Assess the RNA yield and quality by spectrophotometry using a nanodrop. RNA concentration can be assessed by measuring its absorbance at 260 nM.

## *3.6.4. Reverse transcription*

**3.** Use a cell lifter to detach the cells from the bottom of the well and homogenize the cell

258 Gene Expression and Regulation in Mammalian Cells - Transcription Toward the Establishment of Novel Therapeutics

**4.** Pipette the cell lysate into labeled microcentrifuge tubes and pipette up and down 2–3 more times to further homogenize the cell lysate. At this point store the homogenates at

**1.** Take the samples out of the −80°C freezer and allow them to thaw at room temperature

**5.** Centrifuge at 12000 g for 10–15 min at 4°C. Meanwhile, label a new set of microcentrifuge

**6.** Transfer the aqueous phase (top layer) to a new tube. RNA remains in the aqueous phase

**10.** Centrifuge at 12000 g for 8 min at 4–25°C. At this point, a translucent (gel-like) to white

whereas DNA is in the interphase and proteins remain in the organic phase.

lysate.

*3.6.2. RNA extraction*

for 10 min.

tubes.

**8.** Vortex for 10 s.

−80°C before proceeding to RNA extraction.

**2.** Add 10 μL of bromochloropropane/100 μL of TRI reagent.

**3.** Cap the tubes tightly and vortex vigorously for 15 s.

**4.** Incubate the mixture at room temperature for 10 min.

**7.** Add 50 μL of isopropanol/100 μL of TRI reagent solution.

pellet should become visible at the bottom of the tube.

**11.** Carefully remove the supernatant without disturbing the pellet.

**14.** Remove all the ethanol carefully without disturbing the pellet.

**12.** Add 100 μL of 75% ethanol/100 μL of TRI reagent to wash the pellet.

**16.** Dissolve the RNA in 20 μL of nuclease-free water and mix vigorously.

**17.** Store on ice for immediate analysis, or place at −80°C for long-term storage.

Assess the RNA yield and quality by spectrophotometry using a nanodrop. RNA concentra-

**9.** Incubate at room temperature for 10 min.

**13.** Centrifuge at 7500 g for 5 min at 4–25°C.

*3.6.3. Assessment of RNA concentration and quality*

tion can be assessed by measuring its absorbance at 260 nM.

**15.** Air dry the pellet for 5 min.

To create cDNAs form the extracted mRNAs, use the Origene First Strand cDNA synthesis system for qPCR, following the manufacturer's instructions. Keep your mRNAs and all the components of the kit (except the enzyme) on ice.

## *3.6.5. Real-time quantitative PCR*

To perform real-time quantitative PCR, you will need to design a set of primers specific to the transcript of the gene(s) of interest. Primer design is beyond the scope of this review and will not be described here.12

12Note that several programs for primer designing are freely available on the web such as primer blast: http://www.ncbi. nlm.nih.gov/tools/primer-blast.

*3.7.2. Assessment of protein concentration and sample preparation*

tific #23225).

ddH2

ddH2 O).

*3.7.3. Gel electrophoresis*

polyacrylamide gels.13

*3.7.4. Protein transfer*

and 1600 mL of ddH20).

late with caution and wear gloves at all time.

**3.** Boil protein samples for 5 min.

samples are not running out of the gel.

col for further information.

O up to the desired volume.

**4.** Samples can be stored at −20°C at this point.

**1.** Use a technique of your choice to measure protein concentration. We recommend using Thermo Scientific's BCA assay following the manufacturer's instructions (Thermo Scien-

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261

**2.** Label a new set of tubes in order to prepare the samples in loading buffer. The following steps (sample preparation, gel electrophoresis and protein transfer) are made using the NuPAGE Novex system from Life Technologies. Please refer to the manufacturer's proto-

**3.** In each tube add: Protein sample (to achieve a final concentration of 1–2 μg/μL of protein), 10× dithiothreitol (NuPAGE reducing agent, 500 mM dithiothreitol), 4× loading dye and

Gel electrophoresis is achieved with the NuPAGE Novex Mini system with Bis-Tris precast

**1.** Prepare 1 L of 1× MOPS running buffer (50 mL of 20× MOPS running buffer in 950 mL of

**2.** Insert the precast gel in the gel box and fill the inside chamber with 1× running buffer.

**5.** Load 4 μL of molecular weight markers and the protein samples in the desired order.

**7.** Monitor frequently to assess the degree of separation and to make sure that the protein

**1.** Prepare 2 L of 1× transfer buffer (100 mL of 20× transfer buffer with 15% ethanol (300 mL)

**2.** Cut a polyvinylidene fluoride (PVDF) membrane (pore size 0.2 μm) and 2 pieces of filter paper to the appropriate dimensions of a transfer cassette, or use a premade sandwich (Biorad).

13This system is optimized for separation and resolution of small- to medium-sized proteins (1–200 kDa) under denaturing gel electrophoresis conditions. These precast gels are available in different polyacrylamide percentages, well formats and thicknesses and must be chosen according to specific experimental needs. Acrylamide is a toxic compound: manipu-

**6.** Run the gel under constant voltage at 100–140 V (400 mAmps) for 30–45 min.

**4.** Remove bubbles and residues from the gel wells by pipetting.

**7.** Repeat the same procedure with a set of primers designed to amplify a control housekeeping gene or other species that should not be affected by your experimental conditions. In studies involving cell death induced by PD toxins (6-OHDA, MPP+), we found that 18S rRNA is a good control and can be used for normalization. At the end of the reaction, proceed to quantification and analysis.

