underwent mineralization. These same cells formed cartilage-like tissues when cultured in chondrogenic medium, with concomitant upregulation of cartilage-specific genes. However, these tumor endothelial cells could not be differentiated to form adipocytes, distinguishing them from normal

IHs remain poorly understood but may have serious clinical consequences. While many details of their pathogenesis and regulatory mechanisms are still not known, they most likely originate from a hemangioma stem cell (HemSC) that abnormally differentiate into hemangioma endothelial cells (HemEC). These HemECs are different from mature endothelial cells in that they retain features of immature endothelial progenitor cells, suggesting an abnormality in stem cell differentiation. In addition, many different signaling mechanisms have been studied as potential candidates for regulating the transition of IH

Understanding the mechanisms that regulate IHs will allow directed therapy for affected infants. Moreover, IHs also serve as a model of aberrant vasculogenesis and allow for greater understanding of vascular development, including the role of the VEGF, Notch, and renin-angiotensin signaling pathways. These mechanisms may give insight into

mesenchymal stem cells and HemSCs (Dudley et al., 2008).

from the proliferating to involuting phase, and from HemSC to HemEC.

cells.

cells (Xiong et al., 2009).

**7. Conclusion**

Infantile Hemangiomas: A Disease Model in the Study

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**11**

*USA*

Munekazu Yamakuchi

*Aab Cardiovascular Research Institute,*

*University of Rochester School of Medicine and Dentistry,*

**MicroRNAs Regulation of Tumor Angiogenesis**

In the 1970s, Dr. Judah Folkman proposed that many solid tumors require angiogenesis to grow beyond 2 mm in size (Folkman, 1971). His theory held that solid tumors could grow without additional blood vessels up to 1-2 mm, obtaining oxygen and nutrients by diffusion. However, these tumors could undergo an angiogenic switch, releasing substances that would induce new capillary sproutings from existing blood vessels, which would increase oxygen and nutrient delivery to the tumor, permitting tumor expansion. Since Dr. Folkman's hypothesis, assays for angiogenesis have been developed, new angiogenic molecules such as VEGF and VEGF receptors have been discovered, endogenous inhibitors of angiogenesis such as angiostatin have been defined, and anti-angiogenic drugs have been developed to treat cancers (Ferrara, 2002; Ferrara and Kerbel, 2005; Kerbel, 2008). However, the biology of human tumor angiogenesis is poorly understood, and the molecular

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression in a posttranscriptional manner (Bartel, 2004). miRNAs regulate physiological processes such as cell proliferation, apoptosis, and differentiation (Ambros, 2004; Bartel, 2004; He and Hannon, 2004). However, miRNAs are also involved in pathophysiological processes such as oncogenesis (Croce, 2008, 2009). Recent studies unveiled new functions of miRNAs that control angiogenesis and those miRNAs are thought to be new tools for manipulating tumor angiogenic balance. In this chapter, I summarize how a set of miRNAs can regulate tumor

I first describe the biogenesis and function of miRNAs and briefly review general roles of miRNAs in cancer. Next I classify a set of miRNAs that positively or negatively regulate tumor angiogenesis and add current progress in this area. Lastly I focus on hypoxia inducible factors as key molecule of hypoxia signaling and tumor angiogenesis in a view of

Non-coding RNA (ncRNA), lin-4 and lin-14, were first identified to regulate larval developmental timing in *C. elegance* in 1993 (Lee et al., 1993; Wightman et al., 1993). By now, thousands of miRNAs have been discovered in many organisms and mammalian miRNAs are predicted to alter the activity of about 50% of all protein coding genes (Filipowicz et al., 2008). miRNAs control gene expression by destabilizing or inhibiting transcription,

mechanisms that modulate the angiogenic switch are not well characterized.

angiogenesis, reviewing the current findings and our data.

**1. Introduction**

regulation by miRNAs.

**2. microRNAs (miRNAs)**

nude mice. *Plastic and Reconstructive Surgery*, Vol. 120, No. 4, September 2007, pp. 869-878.

## **MicroRNAs Regulation of Tumor Angiogenesis**

#### Munekazu Yamakuchi

*Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, USA*

#### **1. Introduction**

230 Tumor Angiogenesis

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Xiong, Y.Q.; Sun, H.C.; Zhang, W.; Zhu, X.D.; Zhuang, P.Y.; Zhang, J.B.; Wang, L.; Wu, W.Z.;

Xu, Q.; Chen, W.; Wang, Z.; Zheng, J. & Zhang, Z. (2009). Mice transgenic with SV40-late-

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23.

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159, No. 6, December 2001, pp. 2271-2280.

nude mice. *Plastic and Reconstructive Surgery*, Vol. 120, No. 4, September 2007, pp.

treatment of airway hemangiomas: a case series and treatment algorithm. *International Journal of Pediatric Otorhinolaryngology*, Vol. 74, No. 9, September 2010,

nonrandom distribution of facial hemangiomas. *Archives of Dermatology*, Vol. 139,

(2010). A switch in notch gene expression parallels stem cell to endothelial cell transition in infantile hemangioma. *Angiogenesis*, Vol. 13, No. 1, March 2010, pp. 15-

and regulation of hemangiomas. *The Journal of Craniofacial Surgery*, Vol. 20, Suppl 1,

Qin, L.X. &Tang, Z.Y. (2009). Human hepatocellular carcinoma tumor-derived endothelial cells manifest increased angiogenesis capability and drug resistance compared with normal endothelial cells. *Clinical Cancer Research*, Vol. 15, No. 15,

promoter-driven polyomavirus middle T oncogene exclusively develop

enhanced response to angiopoietin-1, and dysregulated angiopoietin-2 expression in hemangioma-derived endothelial cells. *The American Journal of Pathology*, Vol. In the 1970s, Dr. Judah Folkman proposed that many solid tumors require angiogenesis to grow beyond 2 mm in size (Folkman, 1971). His theory held that solid tumors could grow without additional blood vessels up to 1-2 mm, obtaining oxygen and nutrients by diffusion. However, these tumors could undergo an angiogenic switch, releasing substances that would induce new capillary sproutings from existing blood vessels, which would increase oxygen and nutrient delivery to the tumor, permitting tumor expansion. Since Dr. Folkman's hypothesis, assays for angiogenesis have been developed, new angiogenic molecules such as VEGF and VEGF receptors have been discovered, endogenous inhibitors of angiogenesis such as angiostatin have been defined, and anti-angiogenic drugs have been developed to treat cancers (Ferrara, 2002; Ferrara and Kerbel, 2005; Kerbel, 2008). However, the biology of human tumor angiogenesis is poorly understood, and the molecular mechanisms that modulate the angiogenic switch are not well characterized.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression in a posttranscriptional manner (Bartel, 2004). miRNAs regulate physiological processes such as cell proliferation, apoptosis, and differentiation (Ambros, 2004; Bartel, 2004; He and Hannon, 2004). However, miRNAs are also involved in pathophysiological processes such as oncogenesis (Croce, 2008, 2009). Recent studies unveiled new functions of miRNAs that control angiogenesis and those miRNAs are thought to be new tools for manipulating tumor angiogenic balance. In this chapter, I summarize how a set of miRNAs can regulate tumor angiogenesis, reviewing the current findings and our data.

I first describe the biogenesis and function of miRNAs and briefly review general roles of miRNAs in cancer. Next I classify a set of miRNAs that positively or negatively regulate tumor angiogenesis and add current progress in this area. Lastly I focus on hypoxia inducible factors as key molecule of hypoxia signaling and tumor angiogenesis in a view of regulation by miRNAs.