#### *3.6.6. Quantification and analysis*

Real-time monitoring of the PCR reaction displays the amount of fluorescence signal emitted from the SYBR green dye. During the amplification reaction, the SYBR green dye is progressively inserted in the newly synthesized double-stranded DNA fragments. The amplified fragments correspond to the region of the cDNA from the gene of interest flanked by the forward/reverse primers binding sites. The initial copy number can be quantitated during real-time PCR analysis based on the threshold cycle (Ct). Ct is defined as the cycle at which fluorescence is determined to be statistically significant above background. The more template is initially present, the fewer number of cycles is needed to reach a threshold at which the fluorescence is statistically significant above background. To calculate the fold induction of your gene of interest (GI) normalized to a control housekeeping gene (HKG) in an experimental condition (Exp) compared to a control condition (Ctl), use the following formula:

$$\text{fold involution} = \text{2}^{\text{-}}[(\text{c}\_{\text{an}\_{\theta}} \text{-} \text{c}\_{\text{can}\_{\theta}}) \cdot (\text{c}\_{\text{can}} \text{-} \text{c}\_{\text{can}})] \tag{1}$$

#### **3.7. Downstream application: Western blot**

#### *3.7.1. Harvesting cells and extracting proteins*

## *3.7.2. Assessment of protein concentration and sample preparation*

## *3.7.3. Gel electrophoresis*

**5.** Set the detection channel to SYBR Green and the reaction volume at 25 μL.

260 Gene Expression and Regulation in Mammalian Cells - Transcription Toward the Establishment of Novel Therapeutics

must be determined for each primer set) for 30–60 s, 72°C for 30–60 s.

ceed to quantification and analysis.

fold induction = 2−[(CtGIExp − CtHGKExp)−(Ct

**1.** Take the cells out of the incubator and place them on ice.

point, the homogenate should be clear of debris.

**3.** Use a cell lifter to detach the cells from the bottom of the well.

**3.7. Downstream application: Western blot**

*3.7.1. Harvesting cells and extracting proteins*

hibitor (30 μL of buffer/cm2

samples.

*3.6.6. Quantification and analysis*

**6.** Run the reaction as follow: 1 cycle at 95°C for 10 min to activate the Taq DNA polymerase, followed by 40 cycles of amplification: 95°C for 15 s, 58–60°C (the optimum temperature

**7.** Repeat the same procedure with a set of primers designed to amplify a control housekeeping gene or other species that should not be affected by your experimental conditions. In studies involving cell death induced by PD toxins (6-OHDA, MPP+), we found that 18S rRNA is a good control and can be used for normalization. At the end of the reaction, pro-

Real-time monitoring of the PCR reaction displays the amount of fluorescence signal emitted from the SYBR green dye. During the amplification reaction, the SYBR green dye is progressively inserted in the newly synthesized double-stranded DNA fragments. The amplified fragments correspond to the region of the cDNA from the gene of interest flanked by the forward/reverse primers binding sites. The initial copy number can be quantitated during real-time PCR analysis based on the threshold cycle (Ct). Ct is defined as the cycle at which fluorescence is determined to be statistically significant above background. The more template is initially present, the fewer number of cycles is needed to reach a threshold at which the fluorescence is statistically significant above background. To calculate the fold induction of your gene of interest (GI) normalized to a control housekeeping gene (HKG) in an experimental condition (Exp) compared to a control condition (Ctl), use the following formula:

**2.** Remove the medium gently and add 1× cell lysis buffer supplemented with protease in-

**5.** Pipette up and down 2–3 more times and briefly vortex to further homogenize the cell lysate. Check the homogenate: if cellular debris is still visible, proceed to sonicate the

**6.** Sonicate for 20 s (10 pulses over 10 s followed by one continuous 10-s pulse) on ice. At this

**7.** Store the samples at −80°C before measuring the protein concentration in each sample.

of culture dish area).

**4.** Pipette the cell lysate into labeled microcentrifuge tubes and place them on ice.

GICtl − Ct

HKGCtl)] (1)

Gel electrophoresis is achieved with the NuPAGE Novex Mini system with Bis-Tris precast polyacrylamide gels.13

13This system is optimized for separation and resolution of small- to medium-sized proteins (1–200 kDa) under denaturing gel electrophoresis conditions. These precast gels are available in different polyacrylamide percentages, well formats and thicknesses and must be chosen according to specific experimental needs. Acrylamide is a toxic compound: manipulate with caution and wear gloves at all time.

**3.** Soak the PVDF membrane for few seconds in 95% ethanol then soak in transfer buffer for 5 min.

death induced by PD toxins (6-OHDA, MPP+), we found that extracellular signal–regulated kinases (ERK) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are good

Manipulation and Study of Gene Expression in Neurotoxin-Treated Neuronal PC12 and SH-SY5Y…

The quantification of viable cells following treatment with PD-toxins and/or gene manipulation can be achieved by several methods. A number of methods (such as MTT or LDH assays) rely on measuring the metabolic activity of the cultured cells and give an indirect and potentially biased measurement of cell viability. We consider that the most accurate and robust measure of cell death is an absolute count of the numbers of surviving cells remaining in the cell culture dish following toxin treatment and/or gene manipulations. We routinely use a method in which the nuclei of the remaining cells are counted by incubating the cell cultures with a detergent solution that lyses the plasma membrane and leaves the nuclei intact.

**1.** Prepare 100 mL of 10× counting lysis buffer by combining: 5 g of cetyldimethyl-ethanolammonium bromide, 0.165 g of NaCl, 2.8 mL of glacial acetic acid, 50 mL of 10% Triton-X,

**4.** Pipette 10 μL of the suspended nuclei into a hemacytometer and count at least 100 nuclei. **5.** Cell survival in a given condition is expressed as the percentage of remaining nuclei in

Application of the above techniques by our and other groups has identified a variety of transcriptionally regulated genes with potential relevance to the pathophysiology and treatment

An early serial analysis of gene expression (SAGE) study of transcriptional responses of PC12 cells to 6-OHDA treatment revealed a strong endoplasmic reticulum (ER) stress response signature [19, 20]. Such work contributed to the current focus on the role of ER stress in PD [21, 22]. Among the upregulated stress, genes were the transcription factors *ATF4* (Activating transcription factor 4) and *Ddit3* (DNA damage-inducible transcript 3, which encode the ATF4 and CHOP (C/EBP homologous protein) proteins, respectively). *In vitro* work, including with PC12 cells as well as *in vivo* studies have indicated that CHOP is death promoting in the

O.

of culture dish area) and leave sit

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263

, 10 mL of 10× PBS, 35.2 mL of H2

**4. Outcomes: examples of findings using the above techniques**

of PD. Here, we provide a few examples arising from our own studies (**Figure 2**).

**2.** Take the cells out of the incubator and aspirate all the medium.

**3.** Add 1× of counting lysis buffer to the wells (250 μL/cm2

experimental cultures compared to control cultures.

loading controls and can be used for normalization.

**3.8. Downstream application: survival assay**

2 mL of 1 M MgCl2

**4.1. Published findings**

for 5 min.

#### *3.7.5. Protein detection*

14The final concentration of primary antibody as well as the duration and temperature of incubation should be determined empirically and according to the manufacturer's guidelines.

death induced by PD toxins (6-OHDA, MPP+), we found that extracellular signal–regulated kinases (ERK) or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are good loading controls and can be used for normalization.

## **3.8. Downstream application: survival assay**

**3.** Soak the PVDF membrane for few seconds in 95% ethanol then soak in transfer buffer for

262 Gene Expression and Regulation in Mammalian Cells - Transcription Toward the Establishment of Novel Therapeutics

**5.**  Keep the sandwich wet at all times and avoid the formation of bubbles in

**6.** Place the sandwich in a transfer cassette and fill up the gel box with 1× transfer buffer. Apply a constant voltage at 40 V (400 mAmps) for 94 min to transfer the proteins form the gel

**1.** Check the uniformity and overall efficiency of the transfer by staining the membrane

**2.** Wash the membrane with TBST (1× TBS + 0.1% Tween) for 5 min. Wash again quickly

**3.** Block non-specific protein binding by incubating the membrane with TBST +5% pow-

**5.** Incubate the membrane with a solution of primary antibody in blocking solution under

**7.** Incubate the membrane with a secondary antibody targeted to the primary antibody and

**11.** Expose a light-sensitive autoradiographic film against the membrane and develop to re-

**12.** Repeat the same procedure with a primary antibody against a control housekeeping protein that should not be affected by your experimental conditions. In studies involving cell

14The final concentration of primary antibody as well as the duration and temperature of incubation should be deter-

O).

with Ponceau S dye (0.1% w/v Ponceau, 5% acetic acid in ddH2

with TBST to discard the excess of Ponceau.

gentle agitation at 4–25°C from 30 min to overnight.14

**6.** Wash the membrane with TBST 3 times for 5–15 min.

**8.** Incubate at room temperature for 1–2 h on a shaker.

mined empirically and according to the manufacturer's guidelines.

**9.** Wash the membrane with TBST three times for 5–15 min. **10.** Incubate the membrane with a chemiluminescent agent.

**4.** Soak filter paper and sponges in transfer buffer for 5 min.

5 min.

between layers.

to the membrane.

*3.7.5. Protein detection*

dered milk for 45 min.

veal the protein signal.

**4.** Rinse 3 times for 5 min with TBST.

bound with horseradish peroxidase.

The quantification of viable cells following treatment with PD-toxins and/or gene manipulation can be achieved by several methods. A number of methods (such as MTT or LDH assays) rely on measuring the metabolic activity of the cultured cells and give an indirect and potentially biased measurement of cell viability. We consider that the most accurate and robust measure of cell death is an absolute count of the numbers of surviving cells remaining in the cell culture dish following toxin treatment and/or gene manipulations. We routinely use a method in which the nuclei of the remaining cells are counted by incubating the cell cultures with a detergent solution that lyses the plasma membrane and leaves the nuclei intact.

## **4. Outcomes: examples of findings using the above techniques**

## **4.1. Published findings**

Application of the above techniques by our and other groups has identified a variety of transcriptionally regulated genes with potential relevance to the pathophysiology and treatment of PD. Here, we provide a few examples arising from our own studies (**Figure 2**).

An early serial analysis of gene expression (SAGE) study of transcriptional responses of PC12 cells to 6-OHDA treatment revealed a strong endoplasmic reticulum (ER) stress response signature [19, 20]. Such work contributed to the current focus on the role of ER stress in PD [21, 22]. Among the upregulated stress, genes were the transcription factors *ATF4* (Activating transcription factor 4) and *Ddit3* (DNA damage-inducible transcript 3, which encode the ATF4 and CHOP (C/EBP homologous protein) proteins, respectively). *In vitro* work, including with PC12 cells as well as *in vivo* studies have indicated that CHOP is death promoting in the

**Figure 2.** Example of transcriptionally regulated genes in PD cellular models identified by our group using the methods described in this chapter.

context of PD [23–25]. The potential role of ATF4 in contrast has proved to be more complex, and here again, studies with PC12 cells have proved useful for enlightenment. On one hand, ATF4 appears to have protective actions in PD models and does so by causing stabilization of the anti-death protein Parkin [13]. On the other hand, ATF4 appears to cooperate with CHOP in transcriptional induction of Trib3, a protein with pro-apoptotic actions in neurons [25, 26]. *Trib3* was among the genes found to be induced by 6-OHDA [19, 20, 25] in PC12 cells, and subsequently, Trib3 protein was found to be elevated in dopaminergic neurons of PD patients [25]. Down-regulation of Trib3 or inhibition of its transcription is highly protective in multiple cellular PD models including PC12 cells, suggesting it as a potential therapeutic target for PD treatment [25]. An additional transcriptionally regulated gene of interest to arise from the initial SAGE study was *Ddit4* (DNA-damage-inducible transcript 4), which encodes the pro-death RTP801 protein. A series of studies has indicated that like Trib3, RTP801 is elevated in dopaminergic neurons of PD patients and that eliminating its expression is protective in cellular models of PD such as PC12 cells [27–29]. Interference with RTP801 induction in an animal model of PD proved to be protective, thus identifying this as an additional potential target for PD therapy [29].

Consistent with the upregulation of Nupr1 seen in apoptotic cancer cells [30, 31], we found that 6-OHDA induced a significant 1.6 fold increase in Nupr1. Under these conditions, we measured a 2.1-fold increase in Trib3 mRNA levels, as reported previously [25]. To assess whether Nupr1 is required for this Trib3 induction, we employed lentivirally delivered Nupr1 shRNAs as described in this chapter. Although both shRNA constructs achieved a 70% reduction of Nupr1 mRNA levels, Nupr1 knockdown had no effect on Trib3 mRNA levels neither at baseline nor under 6-OHDA treatment. These results suggest that although Nupr1 is upregulated in PD cellular models, it is not one of the transcription factors responsible for

p < 0.05 comparing shSCR/6OHDA relative to shSCR/control; ##p < 0.005

**Figure 3.** Nupr1 is upregulated but does not regulate Trib3 expression in PD cellular models. PC12 cells were differentiated with NGF for 3 days and infected with three different lentiviruses: two lentiviruses carrying constructs expressing shRNAs directed against two distinct regions of the Nupr1 mRNA (shNUPR1#1 and shNUPR1#2) or a control lentivirus carrying a scrambled shRNA sequence (shSCR). 4 days later, the cells were treated with sterile water (control) or 150 μM 6-OHDA for 8 h (6OHDA). At the end of the treatment, total mRNAs were extracted and RT-qPCR was performed to measure Nupr1 and Trib3 mRNA levels. Nupr1 and Trib3 mRNA levels were normalized to GAPDH. For Nupr1 RT-qPCR primers: (forward) GGGCAAGTTAGGAGCGAGAA and (reverse) GGGCATCCAGTTTTTCCCAC. For Trib3 RT-qPCR primers: (forward) GTTGCGTCGATTTGTCTTCA and (reverse) CGGGAGCTGAGTATCTCTGG. Values are expressed as mean ± SEM of three independent experiments. Statistical analysis was performed using two-way ANOVA