#### **2. microRNAs (miRNAs)**

Non-coding RNA (ncRNA), lin-4 and lin-14, were first identified to regulate larval developmental timing in *C. elegance* in 1993 (Lee et al., 1993; Wightman et al., 1993). By now, thousands of miRNAs have been discovered in many organisms and mammalian miRNAs are predicted to alter the activity of about 50% of all protein coding genes (Filipowicz et al., 2008). miRNAs control gene expression by destabilizing or inhibiting transcription,

MicroRNAs Regulation of Tumor Angiogenesis 233

miRNA perfectly matches the sequence of a target mRNA, then the target mRNA is degraded. Sequence identity between micro-RNA and mRNA often occur in plants. In mammalian cells, there are very few reports about miRNA cleavage of target mRNA (Davis et al., 2005; Jones-Rhoades et al., 2006). Instead, the seed sequence of miRNA usually has homology but not identity to target mRNA in mammalian cells. This imperfect match represses translation by decreasing mRNA stability. The match sequences of 2nd to 6th nucleotide position in 5' end of miRNAs (seed sequence) are sufficient to suppress gene expressions. Some miRNAs suppress target mRNAs expression unless the seed sequence does not completely match to target mRNA target (Bartel, 2009; Shin et al.). Moreover miRNAs can reduce not only the expressions of protein from the target mRNAs, but also the

To accomplish miRNA searches, accurate methods for target prediction are required. There are many miRNA target prediction programs available on-line (Hofacker, 2007; Sethupathy et al., 2006). These prediction systems depend on algorithms of miRNA and mRNA (especially region of seed sequence) and evolutionarily conservation. Targetscan, miRanda, and PicTar are commonly used tools for target prediction. Those programs provided a lot of information for potential target genes, but the false positive rate were calculated about 24- 70% (Thomson et al.). To avoid false positive target genes, overlapped targets from those three prediction programs are generally chosen as more convincing potential target genes.

Genomic alterations of miRNAs are associated with human cancers (Calin et al., 2004a; Iorio et al., 2005). MiR-15a and miR-16a are frequently downregulated in patients with B cell chronic lymphocytic leukemia (Calin et al., 2004b). Lu et al. performed miRNA expression profiling of 334 human cancer samples and showed the feasibility of classification tumor types and stages on miRNA profiles (Lu et al., 2005). This suggested that monitoring the expression of miRNAs in human cancer is very informative for cancer diagnosis. There have been many studies focusing on this diagnostic role of miRNAs and on the association between cancer specific miRNAs and their function. Let-7 family members regulate the human RAS oncogene; and human lung tumor tissues displayed reduced levels of let-7 and increased levels of RAS protein compared to normal lung tissues, suggesting that let-7 might control oncogenesis in humans (Johnson et al., 2005). Furthermore, p53 upregulated the expression of miR-34 family members, which functions as a tumor suppressor (Chang et al.,

Distinct miRNAs regulate tumor angiogenesis in diverse pathways. These miRNAs that regulate angiogenesis are classified into three groups; (1) miRNAs that are not altered under hypoxic condition, (2) hypoxia regulated miRNAs, and (3) miRNAs that regulate HIF-1 signaling molecules. I address functions of each miRNA and discuss two types of angiogenesis miRNAs; pro-angiogenic miRNAs and anti-angiogenic miRNAs (Figure 2).

Several studies of miRNA profiling revealed that miR-126 is expressed specifically in endothelial cells (Harris et al., 2008; Kuehbacher et al., 2007).

**3. miRNAs and tumor angiogenesis**

**3.1 Hypoxia independent miRNAs**

**3.1.1 miR-126**

**2.2 miRNAs function in cancer**

involving in a variety of biological processes. According to mapping of 186 miRNAs and comparison of locations of previous reported nonrandom genetic alterations, Calin et al. discovered that miRNA genes are frequently (more than 50%) located at fragile sites, in minimal regions of loss of heterozygosity, in minimal regions of amplification or in common breakpoint regions (Calin et al., 2004b). As the diverse functions of miRNAs are further studied, our understanding of the molecular pathways of cancers becomes more complicated (Nana-Sinkam and Croce).

#### **2.1 miRNA biogenesis**

Figure 1 illustrates intergenic miRNA biogenesis. First mRNAs are transcribed into primary miRNAs (pri-miRNAs) by RNA polymerase II. These long pri-miRNAs contain stem-loop hairpin structures, which are cleaved into smaller pieces (pre-miRNA) by RNase III Drosha and DiGeorge syndrome critical region 8 (DGCR8) complex. Pre-miRNA is transported from nucleus into cytoplasm by exportin 5 and then diced into double stranded mature miRNAs by Dicer and TAR RNA-binding protein 2 (TRBP2) complex. Both or one strand of single mature miRNAs is incorporated into Argonaute containing RNA-induced silencing complex (RISC), which bind to target mRNAs and dampen their expression.

Fig. 1. miRNA biogenesis. DNA is transcribed into a primary micro-RNA (pri-miR). The primiR is processed into a 70 nt precursor micro-RNA (pre-miR). The pre-miR is processed into a 22 nt long micro-RNA (miRNA). A single strand of the mature miRNA is loaded onto a nucleoprotein RISC complex that silences target messenger RNAs.

MiRNAs suppress target mRNA expression in two ways, Argonaute-catalyzed cleavage of mRNA or mRNA destabilization (Filipowicz et al., 2008). If the seed sequence of a particular

involving in a variety of biological processes. According to mapping of 186 miRNAs and comparison of locations of previous reported nonrandom genetic alterations, Calin et al. discovered that miRNA genes are frequently (more than 50%) located at fragile sites, in minimal regions of loss of heterozygosity, in minimal regions of amplification or in common breakpoint regions (Calin et al., 2004b). As the diverse functions of miRNAs are further studied, our understanding of the molecular pathways of cancers becomes more

Figure 1 illustrates intergenic miRNA biogenesis. First mRNAs are transcribed into primary miRNAs (pri-miRNAs) by RNA polymerase II. These long pri-miRNAs contain stem-loop hairpin structures, which are cleaved into smaller pieces (pre-miRNA) by RNase III Drosha and DiGeorge syndrome critical region 8 (DGCR8) complex. Pre-miRNA is transported from nucleus into cytoplasm by exportin 5 and then diced into double stranded mature miRNAs by Dicer and TAR RNA-binding protein 2 (TRBP2) complex. Both or one strand of single mature miRNAs is incorporated into Argonaute containing RNA-induced silencing complex

Fig. 1. miRNA biogenesis. DNA is transcribed into a primary micro-RNA (pri-miR). The primiR is processed into a 70 nt precursor micro-RNA (pre-miR). The pre-miR is processed into a 22 nt long micro-RNA (miRNA). A single strand of the mature miRNA is loaded onto a

**Regulation of Stability or Translation**

**5'Cap 3' end**

Ago2

**Drosha / DGCR8**

**RNA polymerase II**

**Dicer / TRBP**

**RISC/miRNA**

**AAAAA**

**Pre-miRNA**

MiRNAs suppress target mRNA expression in two ways, Argonaute-catalyzed cleavage of mRNA or mRNA destabilization (Filipowicz et al., 2008). If the seed sequence of a particular

nucleoprotein RISC complex that silences target messenger RNAs.

**Target mRNA**

(RISC), which bind to target mRNAs and dampen their expression.

complicated (Nana-Sinkam and Croce).

**Pri-miRNA**

**DNA**

**miRNA duplex**

**2.1 miRNA biogenesis**

miRNA perfectly matches the sequence of a target mRNA, then the target mRNA is degraded. Sequence identity between micro-RNA and mRNA often occur in plants. In mammalian cells, there are very few reports about miRNA cleavage of target mRNA (Davis et al., 2005; Jones-Rhoades et al., 2006). Instead, the seed sequence of miRNA usually has homology but not identity to target mRNA in mammalian cells. This imperfect match represses translation by decreasing mRNA stability. The match sequences of 2nd to 6th nucleotide position in 5' end of miRNAs (seed sequence) are sufficient to suppress gene expressions. Some miRNAs suppress target mRNAs expression unless the seed sequence does not completely match to target mRNA target (Bartel, 2009; Shin et al.). Moreover miRNAs can reduce not only the expressions of protein from the target mRNAs, but also the levels of their target transcripts (Lim et al., 2005).