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Our PC12 cell experiments indicate upregulation of ATF4 in multiple PD models, and that while ATF4 can play a protective role in PD by reducing loss of the anti-death protein Parkin, it also contributes to induction of the pro-apoptotic protein Trib3 (**Figure 2**). An additional way that ATF4 could affect cell survival or death is by transcriptional regulation of xCT protein (product of the *SLC7A11* gene) [32]. xCT protein levels are reported to be increased in animal PD models such as 6-OHDA and chronic MPTP treatment [33, 34]. These findings are capitulated in our PC12 cell studies, in which we found massive upregulation of xCT mRNA

xCT is a subunit of the cystine/glutamate antiporter system that transports cystine into cells in exchange for exported glutamate. This property has suggested the possibility of both pro-survival

orchestrating Trib3 upregulation in these models.

with Sidak's multiple comparisons test. \*

**4.3. xCT/SLC7A11 is induced in PD cellular models**

comparing shNUPR1#1/control or shNUPR1#2/control relative to shSCR/control.

in both the MPTP and 6-OHDA models (**Figure 4**).

#### **4.2. Nupr1 is induced but does not regulate Trib3 upregulation in a PD cellular model**

As noted above, we recently described Trib3 as a gene-mediating cell death and degeneration in PD and identified two transcription factors, ATF4 and CHOP, responsible for Trib3 upregulation in PD cellular models, including neuronal PC12 cells treated with 6-OHDA [25]. However, ATF4 and CHOP downregulation only partially abrogated Trib3 induction in response to PD toxin mimetics, indicating that other transcription factors might be responsible for Trib3 induction in these models. Several reports indicate that Nupr1 (also known as p8) is upregulated and leads to apoptosis of cancer cells through activation of the ATF4/ CHOP-Trib3 pathway [30, 31]. Therefore, we tested whether Nupr1 was also upregulated in PD cellular models and, if so, whether it could regulate Trib3 expression (**Figure 3**).

**Figure 3.** Nupr1 is upregulated but does not regulate Trib3 expression in PD cellular models. PC12 cells were differentiated with NGF for 3 days and infected with three different lentiviruses: two lentiviruses carrying constructs expressing shRNAs directed against two distinct regions of the Nupr1 mRNA (shNUPR1#1 and shNUPR1#2) or a control lentivirus carrying a scrambled shRNA sequence (shSCR). 4 days later, the cells were treated with sterile water (control) or 150 μM 6-OHDA for 8 h (6OHDA). At the end of the treatment, total mRNAs were extracted and RT-qPCR was performed to measure Nupr1 and Trib3 mRNA levels. Nupr1 and Trib3 mRNA levels were normalized to GAPDH. For Nupr1 RT-qPCR primers: (forward) GGGCAAGTTAGGAGCGAGAA and (reverse) GGGCATCCAGTTTTTCCCAC. For Trib3 RT-qPCR primers: (forward) GTTGCGTCGATTTGTCTTCA and (reverse) CGGGAGCTGAGTATCTCTGG. Values are expressed as mean ± SEM of three independent experiments. Statistical analysis was performed using two-way ANOVA with Sidak's multiple comparisons test. \* p < 0.05 comparing shSCR/6OHDA relative to shSCR/control; ##p < 0.005 comparing shNUPR1#1/control or shNUPR1#2/control relative to shSCR/control.

Consistent with the upregulation of Nupr1 seen in apoptotic cancer cells [30, 31], we found that 6-OHDA induced a significant 1.6 fold increase in Nupr1. Under these conditions, we measured a 2.1-fold increase in Trib3 mRNA levels, as reported previously [25]. To assess whether Nupr1 is required for this Trib3 induction, we employed lentivirally delivered Nupr1 shRNAs as described in this chapter. Although both shRNA constructs achieved a 70% reduction of Nupr1 mRNA levels, Nupr1 knockdown had no effect on Trib3 mRNA levels neither at baseline nor under 6-OHDA treatment. These results suggest that although Nupr1 is upregulated in PD cellular models, it is not one of the transcription factors responsible for orchestrating Trib3 upregulation in these models.

## **4.3. xCT/SLC7A11 is induced in PD cellular models**

context of PD [23–25]. The potential role of ATF4 in contrast has proved to be more complex, and here again, studies with PC12 cells have proved useful for enlightenment. On one hand, ATF4 appears to have protective actions in PD models and does so by causing stabilization of the anti-death protein Parkin [13]. On the other hand, ATF4 appears to cooperate with CHOP in transcriptional induction of Trib3, a protein with pro-apoptotic actions in neurons [25, 26]. *Trib3* was among the genes found to be induced by 6-OHDA [19, 20, 25] in PC12 cells, and subsequently, Trib3 protein was found to be elevated in dopaminergic neurons of PD patients [25]. Down-regulation of Trib3 or inhibition of its transcription is highly protective in multiple cellular PD models including PC12 cells, suggesting it as a potential therapeutic target for PD treatment [25]. An additional transcriptionally regulated gene of interest to arise from the initial SAGE study was *Ddit4* (DNA-damage-inducible transcript 4), which encodes the pro-death RTP801 protein. A series of studies has indicated that like Trib3, RTP801 is elevated in dopaminergic neurons of PD patients and that eliminating its expression is protective in cellular models of PD such as PC12 cells [27–29]. Interference with RTP801 induction in an animal model of PD proved to be protective, thus identifying this as an additional potential

**Figure 2.** Example of transcriptionally regulated genes in PD cellular models identified by our group using the methods

264 Gene Expression and Regulation in Mammalian Cells - Transcription Toward the Establishment of Novel Therapeutics

**4.2. Nupr1 is induced but does not regulate Trib3 upregulation in a PD cellular**

PD cellular models and, if so, whether it could regulate Trib3 expression (**Figure 3**).