To accomplish miRNA searches, accurate methods for target prediction are required. There are many miRNA target prediction programs available on-line (Hofacker, 2007; Sethupathy et al., 2006). These prediction systems depend on algorithms of miRNA and mRNA (especially region of seed sequence) and evolutionarily conservation. Targetscan, miRanda, and PicTar are commonly used tools for target prediction. Those programs provided a lot of information for potential target genes, but the false positive rate were calculated about 24- 70% (Thomson et al.). To avoid false positive target genes, overlapped targets from those three prediction programs are generally chosen as more convincing potential target genes.

#### **2.2 miRNAs function in cancer**

Genomic alterations of miRNAs are associated with human cancers (Calin et al., 2004a; Iorio et al., 2005). MiR-15a and miR-16a are frequently downregulated in patients with B cell chronic lymphocytic leukemia (Calin et al., 2004b). Lu et al. performed miRNA expression profiling of 334 human cancer samples and showed the feasibility of classification tumor types and stages on miRNA profiles (Lu et al., 2005). This suggested that monitoring the expression of miRNAs in human cancer is very informative for cancer diagnosis. There have been many studies focusing on this diagnostic role of miRNAs and on the association between cancer specific miRNAs and their function. Let-7 family members regulate the human RAS oncogene; and human lung tumor tissues displayed reduced levels of let-7 and increased levels of RAS protein compared to normal lung tissues, suggesting that let-7 might control oncogenesis in humans (Johnson et al., 2005). Furthermore, p53 upregulated the expression of miR-34 family members, which functions as a tumor suppressor (Chang et al., 2007; He et al., 2007; Raver-Shapira et al., 2007).

#### **3. miRNAs and tumor angiogenesis**

Distinct miRNAs regulate tumor angiogenesis in diverse pathways. These miRNAs that regulate angiogenesis are classified into three groups; (1) miRNAs that are not altered under hypoxic condition, (2) hypoxia regulated miRNAs, and (3) miRNAs that regulate HIF-1 signaling molecules. I address functions of each miRNA and discuss two types of angiogenesis miRNAs; pro-angiogenic miRNAs and anti-angiogenic miRNAs (Figure 2).

#### **3.1 Hypoxia independent miRNAs**

#### **3.1.1 miR-126**

Several studies of miRNA profiling revealed that miR-126 is expressed specifically in endothelial cells (Harris et al., 2008; Kuehbacher et al., 2007). MiR-126 is encoded on an

cells.

**3.1.3 miR-296**

angiogenesis.

**3.1.4 miR-378**

activity in endothelial cells.

**3.1.5 miR-17-92 in tumors**

MicroRNAs Regulation of Tumor Angiogenesis 235

decrease in p120RasGAP expression and a corresponding increase in miR-132 level (Anand and Cheresh). These findings suggest that miR-132 controls an angiogenic switch in tumor

The upregulation of growth factor receptors on endothelial cells is an important step in angiogenesis. Angiogenic growth factors increased the expression of miR-296 in human brain microvascular endothelial cells and knockdown of miR-296 inhibited endothelial cell migration and tube formation by modulating hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) (Wurdinger et al., 2008). HGS mediated the sorting of growth factor receptors (PDGFR-ß and VEGFR2) to lysosomes to degrade them (Ewan et al., 2006; Takata et al., 2000). Inhibition of miR-296 showed significant decrease glioma angiogenesis in vivo,

Loss of miR-296 expression was observed during tumor progression in human cancers (Hong et al.). One of potential target proteins was Scribble (Scrib) and overexpression of Scrib in human mammary epithelial cells (HMEC) inhibited cell migration and invasion (Vaira et al.). The levels of Scrib were upregulated in primary lesions and distant metastases of liver, colon, lung, breast and stomach cancer, but the exact function in tumors has been not determined. Another target, high-motility group AT-hook gene 1 (HMGA1) was identified in prostate cancer (Wei et al.). HMGA1 expression was associated with high-grade prostate cancer and proliferative and metastatic potential in vitro (Diana et al., 2005). These findings indicate that inhibition of miR-296 cell-autonomously downregulates tumor

MiR-378 enhanced cell survival and promotes tumor growth and angiogenesis. Knockdown of miR-378 decreased cell survival in U87 glioma cells. Injection of miR-378 overexpressed U87 cells into nude mice increased tumor volume and elevated the size of blood vessels (Lee et al., 2007). At least two potential targets of miR-378 were identified; suppressor of fused (Sufu) and tumor suppressor candidate 2 (Fus-1). Sufu is a negative regulator of sonic hedgehog signaling pathway (Yue et al., 2009). Sonic hedgehog pathway is a crucial regulator of angiogenesis and metastasis and increases angiogenic factors such as angiopoietin-1 and angiopoietin-2 (Pola et al., 2001). Sonic hedgehog promotes capillary morphogenesis, induced endothelial cell migration, and increased the expression of MMP-9 and osteopontin in endothelial cells via Rho kinase (Renault et al.). These data suggested that miR-378 might control not only angiogenic potential in tumor cells but also angiogenic

The miR-17-92 cluster is a polycistronic miRNA gene, containing six mature miRNAs (miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a, and miR-92) on *c13orf25* transcript (He et al., 2005; Tanzer and Stadler, 2004). These six miRNAs and two miR-17-92 cluster paralogs (miR-106a-363 and miR-106b-25) are categorized into four families by their seed sequences; (1) miR-17 family (miR-17, -20a, -20b, -106a, -106b, and -93), (2) miR-18 family (miR-18a and miR-18b), (3) miR-19 family (miR-19a, -19b-1, and -19b-2), and (4) miR-92 family (miR-92a-1,

suggesting that miR-296 acts as a pro-angiogenic miRNA in endothelial cells.

Fig. 2. Role of miRNAs in tumor angiogenesis. Different miRNAs contribute to angiogenic response in tumor cells and endothelial cells.

intron of Egfl7 gene and Egfl7 knockout mice displays vascular abnormalities (Schmidt et al., 2007). Endothelial outgrowth was impaired in aortic rings from *miR-126*−/− mice and endothelial cells from *miR-126*−/− mice diminished angiogenic response to FGF-2, suggesting that miR-126 knockout endothelial cells are defective in angiogenesis (Fish et al., 2008; Wang et al., 2008). Moreover Sprouty-related EVH domain-containing protein (Spred-1) and PI3K regulatory subunit 2 (PIK3R2) were defined as target gene of miR-126. Therefore, proangiogenic activity of miR-126 is mediated via the increase of MAP kinase and PI3K signaling in response to angiogenic growth factors. In cancer cells, miR-126 behaves as an anti-tumor miRNA. MiR-126 inhibited metastasis in human breast cancer and tumorigenesis in colon cancer and lung cancer (Crawford et al., 2008; Guo et al., 2008; Tavazoie et al., 2008), but the non-cell-autonomous effects of miR-126 in tumor on angiogenesis are still unclear.

#### **3.1.2 miR-132**

MiR-132 has been recognized as an angiogenic growth factor inducible miRNA in endothelial cells. In normal endothelial cells, the level of miR-132 is very low, but miR-132 is highly expressed in human tumors and hemangiomas. VEGF and bFGF or conditioned media from tumors activated cAMP-response element binding protein (CREB) and increased miR-132 levels in various types of endothelial cells (Anand et al.; Lagos et al.). Interestingly neurotropic growth factors also induced miR-132, promoting dendritic growth and spine formation in neuronal culture (Magill et al.). MiR-132 facilitated pathological angiogenesis by suppressing p120RasGAP, a molecular brake for RAS. Knockdown of miR-132 decreased angiogenesis in tumor models (Anand et al.). Moreover, endothelial cells from several pathological human and mouse tissues including tumors had a dramatic decrease in p120RasGAP expression and a corresponding increase in miR-132 level (Anand and Cheresh). These findings suggest that miR-132 controls an angiogenic switch in tumor cells.

#### **3.1.3 miR-296**

234 Tumor Angiogenesis

**miR-210**

**GPD1L NDUFA4 SDHD**

**HIF1**

**miR-424**

**CLU2**

**miR-22 miR-17 miR-519c**

**HIF1**

**miR-126**

**PI3KR2 SPRED1**

**miR-132**

**p120Ras GAP**

**miR-92**

**c-KIT ITG-a5**

**Jak1**

**miR-17**

**miR-296**

**miR-210**

**EphrinA3**

**miR-130**

**GAX HOX5A**

**HGS**

**VEGFR2**

**Pro-angiogenic**

**Anti-angiogenic**

**miR-221 miR-222**

Fig. 2. Role of miRNAs in tumor angiogenesis. Different miRNAs contribute to angiogenic

**Tumor cell Endothelial cell**

intron of Egfl7 gene and Egfl7 knockout mice displays vascular abnormalities (Schmidt et al., 2007). Endothelial outgrowth was impaired in aortic rings from *miR-126*−/− mice and endothelial cells from *miR-126*−/− mice diminished angiogenic response to FGF-2, suggesting that miR-126 knockout endothelial cells are defective in angiogenesis (Fish et al., 2008; Wang et al., 2008). Moreover Sprouty-related EVH domain-containing protein (Spred-1) and PI3K regulatory subunit 2 (PIK3R2) were defined as target gene of miR-126. Therefore, proangiogenic activity of miR-126 is mediated via the increase of MAP kinase and PI3K signaling in response to angiogenic growth factors. In cancer cells, miR-126 behaves as an anti-tumor miRNA.

MiR-132 has been recognized as an angiogenic growth factor inducible miRNA in endothelial cells. In normal endothelial cells, the level of miR-132 is very low, but miR-132 is highly expressed in human tumors and hemangiomas. VEGF and bFGF or conditioned media from tumors activated cAMP-response element binding protein (CREB) and increased miR-132 levels in various types of endothelial cells (Anand et al.; Lagos et al.). Interestingly neurotropic growth factors also induced miR-132, promoting dendritic growth and spine formation in neuronal culture (Magill et al.). MiR-132 facilitated pathological angiogenesis by suppressing p120RasGAP, a molecular brake for RAS. Knockdown of miR-132 decreased angiogenesis in tumor models (Anand et al.). Moreover, endothelial cells from several pathological human and mouse tissues including tumors had a dramatic

response in tumor cells and endothelial cells.

**miR-378**

**Sufu Fus-1**

**Anti-angiogenic**

**Pro-angiogenic**

**p53**

**miR-107**

**HIF1**

**3.1.2 miR-132**

**c-Myc**

**miR-200b**

**ZEB1/2**

**miR-18 miR-19**

**Tsp-1 CTGF** The upregulation of growth factor receptors on endothelial cells is an important step in angiogenesis. Angiogenic growth factors increased the expression of miR-296 in human brain microvascular endothelial cells and knockdown of miR-296 inhibited endothelial cell migration and tube formation by modulating hepatocyte growth factor-regulated tyrosine kinase substrate (HGS) (Wurdinger et al., 2008). HGS mediated the sorting of growth factor receptors (PDGFR-ß and VEGFR2) to lysosomes to degrade them (Ewan et al., 2006; Takata et al., 2000). Inhibition of miR-296 showed significant decrease glioma angiogenesis in vivo, suggesting that miR-296 acts as a pro-angiogenic miRNA in endothelial cells.

Loss of miR-296 expression was observed during tumor progression in human cancers (Hong et al.). One of potential target proteins was Scribble (Scrib) and overexpression of Scrib in human mammary epithelial cells (HMEC) inhibited cell migration and invasion (Vaira et al.). The levels of Scrib were upregulated in primary lesions and distant metastases of liver, colon, lung, breast and stomach cancer, but the exact function in tumors has been not determined. Another target, high-motility group AT-hook gene 1 (HMGA1) was identified in prostate cancer (Wei et al.). HMGA1 expression was associated with high-grade prostate cancer and proliferative and metastatic potential in vitro (Diana et al., 2005). These findings indicate that inhibition of miR-296 cell-autonomously downregulates tumor angiogenesis.

#### **3.1.4 miR-378**

MiR-378 enhanced cell survival and promotes tumor growth and angiogenesis. Knockdown of miR-378 decreased cell survival in U87 glioma cells. Injection of miR-378 overexpressed U87 cells into nude mice increased tumor volume and elevated the size of blood vessels (Lee et al., 2007). At least two potential targets of miR-378 were identified; suppressor of fused (Sufu) and tumor suppressor candidate 2 (Fus-1). Sufu is a negative regulator of sonic hedgehog signaling pathway (Yue et al., 2009). Sonic hedgehog pathway is a crucial regulator of angiogenesis and metastasis and increases angiogenic factors such as angiopoietin-1 and angiopoietin-2 (Pola et al., 2001). Sonic hedgehog promotes capillary morphogenesis, induced endothelial cell migration, and increased the expression of MMP-9 and osteopontin in endothelial cells via Rho kinase (Renault et al.). These data suggested that miR-378 might control not only angiogenic potential in tumor cells but also angiogenic activity in endothelial cells.

#### **3.1.5 miR-17-92 in tumors**

The miR-17-92 cluster is a polycistronic miRNA gene, containing six mature miRNAs (miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a, and miR-92) on *c13orf25* transcript (He et al., 2005; Tanzer and Stadler, 2004). These six miRNAs and two miR-17-92 cluster paralogs (miR-106a-363 and miR-106b-25) are categorized into four families by their seed sequences; (1) miR-17 family (miR-17, -20a, -20b, -106a, -106b, and -93), (2) miR-18 family (miR-18a and miR-18b), (3) miR-19 family (miR-19a, -19b-1, and -19b-2), and (4) miR-92 family (miR-92a-1, -92a-2, -383, and -25) (Figure 3A).

MicroRNAs Regulation of Tumor Angiogenesis 237

The regulation and function of miR-17-92 have become more complicated. Each miRNAs in this cluster works cooperatively and independently. Each individual miRNA has different target genes because of their own seed sequences. For example, miR-19, which is thought to be a major oncogenic component of miR-17-92 cluster, suppressed PTEN expression (Olive et al., 2009). In the Eμ-myc model of Burkitt's lymphoma, miR-19 was necessary and sufficient for mir-17-92 to promote c-Myc-induced B lymphomagenesis. MiR-17, miR-20, and miR106b regulate cell cycle progression through the inhibition of cyclin dependent

The miR-17-92 cluster is highly expressed in endothelial cells (Kuehbacher et al., 2007). Overexpression of miR-92a inhibited angiogenesis in endothelial cells in part by repressing integrin α5 (ITGa5). Inhibition of miR-92a in a mouse model of hind limb ischemia augmented neovascularization and functional recovery of damaged tissue (Bonauer et al., 2009). The same research group has explored the role of the other miRNAs of miR-17-92 cluster in endothelial cells as well (Doebele et al.). Overexpression of all individual members of miR-17-92 cluster (miR-17, miR-18a, miR-19a, and miR-20a) inhibited endothelial proliferation and migration in vitro, in contrast, and individual knockdown enhanced migration and proliferation. MiR-17 and miR-20 were particularly potent inhibitors of endothelial migration, and their effects were

These data indicate that individual miRNAs in miR-17-92 cluster suppress angiogenesis of normal endothelial cells in vivo. What about their effect upon angiogenesis of endothelial cells in tumors in vivo? Although inhibitors of miR-17 and miR-20 increased vascularization of Matrigel plugs in mice, but these same inhibitors did not affect tumor angiogenesis (Doebele et al.). These results show that specific miRNAs can regulate angiogenesis in one context (Matrigel plugs) but have no effect upon angiogenesis in another context (tumor angiogenesis). One possible explanation is that different sets of genes modulate angiogenesis in different physiological situations (Figure 3B). Suarez et al. showed that reduction of miRNAs by knockdown of Dicer in endothelial cells impaired angiogenic response and reconstitution of individual miRNAs in miR-17-92 cluster rescued angiogenic inhibition (Suarez et al., 2008).