As noted above, we recently described Trib3 as a gene-mediating cell death and degeneration in PD and identified two transcription factors, ATF4 and CHOP, responsible for Trib3 upregulation in PD cellular models, including neuronal PC12 cells treated with 6-OHDA [25]. However, ATF4 and CHOP downregulation only partially abrogated Trib3 induction in response to PD toxin mimetics, indicating that other transcription factors might be responsible for Trib3 induction in these models. Several reports indicate that Nupr1 (also known as p8) is upregulated and leads to apoptosis of cancer cells through activation of the ATF4/ CHOP-Trib3 pathway [30, 31]. Therefore, we tested whether Nupr1 was also upregulated in

target for PD therapy [29].

described in this chapter.

**model**

Our PC12 cell experiments indicate upregulation of ATF4 in multiple PD models, and that while ATF4 can play a protective role in PD by reducing loss of the anti-death protein Parkin, it also contributes to induction of the pro-apoptotic protein Trib3 (**Figure 2**). An additional way that ATF4 could affect cell survival or death is by transcriptional regulation of xCT protein (product of the *SLC7A11* gene) [32]. xCT protein levels are reported to be increased in animal PD models such as 6-OHDA and chronic MPTP treatment [33, 34]. These findings are capitulated in our PC12 cell studies, in which we found massive upregulation of xCT mRNA in both the MPTP and 6-OHDA models (**Figure 4**).

xCT is a subunit of the cystine/glutamate antiporter system that transports cystine into cells in exchange for exported glutamate. This property has suggested the possibility of both pro-survival

BDNF: brain-derived neurotrophic factor

O: double distilled or deionized water

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dibutyryl cAMP: dibutyryl cyclic adenosine monophosphate

DDIT3: DNA damage-inducible transcript 3

DDIT4: DNA-damage-inducible transcript 4

ERK: extracellular signal–regulated kinases

GAPDH: glyceraldehyde 3-phosphate dehydrogenase

MPTP: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

BSA: bovine serum albumin

DMSO: dimethyl sulfoxide

FBS: fetal bovine serum

GSH: glutathione

ER: endoplasmic reticulum

GFP: green fluorescent protein

HBSS: Hank's balanced salt solution

MPP+: 1-methyl-4-phenylpyridinium

PBS: phosphate-buffered saline

PCR: polymerase chain reaction

NGF: nerve growth factor

PD: Parkinson's disease

pen/strep: penicillin/streptomycin

PVDF: polyvinylidene fluoride

qPCR: quantitative polymerase chain reaction

SAGE: serial analysis of gene expression

rhBDNF: recombinant human brain-derived neurotrophic factor

RT-PCR: reverse transcription polymerase chain reaction

ddH2

CHOP: C/EBP homologous protein

**Figure 4.** xCT/SLC7A11 mRNA is upregulated in differentiated PC12 cells by PD-mimetic toxin treatment. Neuronally differentiated PC12 cells were treated with 100-μM 6-OHDA for different times as indicated (A) or with 150 μM 6-OHDA for 10 h or 1 mM MPP+ for 16 h (B). Total mRNA was extracted, and RT-qPCR was performed to measure SLC7A11 mRNA levels (normalized to tubulin). For xCT RT-qPCR primers: (forward) GACAGTGTGTGCATCCCCTT and GCATGCATTTCTTGCACAGTTC (reverse). Values are expressed as mean ± SEM. In B, statistical analysis was performed using one-way ANOVA with Tukey's multiple comparisons test. \* p < 0.05 comparing 6-OHDA or MPP+ relative to control.

and pro-apoptotic activities in the context of PD. By promoting cystine uptake, xCT increases synthesis of glutathione (GSH), an intracellular antioxidant that has been suggested to play a protective role in PD [35, 36]. One possibility is that the upregulation of xCT might be a compensatory protective reaction to increased oxidative stress. Consistent with this idea, treatments that elevate neuronal levels of GSH show protection in a variety of PD models [36, 37]. On the other hand, as an exchanger, xCT elevates extracellular levels of glutamate that in turn may have toxic effects on dopaminergic neurons via glutamate receptors. In agreement with this possibility, 6-OHDAtreated xCT−/− mice were reported to have less striatal extracellular glutamate than wt mice, and their dopaminergic neurons showed substantial protection from 6-OHDA [38]. Such findings raise the challenge of sorting out and appropriately manipulating the pro- and anti-apoptotic actions of xCT for therapeutic advantage in PD. Cellular models of PD with induction of xCT such as described here, have the potential to serve as convenient, first-line screening systems to this end.

## **Acknowledgements**

Support for the authors during preparation of this review was provided in part by the NIH-NIDS (Columbia University Center for excellence in Parkinson's disease research, 5P50 NS38370), the Parkinson's Disease Foundation, the William N. & Bernice E. Bumpus Foundation and the NIH-NINDS (NS072050).

## **Abbreviations**

6-OHDA: 6-hydroxydopamine

ATF4: activating transcription factor 4

and pro-apoptotic activities in the context of PD. By promoting cystine uptake, xCT increases synthesis of glutathione (GSH), an intracellular antioxidant that has been suggested to play a protective role in PD [35, 36]. One possibility is that the upregulation of xCT might be a compensatory protective reaction to increased oxidative stress. Consistent with this idea, treatments that elevate neuronal levels of GSH show protection in a variety of PD models [36, 37]. On the other hand, as an exchanger, xCT elevates extracellular levels of glutamate that in turn may have toxic effects on dopaminergic neurons via glutamate receptors. In agreement with this possibility, 6-OHDAtreated xCT−/− mice were reported to have less striatal extracellular glutamate than wt mice, and their dopaminergic neurons showed substantial protection from 6-OHDA [38]. Such findings raise the challenge of sorting out and appropriately manipulating the pro- and anti-apoptotic actions of xCT for therapeutic advantage in PD. Cellular models of PD with induction of xCT such as described here, have the potential to serve as convenient, first-line screening systems to this end.

p < 0.05 comparing 6-OHDA or MPP+ relative to control.