In summary, contradictory data show that miR-17 inhibits angiogenesis following hind limb ischemia, but all miRNAs together promote angiogenesis in hind limb ischemia. The individual contribution to angiogenesis of distinct miRNA inside endothelial cells is not

miR-221 and miR-222 are abundant in endothelial cells, and they affect angiogenic activity of stem cell factor (SCF) by modulating the level of its receptor c-Kit (Poliseno et al., 2006). miR-221 strongly upregulated GAX expression by inhibiting ZEB2, a modulator of the epithelialmesenchymal transition (Chen et al.). GAX is a regulatory gene controlling the angiogenic phenotype in endothelial cells. In several solid tumors, miR-221/222 is highly expressed (le Sage et al., 2007; Medina et al., 2008) and regulated by proto-oncogene ETS-1, involving in the pathogenesis of cancers (Mattia et al.). In melanoma progression, ETS-1 is directly targeted by miR-222, but not by miR-221, which makes a complex ETS-1/miR-222 co-regulatory loop.

miR-222 might inhibit angiogenesis by repressing Ets signaling in endothelial cells.

kinase inhibitor, p21 (Ivanovska et al., 2008).

mediated in part through the tyrosine kinase Jak1 (Doebele et al.).

These results show that endothelial miRNAs are important for angiogenesis.

**3.1.6 miR-17-92 in endothelial cells**

fully known.

**3.1.7 miR-221/222**

The transcripts of region containing miR-17-92 precursor were elevated n various cancers, such as B-cell lymphoma, multiple myeloma, thyroid cancer, and lung cancer (Hayashita et al., 2005; Inomata et al., 2009; Pichiorri et al., 2008; Takakura et al., 2008). This elevation of miR-17-92 contributes to tumor growth partially by enhancing tumor angiogenesis (Dews et al., 2006).

Fig. 3. The pleiotropic functions of miR-17-92 cluster. (A) Primary transcripts of miR-17-92 cluster and its paralogs. (B) Mir-17-92 can promote proliferation and increase angiogenesis in cancer cells. In endothelial cells, miR-17-92 inhibits angiogenesis.

miR-17-92 cluster has been identified as a direct transcriptional target of c-Myc by using a spotted oligonucleotide array (O'Donnell et al., 2005). c-Myc is a helix-loop-helix oncogenic transcription factor that regulates cell proliferation and transformation and activated in many human cancers (Dang, 1999). Myc binds to E-box element with Max, driving cell cycle progression. Myc also negatively regulates some gene transcriptions with Miz-1. Dews et al. have shown that Myc-overexpressing tumors possessed more robust neovascularization and miR-17-92 mediated tumor angiogenesis by Myc (Dews et al., 2006). They also found that miR-17-92 targeted two anti-angiogenic factors, thrombospondin-1 (TSP-1) and connective tissue growth factor (CTGF) expression in RasGfpMyc colonocytes.

Another transcription factor that regulates miR-17-92 cluster has been reported. E2F1 and E2F3 also directly activated miR-17-92 transcription as well as turns on the progression of the cell cycle (Sylvestre et al., 2007; Woods et al., 2007). MiR-17 and miR-20 inhibited the expression of E2F1, E2F2, and E2F3 (O'Donnell et al., 2005). These data suggested a negative feedback loop between miR-17-92 cluster, E2F, and Myc.

 This elevation of miR-17-92 contributes to tumor growth partially by enhancing tumor angiogenesis (Dews et

Fig. 3. The pleiotropic functions of miR-17-92 cluster. (A) Primary transcripts of miR-17-92 cluster and its paralogs. (B) Mir-17-92 can promote proliferation and increase angiogenesis

**Angiogenesis**

**miR-17-92**

**ITGa5**

**Jak1**

**Tumor cells Endothelial Cells**

**106b 93 25**

**17 18a 19a 20a 19b-1 92a-1**

**106a 18b 20b 19b-2 92a-2 363**

**Apoptosis**

miR-17-92 cluster has been identified as a direct transcriptional target of c-Myc by using a spotted oligonucleotide array (O'Donnell et al., 2005). c-Myc is a helix-loop-helix oncogenic transcription factor that regulates cell proliferation and transformation and activated in many human cancers (Dang, 1999). Myc binds to E-box element with Max, driving cell cycle progression. Myc also negatively regulates some gene transcriptions with Miz-1. Dews et al. have shown that Myc-overexpressing tumors possessed more robust neovascularization and miR-17-92 mediated tumor angiogenesis by Myc (Dews et al., 2006). They also found that miR-17-92 targeted two anti-angiogenic factors, thrombospondin-1 (TSP-1) and connective

Another transcription factor that regulates miR-17-92 cluster has been reported. E2F1 and E2F3 also directly activated miR-17-92 transcription as well as turns on the progression of the cell cycle (Sylvestre et al., 2007; Woods et al., 2007). MiR-17 and miR-20 inhibited the expression of E2F1, E2F2, and E2F3 (O'Donnell et al., 2005). These data suggested a negative

in cancer cells. In endothelial cells, miR-17-92 inhibits angiogenesis.

**CTGF**

**PTEN**

**Bim**

**Tsp1**

tissue growth factor (CTGF) expression in RasGfpMyc colonocytes.

feedback loop between miR-17-92 cluster, E2F, and Myc.

al., 2006).

**(A)**

**(B)**

**Myc**

**miR-17-92 cluster**

**miR-106b-25 cluster**

**miR-106a-363 cluster**

**miR-17-92**

The regulation and function of miR-17-92 have become more complicated. Each miRNAs in this cluster works cooperatively and independently. Each individual miRNA has different target genes because of their own seed sequences. For example, miR-19, which is thought to be a major oncogenic component of miR-17-92 cluster, suppressed PTEN expression (Olive et al., 2009). In the Eμ-myc model of Burkitt's lymphoma, miR-19 was necessary and sufficient for mir-17-92 to promote c-Myc-induced B lymphomagenesis. MiR-17, miR-20, and miR106b regulate cell cycle progression through the inhibition of cyclin dependent kinase inhibitor, p21 (Ivanovska et al., 2008).

#### **3.1.6 miR-17-92 in endothelial cells**

The miR-17-92 cluster is highly expressed in endothelial cells (Kuehbacher et al., 2007). Overexpression of miR-92a inhibited angiogenesis in endothelial cells in part by repressing integrin α5 (ITGa5). Inhibition of miR-92a in a mouse model of hind limb ischemia augmented neovascularization and functional recovery of damaged tissue (Bonauer et al., 2009). The same research group has explored the role of the other miRNAs of miR-17-92 cluster in endothelial cells as well (Doebele et al.). Overexpression of all individual members of miR-17-92 cluster (miR-17, miR-18a, miR-19a, and miR-20a) inhibited endothelial proliferation and migration in vitro, in contrast, and individual knockdown enhanced migration and proliferation. MiR-17 and miR-20 were particularly potent inhibitors of endothelial migration, and their effects were mediated in part through the tyrosine kinase Jak1 (Doebele et al.).

These data indicate that individual miRNAs in miR-17-92 cluster suppress angiogenesis of normal endothelial cells in vivo. What about their effect upon angiogenesis of endothelial cells in tumors in vivo? Although inhibitors of miR-17 and miR-20 increased vascularization of Matrigel plugs in mice, but these same inhibitors did not affect tumor angiogenesis (Doebele et al.). These results show that specific miRNAs can regulate angiogenesis in one context (Matrigel plugs) but have no effect upon angiogenesis in another context (tumor angiogenesis). One possible explanation is that different sets of genes modulate angiogenesis in different physiological situations (Figure 3B). Suarez et al. showed that reduction of miRNAs by knockdown of Dicer in endothelial cells impaired angiogenic response and reconstitution of individual miRNAs in miR-17-92 cluster rescued angiogenic inhibition (Suarez et al., 2008). These results show that endothelial miRNAs are important for angiogenesis.

In summary, contradictory data show that miR-17 inhibits angiogenesis following hind limb ischemia, but all miRNAs together promote angiogenesis in hind limb ischemia. The individual contribution to angiogenesis of distinct miRNA inside endothelial cells is not fully known.

#### **3.1.7 miR-221/222**

miR-221 and miR-222 are abundant in endothelial cells, and they affect angiogenic activity of stem cell factor (SCF) by modulating the level of its receptor c-Kit (Poliseno et al., 2006). miR-221 strongly upregulated GAX expression by inhibiting ZEB2, a modulator of the epithelialmesenchymal transition (Chen et al.). GAX is a regulatory gene controlling the angiogenic phenotype in endothelial cells. In several solid tumors, miR-221/222 is highly expressed (le Sage et al., 2007; Medina et al., 2008) and regulated by proto-oncogene ETS-1, involving in the pathogenesis of cancers (Mattia et al.). In melanoma progression, ETS-1 is directly targeted by miR-222, but not by miR-221, which makes a complex ETS-1/miR-222 co-regulatory loop. miR-222 might inhibit angiogenesis by repressing Ets signaling in endothelial cells.

MicroRNAs Regulation of Tumor Angiogenesis 239

hydroxylation, HIF-1α binds to a complex containing von Hippel-Lindau (VHL) which leads to HIF-1α degradation. However, under hypoxic conditions, HIF-1α is not prolyl hydroxylated. Instead, HIF-1α interacts with its partner HIF-1 beta (HIF-1β), and the HIF-1 complex translocates to the nucleus, binds to hypoxia responsive element (HRE), and regulates transcription of hundreds of genes. Genes transactivated by HIF-1 help to adapt the cells to low oxygen levels, controlling erythropoiesis, glycolysis, and angiogenesis (Harris, 2002; Semenza, 2003). Interest in the role of HIF-1 in cancer has been growing and HIF-1 is thought to be a therapeutic target of cancer progression (Semenza, 2003). Therefore

Hypoxia

VHL

HIF-1 HIF-1

**miR-424** CLU2

Prolyl Hydroxylase (PHD2)

P HO-P

**miR-210**

GPD1L

NDUFA4 SDHD

degradation

Ub

Fig. 4. Regulation of hypoxia signaling via miRNAs. Normally prolyl hydroxylase (PHD) hydroxylates HIF-1α on prolyl residues, marking HIF-1α for destruction through pVHL. However, during hypoxia, HIF-1α is not hydroxylated, and instead HIF-1α binds to HIF-1β, translocates to the nucleus, and activates gene transcription.  There are several miRNAs that regulate HIF-1α. First HIF-1α was reported as the target of miR-17-92 miRNA cluster in lung cancer cells (Taguchi et al., 2008). Taguchi et al. performed proteomic comparison between a miR-17-92– transfected clone and an empty vector–transfected clone from normal human bronchial epithelial cell line and then found that miR-17-92 targets HIF-1α. MiR-20b, located on 106a-

hypoxia signaling.

**miR-107**

**p53**

HIF-1

**3.3.1 HIF-1α**

we demonstrate a set of miRNAs that affect HIF-1 complex (Figure 4).

#### **3.2 Hypoxia-regulated miRNAs 3.2.1 miR-210**

Hypoxia promotes tumor angiogenesis. Hypoxia induced miRNAs are identified both in cancer and endothelial cells. Hypoxia dramatically increased miR-210 expression (Huang et al., 2009). The expression of miR-210 is higher in many solid tumors and correlates with poor survival in cancer patients (Greither et al.; Huang et al., 2009; Puissegur et al.). Overexpression of miR-210 in endothelial cells stimulated angiogenesis and migration and inhibition of miR-210 decreased hypoxia induced tube formation of endothelial cells (Fasanaro et al., 2008). MiR-210 targeted the receptor tyrosine kinase Ephrin-A3 that involves in vascular remodeling. Glycerol-3-phosphate dehydrogenase (GPD1L) was identified as another direct target of miR-210 in cancer cells (Kelly et al.). GPD1L is linked to the stability of HIF-1α protein through the activity of prolyl hydroxylases (PHDs). Hypoxia triggered accumulation of miR-210 and increased miR-210 inactivates PHDs by decreasing GPD1L protein, which blocked HIF-1α degradation. MiR-210 also targeted specific mitochondrial components with consequences on the regulation of cell death and survival and the modulation of HIF-1 activity (Puissegur et al.). Overexpression of miR-210 directly repressed two members of electron transport chain (ETC) complexes: NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 (NDUFA4), a subunit of ETC complex I, and succinate dehydrogenase complex, subunit D (SDHD), a subunit of the ETC complex II and induced mitochondrial dysfunctions, which accumulated succinate, inhibiting PHDs activity, then increasing HIF-1α. These two models represent a positive feedback loop. These data suggest that miR-210 has pro-angiogenic activity. This hypoxia-induced miRNA could be one of promising tumor markers.

#### **3.2.2 miR-424**

Hypoxia increased miR-424 level in endothelial cells and in ischemic tissues undergoing vascular remodeling and angiogenesis. Increased level of miR-424 led to degradation of an ubiquitin ligase scaffold protein cullin-2 (CLU2), which prevented HIF-1α downregulation by destabilizing the E3-ligase assembly (Ghosh et al., 2010). The expression of miR-424 was regulated by C/EBP-α/RUNX-1–mediated transactivation of PU.1. This finding indicates that miR-424 acts as a pro-angiogenic factor.

#### **3.2.3 miR-200b**

MiR-200b, one of five members of miR-200 family inhibited cell migration and epithelialmesenchymal transition (EMT) in epithelial cancer cells by modulating the expression of zinc finger E-box-binding homeobox 1 and 2 (ZEB1 and ZEB2) (Burk et al., 2008; Park et al., 2008). The miR-200 family is regulated by transforming growth factor β 1 (TGF-β1) and platelet-derived growth factor (PDGF) (Gregory et al., 2008). MiR-200b is inducible by hypoxia and down-regulates v-ets erythroblastosis virus E26 oncogene homolog 1 (ETS-1), promoting angiogenesis activity in endothelial cells (Chan et al., 2011).

#### **3.3 HIF-1 and miRNAs**

Continued tumor growth depends upon obtaining oxygen and nutrients. As tumor grows, the center of tumor becomes hypoxic, causing an angiogenic switch (Kaelin, 2005). In the presence of oxygen, prolyl hydroxylase domain protein 2 (PHD2 or EGLN1) hydroxylates prolyl residues on hypoxia-inducible factor-1 alpha (HIF-1α) (Semenza, 2007). After prolyl

Hypoxia promotes tumor angiogenesis. Hypoxia induced miRNAs are identified both in cancer and endothelial cells. Hypoxia dramatically increased miR-210 expression (Huang et al., 2009). The expression of miR-210 is higher in many solid tumors and correlates with poor survival in cancer patients (Greither et al.; Huang et al., 2009; Puissegur et al.). Overexpression of miR-210 in endothelial cells stimulated angiogenesis and migration and inhibition of miR-210 decreased hypoxia induced tube formation of endothelial cells (Fasanaro et al., 2008). MiR-210 targeted the receptor tyrosine kinase Ephrin-A3 that involves in vascular remodeling. Glycerol-3-phosphate dehydrogenase (GPD1L) was identified as another direct target of miR-210 in cancer cells (Kelly et al.). GPD1L is linked to the stability of HIF-1α protein through the activity of prolyl hydroxylases (PHDs). Hypoxia triggered accumulation of miR-210 and increased miR-210 inactivates PHDs by decreasing GPD1L protein, which blocked HIF-1α degradation. MiR-210 also targeted specific mitochondrial components with consequences on the regulation of cell death and survival and the modulation of HIF-1 activity (Puissegur et al.). Overexpression of miR-210 directly repressed two members of electron transport chain (ETC) complexes: NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 4 (NDUFA4), a subunit of ETC complex I, and succinate dehydrogenase complex, subunit D (SDHD), a subunit of the ETC complex II and induced mitochondrial dysfunctions, which accumulated succinate, inhibiting PHDs activity, then increasing HIF-1α. These two models represent a positive feedback loop. These data suggest that miR-210 has pro-angiogenic activity. This hypoxia-induced miRNA could