**Figure 4.** xCT/SLC7A11 mRNA is upregulated in differentiated PC12 cells by PD-mimetic toxin treatment. Neuronally differentiated PC12 cells were treated with 100-μM 6-OHDA for different times as indicated (A) or with 150 μM 6-OHDA for 10 h or 1 mM MPP+ for 16 h (B). Total mRNA was extracted, and RT-qPCR was performed to measure SLC7A11 mRNA levels (normalized to tubulin). For xCT RT-qPCR primers: (forward) GACAGTGTGTGCATCCCCTT and GCATGCATTTCTTGCACAGTTC (reverse). Values are expressed as mean ± SEM. In B, statistical analysis was performed

266 Gene Expression and Regulation in Mammalian Cells - Transcription Toward the Establishment of Novel Therapeutics

Support for the authors during preparation of this review was provided in part by the NIH-NIDS (Columbia University Center for excellence in Parkinson's disease research, 5P50 NS38370), the Parkinson's Disease Foundation, the William N. & Bernice E. Bumpus

**Acknowledgements**

**Abbreviations**

Foundation and the NIH-NINDS (NS072050).

using one-way ANOVA with Tukey's multiple comparisons test. \*

6-OHDA: 6-hydroxydopamine

ATF4: activating transcription factor 4

[10] Nakamura K, Bindokas VP, Marks JD, et al. The selective toxicity of 1-methyl-4-phenylpyridinium to dopaminergic neurons: The role of mitochondrial complex I and reactive

Manipulation and Study of Gene Expression in Neurotoxin-Treated Neuronal PC12 and SH-SY5Y…

http://dx.doi.org/10.5772/intechopen.71811

269

[11] Gimenez-Cassina A, Lim F, Diaz-Nido J. Differentiation of a human neuroblastoma into neuron-like cells increases their susceptibility to transduction by herpesviral vectors.

[12] Cheung YT, Lau WK, Yu MS, et al. Effects of all-trans-retinoic acid on human SH-SY5Y neuroblastoma as in vitro model in neurotoxicity research. Neurotoxicology. 2009;**30**(1):

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[14] Naldini L, Blömer U, Gallay P, et al. In vivo gene delivery and stable transduction of

[15] Zufferey R, Nagy D, Mandel RJ, et al. Multiply attenuated lentiviral vector achieves effi-

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[18] Tiscornia G, Singer O, Verma IM. Production and purification of lentiviral vectors.

[19] Ryu EJ, Harding HP, Angelastro JM, et al. Endoplasmic reticulum stress and the unfolded protein response in cellular models of Parkinson's disease. The Journal of Neuroscience.

[20] Ryu EJ, Angelastro JM, Greene LA. Analysis of gene expression changes in a cellular

[21] Michel PP, Hirsch EC, Hunot S. Understanding dopaminergic cell death pathways in

[22] Xiang C, Wang Y, Zhang H, et al. The role of endoplasmic reticulum stress in neurode-

[23] Silva RM, Ries V, Oo TF, et al. CHOP/GADD153 is a mediator of apoptotic death in substantia nigra dopamine neurons in an in vivo neurotoxin model of parkinsonism.

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127-135

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## **Author details**

Pascaline Aimé\*† , Xiaotian Sun† and Lloyd A. Greene

\*Address all correspondence to<EMAIL_ADDRESS>
Department of Pathology and Cell Biology, Columbia University Medical Center, New York, USA

† These authors contributed equally

## **References**

[10] Nakamura K, Bindokas VP, Marks JD, et al. The selective toxicity of 1-methyl-4-phenylpyridinium to dopaminergic neurons: The role of mitochondrial complex I and reactive oxygen species revisited. Molecular Pharmacology. 2000;**58**(2):271-278

TBS: Tris-buffered saline

**Author details**

Pascaline Aimé\*†

New York, USA

**References**

†

Trib3: Tribbles pseudokinase 3

, Xiaotian Sun†

Academy of Sciences. 2003;**991**:1-14

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These authors contributed equally

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\*Address all correspondence to<EMAIL_ADDRESS>
and Lloyd A. Greene

268 Gene Expression and Regulation in Mammalian Cells - Transcription Toward the Establishment of Novel Therapeutics

Department of Pathology and Cell Biology, Columbia University Medical Center,

[1] Fahn S. Description of Parkinson's disease as a clinical syndrome. Annals of the New York

[2] Fahn S, Sulzer D. Neurodegeneration and neuroprotection in Parkinson disease. NeuroRx.

[3] Davie CA. A review of Parkinson's disease. British Medical Bulletin. 2008;**86**:109-127

[4] Schapira AH. Neurobiology and treatment of Parkinson's disease. Trends in Pharma-

[5] Levy OA, Malagelada C, Greene LA. Cell death pathways in Parkinson's disease: Pro-

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**Section 4**

**Application of Transcription-Based Therapies on**

**Human**

**Application of Transcription-Based Therapies on Human**

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[33] Hashida K, Kitao Y, Sudo H, et al. ATF6alpha promotes astroglial activation and neuronal survival in a chronic mouse model of Parkinson's disease. PLoS One. 2012;**7**(10):e47950

[34] Massie A, Schallier A, Mertens B, et al. Time-dependent changes in striatal xCT protein

[35] Sian J, Dexter DT, Lees AJ, et al. Alterations in glutathione levels in Parkinson's disease and other neurodegenerative disorders affecting basal ganglia. Annals of Neurology.

[36] Smeyne M, Smeyne RJ. Glutathione metabolism and Parkinson's disease. Free Radical

[37] Miyazaki I, Murakami S, Torigoe N, et al. Neuroprotective effects of levetiracetam target xCT in astrocytes in parkinsonian mice. Journal of Neurochemistry. 2016;**136**(1):194-204

[38] Massie A, Schallier A, Kim SW, et al. Dopaminergic neurons of system x(c)(−)-deficient mice are highly protected against 6-hydroxydopamine-induced toxicity. The FASEB

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2006;**66**(13):6748-6755

1994;**36**(3):348-355

**Chapter 12**

**Provisional chapter**

**Transcriptional Regulation of the Intestinal Cancer**

Colorectal cancer (CRC) is one of the most frequent cancers worldwide. Current treatments include surgery and chemotherapy, but disease recurrence occurs frequently. The continuous renewal of intestinal epithelium relies on the presence of intestinal stem cells that are also at the origin of CRC and contribute to therapy resistance and metastatic dissemination. Several nuclear signaling pathways and transcription factors regulate both intestinal cell homeostasis and tumorigenesis. However, the transcriptional events that govern the emergence of aggressive therapy-resistant cancer stem cells are still poorly defined. This review summarizes the relevance of transcription factors in intestinal stem cell biology and their involvement in colon cancer development and drug resistance. **Keywords:** transcription factors, intestinal cancer stem cells, colon cancer, chemotherapy

**Transcriptional Regulation of the Intestinal Cancer**

DOI: 10.5772/intechopen.71859

© 2016 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,

© 2018 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.