Hypoxia increased miR-424 level in endothelial cells and in ischemic tissues undergoing vascular remodeling and angiogenesis. Increased level of miR-424 led to degradation of an ubiquitin ligase scaffold protein cullin-2 (CLU2), which prevented HIF-1α downregulation by destabilizing the E3-ligase assembly (Ghosh et al., 2010). The expression of miR-424 was regulated by C/EBP-α/RUNX-1–mediated transactivation of PU.1. This finding indicates

MiR-200b, one of five members of miR-200 family inhibited cell migration and epithelialmesenchymal transition (EMT) in epithelial cancer cells by modulating the expression of zinc finger E-box-binding homeobox 1 and 2 (ZEB1 and ZEB2) (Burk et al., 2008; Park et al., 2008). The miR-200 family is regulated by transforming growth factor β 1 (TGF-β1) and platelet-derived growth factor (PDGF) (Gregory et al., 2008). MiR-200b is inducible by hypoxia and down-regulates v-ets erythroblastosis virus E26 oncogene homolog 1 (ETS-1),

Continued tumor growth depends upon obtaining oxygen and nutrients. As tumor grows, the center of tumor becomes hypoxic, causing an angiogenic switch (Kaelin, 2005). In the presence of oxygen, prolyl hydroxylase domain protein 2 (PHD2 or EGLN1) hydroxylates prolyl residues on hypoxia-inducible factor-1 alpha (HIF-1α) (Semenza, 2007). After prolyl

promoting angiogenesis activity in endothelial cells (Chan et al., 2011).

**3.2 Hypoxia-regulated miRNAs**

be one of promising tumor markers.

that miR-424 acts as a pro-angiogenic factor.

**3.2.2 miR-424**

**3.2.3 miR-200b**

**3.3 HIF-1 and miRNAs**

**3.2.1 miR-210**

hydroxylation, HIF-1α binds to a complex containing von Hippel-Lindau (VHL) which leads to HIF-1α degradation. However, under hypoxic conditions, HIF-1α is not prolyl hydroxylated. Instead, HIF-1α interacts with its partner HIF-1 beta (HIF-1β), and the HIF-1 complex translocates to the nucleus, binds to hypoxia responsive element (HRE), and regulates transcription of hundreds of genes. Genes transactivated by HIF-1 help to adapt the cells to low oxygen levels, controlling erythropoiesis, glycolysis, and angiogenesis (Harris, 2002; Semenza, 2003). Interest in the role of HIF-1 in cancer has been growing and HIF-1 is thought to be a therapeutic target of cancer progression (Semenza, 2003). Therefore we demonstrate a set of miRNAs that affect HIF-1 complex (Figure 4).

Fig. 4. Regulation of hypoxia signaling via miRNAs. Normally prolyl hydroxylase (PHD) hydroxylates HIF-1α on prolyl residues, marking HIF-1α for destruction through pVHL. However, during hypoxia, HIF-1α is not hydroxylated, and instead HIF-1α binds to HIF-1β, translocates to the nucleus, and activates gene transcription.  There are several miRNAs that regulate HIF-1α. First HIF-1α was reported as the target of miR-17-92 miRNA cluster in lung cancer cells (Taguchi et al., 2008). Taguchi et al. performed proteomic comparison between a miR-17-92– transfected clone and an empty vector–transfected clone from normal human bronchial epithelial cell line and then found that miR-17-92 targets HIF-1α. MiR-20b, located on 106a-

MicroRNAs Regulation of Tumor Angiogenesis 241

miR-107 controlled tumor angiogenesis through suppressing HIF-1β in vivo. HCT116 tumor cells overexpressing miR-107 subcutaneously implanted in nude mice grew slower than control cells. To exclude the effect of p53, p53 genetically knockout HCT116 cells were used. Injection of p53 knockout HCT116 cells obtained bigger tumor compared to p53 wild type HCT116 cells. When p53 knockout HCT116 cells with overexpressing miR-107 were injected, the size of tumor and the number of vessels in tumor were significantly decreased compared

In human colon cancer, there was a negative correlation between the expression of miR-107 and VEGF level. Data from both clinical studies and experimental models suggested that

HIF-2α and HIF-3α has been identified as partners with HiF-1β. HIF-2α is closely related to HIF-1α, and both activate HRE-dependent gene transcription (Wenger, 2002). HIF-3α lacks structures for transactivation existed in the C-termini of HIF-1α and HIF-2α. HIF-2α might contribute to tumor angiogenesis in some cells (Burkitt et al., 2009; Favier et al., 2007; Giatromanolaki et al., 2006). However regulation of HIF-2α or HIF-3α by miRNAs has not

Tumor angiogenesis is a complex process, consisting of a balance between pro-angiogenic factors and anti-angiogenic factors.  Research into miRNA has produced new insights into tumorigenesis. New data on regulation of tumor angiogenesis by miRNAs suggest that

I thank Dr. Charles J. Lowenstein for discussing the details of this work. This work was supported by Scientist Development Grant 835446N from American Heart Association.

Anand, S., and Cheresh, D.A. MicroRNA-mediated regulation of the angiogenic switch.

Anand, S., Majeti, B.K., Acevedo, L.M., Murphy, E.A., Mukthavaram, R., Scheppke, L.,

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miRNAs are promising therapeutic molecules to prevent and treat cancer.

Ambros, V. (2004). The functions of animal microRNAs. Nature *431*, 350-355.

Curr Opin Hematol *18*, 171-176.

angiogenesis. Nat Med *16*, 909-914.

to p53 knockout HCT116 cells transduced with scramble miRNA.

miR-107 is an anti-angiogenic miRNA.

**3.3.3 HIF-2α and HIF-3α**

reported yet.

**4. Conclusion**

**5. Acknowledgment**

281-297.

233.

**6. References**

363 cluster, a member of miR-17-92 family, was also shown to regulate HIF-1α expression (Lei et al., 2009). The inhibition of miR-20b increased HIF-1α protein and VEGF in normoxic tumor cells. In contrast, the increase of miR-20b in hypoxic tumor cells decreased the expression of HIF-1α and VEGF. HIF-1α, VEGF, and STAT3 are identified to be direct targets of miR-20b (Cascio et al.; Lei et al., 2009). As describing above, the miR-17-92 family has a complicated network to control tumor angiogenesis.

MiR-519c reduced tumor angiogenesis through direct binding to the HIF-1α 3' UTR (Cha et al.). Mice injected with lung adenocarcinoma cells overexpressing miR-519c suppressed tumor angiogenesis, growth, and metastasis. Interestingly hepatocyte growth factor (HGF) suppressed miR-519c expression. The expression of miR-519c was closely correlated with tumor progression in human lung cancer tissues.

 Overexpression of miR-22 decreased transactivation of the 3'UTR of HIF-1α. In contrast, miR-22 did not alter transactivation of HIF-1a when the miR-22 binding site within the 3' UTR of HIF-1 is mutated. Thus miR-22 directly regulates expression of HIF-1α. The function of miR-22 varies between different cancer types. The human miR-22 gene is located within a loss of heterozygosity region (LOH) in several cancer cells (Calin et al., 2004b; Xiong et al.). Ectopic expression of miR-22 suppressed proliferation in breast cancer cells (Xiong et al.). In contrast, knockdown of miR-22 increases apoptosis in human bronchial epithelial cells (Liu et al.). We found that miR-22 downregulated VEGF secretion and inhibited endothelial cell migration, suggesting that miR-22 acts as an anti-angiogenesis factor in colon cancer cell lines. Moreover the expression of miR-22 is inversely correlated to the level of VEGF in human colon cancer (Yamakuchi et al.), supporting the idea that miR-22 has an anti-angiogenic effect.