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

Colorectal cancer (CRC) is one of the most frequent cancers worldwide. The current standard-ofcare management includes surgery, radiotherapy and chemotherapy, sometimes in association with targeted agents to block tyrosine kinase receptors or their ligands. However, cancer recurs

The intestinal epithelium is continuously renewing, thanks to the presence of multipotent stem cells (SCs) within the intestinal crypts that give rise to all the differentiated cell types [2]. Different signaling pathways, including Wnt and Notch, and transcription factors are involved in intestinal development, homeostasis and maintenance of the intestinal SC properties [3]. These signaling cascades must be finely controlled because their deregulation is involved in gut

**Stem Cell Phenotype**

**Stem Cell Phenotype**

Marion Lapierre

**Abstract**

**1. Introduction**

in 30–50% of patients [1].

Antoine Gleizes, Vincent Cavaillès and

http://dx.doi.org/10.5772/intechopen.71859

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Antoine Gleizes, Vincent Cavaillès and Marion Lapierre

#### **Transcriptional Regulation of the Intestinal Cancer Stem Cell Phenotype Transcriptional Regulation of the Intestinal Cancer Stem Cell Phenotype**

DOI: 10.5772/intechopen.71859

Antoine Gleizes, Vincent Cavaillès and Marion Lapierre Antoine Gleizes, Vincent Cavaillès and Marion Lapierre Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.71859

#### **Abstract**

Colorectal cancer (CRC) is one of the most frequent cancers worldwide. Current treatments include surgery and chemotherapy, but disease recurrence occurs frequently. The continuous renewal of intestinal epithelium relies on the presence of intestinal stem cells that are also at the origin of CRC and contribute to therapy resistance and metastatic dissemination. Several nuclear signaling pathways and transcription factors regulate both intestinal cell homeostasis and tumorigenesis. However, the transcriptional events that govern the emergence of aggressive therapy-resistant cancer stem cells are still poorly defined. This review summarizes the relevance of transcription factors in intestinal stem cell biology and their involvement in colon cancer development and drug resistance.

**Keywords:** transcription factors, intestinal cancer stem cells, colon cancer, chemotherapy

## **1. Introduction**

Colorectal cancer (CRC) is one of the most frequent cancers worldwide. The current standard-ofcare management includes surgery, radiotherapy and chemotherapy, sometimes in association with targeted agents to block tyrosine kinase receptors or their ligands. However, cancer recurs in 30–50% of patients [1].

The intestinal epithelium is continuously renewing, thanks to the presence of multipotent stem cells (SCs) within the intestinal crypts that give rise to all the differentiated cell types [2]. Different signaling pathways, including Wnt and Notch, and transcription factors are involved in intestinal development, homeostasis and maintenance of the intestinal SC properties [3]. These signaling cascades must be finely controlled because their deregulation is involved in gut

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

© 2016 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, © 2018 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.

tumorigenesis. Importantly, recent studies suggest that tumor-initiating cells or cancer stem cells (CSCs) can regenerate a tumor and might be at the origin of CRC [4, 5]. Thus, a better understanding of CSC function in tumor initiation, progression and resistance to treatment is necessary to improve the screening, prevention and clinical management of patients with CRC.

In this review, we propose an overview of key transcriptional regulations that are involved in intestinal SC/CSC biology. We present the major signaling pathways and the main transcription factors involved in intestinal homeostasis as well as their roles in the transcriptional regulation of intestinal CSCs.

## **2. The intestinal epithelium and the stem cell compartment**

The main functions of the small intestine are food digestion and absorption and production of gastrointestinal hormones. It is subdivided in duodenum, jejunum and ileum, and is one of the most rapidly self-renewing tissues [6]. It is characterized by the presence of villi and Lieberkühn crypts. The large intestine (cecum, colon and rectum) is specialized in compacting stool for rapid excretion, and is arranged in multiple crypts associated with a flat luminal surface. It shows slower renewal capacities than the small intestine [7].

The intestinal epithelium develops from the embryonic endoderm [8] and its cellular composition is quite similar along the entire intestinal tract. The intestine incredible self-renewal capacity is supported by the SC compartment located at the bottom of the crypts. Specifically, transit-amplifying (TA) cells undergo four to five rounds of rapid cell division and then move out of the crypt and terminally differentiate into enterocytes, goblet cells, Tuft cells and enteroendocrine cells (**Figure 1**). These differentiated cells continue to move up along the villus and die by anoikis 2 or 3 days after having reached the villus tip. Paneth cells also derive from intestinal SC, but migrate downwards and settle at the crypt base where they live for 6–8 weeks [9]. Two other cell types have been detected in the intestinal epithelium: M cells that are associated with Peyer's patches and Cup cells that are located in the ileum.

and was described as the only one responsible for the maintenance of intestinal homeostasis, but without counterpart in the colon [10]. They can retain DNA labels (a surrogate SC marker), possibly due to their infrequent replication or selective retention of labeled DNA during division. Their relative quiescence also explains their resistance to radiation. This SC population was identified thanks to its strong and localized expression of the *BMI1* gene that encodes a component of the Polycomb repressor complex [7]. Lineage tracing of these cells revealed strict terminal differentiation toward the Paneth cell lineage. However, following injury, this population can start cycling and show typical intestinal SC activity and multipotency [11]. These features are typical of SCs, despite the fact that, differently from CBC cells,

**Figure 1.** Schematic representation of the intestinal epithelium and the hierarchy of intestinal lineages. Self-renewal of the intestinal epithelium is fueled by small intestinal stem cells (at the bottom of the crypt) that give rise to progenitor

Transcriptional Regulation of the Intestinal Cancer Stem Cell Phenotype

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275

cells. These can subsequently differentiate into the mature cell types required for normal gut function.

Genetic or epigenetic changes can lead to deregulated cell proliferation, resulting in tumor growth [12]. In the intestine, tumors start with the formation of small lesions called aberrant crypt foci (ACF). ACF expansion gives rise to an adenoma that can progress to *in situ* carcinoma

they do not generate all epithelial lineages.