#### **3.3.2 HIF-1β**

We recently reported that miR-107 regulated tumor angiogenesis in colon cancer (Yamakuchi et al.). First we performed miRNAs profiling to identify miRNAs expressed in human colon cancer samples and found several miRNAs that are highly expressed in human colon cancer specimen such as miR-923, miR-1826, and miR-1915. The tumor suppressor gene p53 is mutated in more than half of all human cancer (Vogelstein and Kinzler, 2004). Since anti-angiogenic therapy is more effective in tumors with wild–type p53 than in tumors with mutant p53, p53 plays an important role in the regulation of tumor angiogenesis. Therefore we selected miRNAs regulated by p53. In the colon cancer cell line HCT116, we've identified several miRNAs induced by p53 (Chang et al., 2007). Furthermore we searched for miRNAs that can target HIF-1 signaling genes using prediction programs described above. Finally we've chosen miR-23a, miR-26, miR-103, and miR-107 as candidate regulators of tumor angiogenesis. Among these miRNAs, only miR-107 could regulate hypoxia signaling and was regulated by p53 in vitro. HIF-1β was shown to be a target gene of miR-107 by Western blotting and reporter gene assay. HIF-1β (also called ARNT, dioxin receptor) is a basic helix-loop-helix transcription factor and a counterpart of HIF-1α (Fukunaga et al., 1995; Reisz-Porszasz et al., 1994). Homozygous knockout of HIF-1β in mice was lethal because of poor placentation and decreased branching of the placental vasculature (Kozak et al., 1997; Maltepe et al., 1997). One of the interesting points in our findings is the regulation of HIF-1β. HIF-1β has been thought to be constitutively expressed in many cells (Wang et al., 1995). Our data indicated that miR-107 modulated the expression of HIF-1β, following tumor angiogenesis.

miR-107 controlled tumor angiogenesis through suppressing HIF-1β in vivo. HCT116 tumor cells overexpressing miR-107 subcutaneously implanted in nude mice grew slower than control cells. To exclude the effect of p53, p53 genetically knockout HCT116 cells were used. Injection of p53 knockout HCT116 cells obtained bigger tumor compared to p53 wild type HCT116 cells. When p53 knockout HCT116 cells with overexpressing miR-107 were injected, the size of tumor and the number of vessels in tumor were significantly decreased compared to p53 knockout HCT116 cells transduced with scramble miRNA.

In human colon cancer, there was a negative correlation between the expression of miR-107 and VEGF level. Data from both clinical studies and experimental models suggested that miR-107 is an anti-angiogenic miRNA.

#### **3.3.3 HIF-2α and HIF-3α**

240 Tumor Angiogenesis

363 cluster, a member of miR-17-92 family, was also shown to regulate HIF-1α expression (Lei et al., 2009). The inhibition of miR-20b increased HIF-1α protein and VEGF in normoxic tumor cells. In contrast, the increase of miR-20b in hypoxic tumor cells decreased the expression of HIF-1α and VEGF. HIF-1α, VEGF, and STAT3 are identified to be direct targets of miR-20b (Cascio et al.; Lei et al., 2009). As describing above, the miR-17-92 family

MiR-519c reduced tumor angiogenesis through direct binding to the HIF-1α 3' UTR (Cha et al.). Mice injected with lung adenocarcinoma cells overexpressing miR-519c suppressed tumor angiogenesis, growth, and metastasis. Interestingly hepatocyte growth factor (HGF) suppressed miR-519c expression. The expression of miR-519c was closely correlated with

We found that miR-22 regulated HIF-1α in HCT116, human colon cancer cells (Yamakuchi et al.). Overexpression of miR-22 decreased transactivation of the 3'UTR of HIF-1α. In contrast, miR-22 did not alter transactivation of HIF-1a when the miR-22 binding site within the 3' UTR of HIF-1 is mutated. Thus miR-22 directly regulates expression of HIF-1α. The function of miR-22 varies between different cancer types. The human miR-22 gene is located within a loss of heterozygosity region (LOH) in several cancer cells (Calin et al., 2004b; Xiong et al.). Ectopic expression of miR-22 suppressed proliferation in breast cancer cells (Xiong et al.). In contrast, knockdown of miR-22 increases apoptosis in human bronchial epithelial cells (Liu et al.). We found that miR-22 downregulated VEGF secretion and inhibited endothelial cell migration, suggesting that miR-22 acts as an anti-angiogenesis factor in colon cancer cell lines. Moreover the expression of miR-22 is inversely correlated to the level of VEGF in human colon cancer (Yamakuchi et al.), supporting the idea that miR-22

We recently reported that miR-107 regulated tumor angiogenesis in colon cancer (Yamakuchi et al.). First we performed miRNAs profiling to identify miRNAs expressed in human colon cancer samples and found several miRNAs that are highly expressed in human colon cancer specimen such as miR-923, miR-1826, and miR-1915. The tumor suppressor gene p53 is mutated in more than half of all human cancer (Vogelstein and Kinzler, 2004). Since anti-angiogenic therapy is more effective in tumors with wild–type p53 than in tumors with mutant p53, p53 plays an important role in the regulation of tumor angiogenesis. Therefore we selected miRNAs regulated by p53. In the colon cancer cell line HCT116, we've identified several miRNAs induced by p53 (Chang et al., 2007). Furthermore we searched for miRNAs that can target HIF-1 signaling genes using prediction programs described above. Finally we've chosen miR-23a, miR-26, miR-103, and miR-107 as candidate regulators of tumor angiogenesis. Among these miRNAs, only miR-107 could regulate hypoxia signaling and was regulated by p53 in vitro. HIF-1β was shown to be a target gene of miR-107 by Western blotting and reporter gene assay. HIF-1β (also called ARNT, dioxin receptor) is a basic helix-loop-helix transcription factor and a counterpart of HIF-1α (Fukunaga et al., 1995; Reisz-Porszasz et al., 1994). Homozygous knockout of HIF-1β in mice was lethal because of poor placentation and decreased branching of the placental vasculature (Kozak et al., 1997; Maltepe et al., 1997). One of the interesting points in our findings is the regulation of HIF-1β. HIF-1β has been thought to be constitutively expressed in many cells (Wang et al., 1995). Our data indicated that miR-107 modulated the expression

has a complicated network to control tumor angiogenesis.

tumor progression in human lung cancer tissues.

has an anti-angiogenic effect.

of HIF-1β, following tumor angiogenesis.

**3.3.2 HIF-1β**

HIF-2α and HIF-3α has been identified as partners with HiF-1β. HIF-2α is closely related to HIF-1α, and both activate HRE-dependent gene transcription (Wenger, 2002). HIF-3α lacks structures for transactivation existed in the C-termini of HIF-1α and HIF-2α. HIF-2α might contribute to tumor angiogenesis in some cells (Burkitt et al., 2009; Favier et al., 2007; Giatromanolaki et al., 2006). However regulation of HIF-2α or HIF-3α by miRNAs has not reported yet.

#### **4. Conclusion**

Tumor angiogenesis is a complex process, consisting of a balance between pro-angiogenic factors and anti-angiogenic factors.  Research into miRNA has produced new insights into tumorigenesis. New data on regulation of tumor angiogenesis by miRNAs suggest that miRNAs are promising therapeutic molecules to prevent and treat cancer.

#### **5. Acknowledgment**

I thank Dr. Charles J. Lowenstein for discussing the details of this work. This work was supported by Scientist Development Grant 835446N from American Heart Association.

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*USA*

**New Molecular Targets for**

**Anti-Angiogenic Therapeutic Strategies**

*1Neuro-Oncology Branch, National Cancer Institute, National Institutes of Health 2Department of Medicine, Vanderbilt-Ingram Cancer Center, Vanderbilt University*

The concept of targeting tumor blood vessel formation, i.e. tumor angiogenesis, has long been accepted as a potential strategy for controlling tumor growth. Characterized by the ability to supply oxygen, growth factors, hormones and nutrients, tumor vasculature has been identified as a key factor for the maintenance and progression of many solid tumors. The development of tumor vasculature is regulated by a highly complex network of signal transduction pathways involving pro- and anti-angiogenic factors. This delicate, yet dynamic balance between the promotion and inhibition of vascularization provides an