**3.1. Colorectal cancer**

**3. Colorectal cancer and intestinal cancer stem cells**

To date, two SC populations have been identified in the crypts, highlighting the high plasticity of the intestinal epithelial SC compartment. The first one corresponds to crypt-based columnar (CBC) cells that express the leucine-rich receptor, LGR5 and are interspersed between Paneth cells (**Figure 1**). CBC cells are required for the long-term maintenance of the self-renewing epithelium. Indeed, they cycle steadily to produce the rapidly proliferating TA cells that can differentiate into all lineages [6]. In the colon, LGR5+ cells are considered to be SCs because they are pluripotent and can maintain epithelial cell self-renewal over long periods of time. However, LGR5+ cells in the small intestine seem to divide more actively than in the colon, possibly due to differences in the epithelial turnover rates [6].

The second crucial SC population corresponds to 'reserve' SCs that can be rapidly recruited to maintain epithelial homeostasis following injury [7]. They are located at position four from the crypt base (hence, the name of +4 SCs) and are generally considered to be relatively quiescent and resistant to acute injury (**Figure 1**). This population was discovered by Potten et al.

**Figure 1.** Schematic representation of the intestinal epithelium and the hierarchy of intestinal lineages. Self-renewal of the intestinal epithelium is fueled by small intestinal stem cells (at the bottom of the crypt) that give rise to progenitor cells. These can subsequently differentiate into the mature cell types required for normal gut function.

and was described as the only one responsible for the maintenance of intestinal homeostasis, but without counterpart in the colon [10]. They can retain DNA labels (a surrogate SC marker), possibly due to their infrequent replication or selective retention of labeled DNA during division. Their relative quiescence also explains their resistance to radiation. This SC population was identified thanks to its strong and localized expression of the *BMI1* gene that encodes a component of the Polycomb repressor complex [7]. Lineage tracing of these cells revealed strict terminal differentiation toward the Paneth cell lineage. However, following injury, this population can start cycling and show typical intestinal SC activity and multipotency [11]. These features are typical of SCs, despite the fact that, differently from CBC cells, they do not generate all epithelial lineages.

## **3. Colorectal cancer and intestinal cancer stem cells**

#### **3.1. Colorectal cancer**

tumorigenesis. Importantly, recent studies suggest that tumor-initiating cells or cancer stem cells (CSCs) can regenerate a tumor and might be at the origin of CRC [4, 5]. Thus, a better understanding of CSC function in tumor initiation, progression and resistance to treatment is necessary to improve the screening, prevention and clinical management of patients with CRC. In this review, we propose an overview of key transcriptional regulations that are involved in intestinal SC/CSC biology. We present the major signaling pathways and the main transcription factors involved in intestinal homeostasis as well as their roles in the transcriptional

274 Gene Expression and Regulation in Mammalian Cells - Transcription Toward the Establishment of Novel Therapeutics

The main functions of the small intestine are food digestion and absorption and production of gastrointestinal hormones. It is subdivided in duodenum, jejunum and ileum, and is one of the most rapidly self-renewing tissues [6]. It is characterized by the presence of villi and Lieberkühn crypts. The large intestine (cecum, colon and rectum) is specialized in compacting stool for rapid excretion, and is arranged in multiple crypts associated with a flat luminal

The intestinal epithelium develops from the embryonic endoderm [8] and its cellular composition is quite similar along the entire intestinal tract. The intestine incredible self-renewal capacity is supported by the SC compartment located at the bottom of the crypts. Specifically, transit-amplifying (TA) cells undergo four to five rounds of rapid cell division and then move out of the crypt and terminally differentiate into enterocytes, goblet cells, Tuft cells and enteroendocrine cells (**Figure 1**). These differentiated cells continue to move up along the villus and die by anoikis 2 or 3 days after having reached the villus tip. Paneth cells also derive from intestinal SC, but migrate downwards and settle at the crypt base where they live for 6–8 weeks [9]. Two other cell types have been detected in the intestinal epithelium: M cells that are associated

To date, two SC populations have been identified in the crypts, highlighting the high plasticity of the intestinal epithelial SC compartment. The first one corresponds to crypt-based columnar (CBC) cells that express the leucine-rich receptor, LGR5 and are interspersed between Paneth cells (**Figure 1**). CBC cells are required for the long-term maintenance of the self-renewing epithelium. Indeed, they cycle steadily to produce the rapidly proliferating TA cells that can differentiate into all lineages [6]. In the colon, LGR5+ cells are considered to be SCs because they are pluripotent and can maintain epithelial cell self-renewal over long periods of time. However, LGR5+ cells in the small intestine seem to divide more actively than in

The second crucial SC population corresponds to 'reserve' SCs that can be rapidly recruited to maintain epithelial homeostasis following injury [7]. They are located at position four from the crypt base (hence, the name of +4 SCs) and are generally considered to be relatively quiescent and resistant to acute injury (**Figure 1**). This population was discovered by Potten et al.

**2. The intestinal epithelium and the stem cell compartment**

surface. It shows slower renewal capacities than the small intestine [7].

with Peyer's patches and Cup cells that are located in the ileum.

the colon, possibly due to differences in the epithelial turnover rates [6].

regulation of intestinal CSCs.

Genetic or epigenetic changes can lead to deregulated cell proliferation, resulting in tumor growth [12]. In the intestine, tumors start with the formation of small lesions called aberrant crypt foci (ACF). ACF expansion gives rise to an adenoma that can progress to *in situ* carcinoma and finally to invasive adenocarcinoma [12]. Studies in humans and in animal models suggest that intestinal tumor development is a process where each successive genetic change confers growth advantage to tumor cells. Collectively, these genetic changes in cancer cells allow tumor progression through different stages [12]. Indeed, CRC development is considered as a paradigm of stepwise tumorigenesis with subsequent histopathological stages that precede invasive neoplastic growth and are associated with a progressively increasing number of specific genetic aberrations [11].

#### **3.2. Intestinal cancer stem cells**

Intriguingly, the biology of intestinal SCs and CRCs is highly interconnected. In many intestinal malignancies, it is assumed that the 'cell of origin' is a SC that acquired the initial mutation(s) necessary for malignant conversion [11]. These genetic alterations promote self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, programmed cell death evasion, limitless replicative potential, sustained angiogenesis, tissue invasion and ultimately metastasis formation [13]. Additionally, heterogeneously differentiated cell types are found in individual CRC specimens, contributing to the idea that such tumors are 'caricatures' of the normal intestinal tissue. This notion is further strengthened by the discovery of SC-like cancer cells that express intestinal SC markers and display multipotency and self-renewal capacities.

It is thought that intestinal CSCs are the cells that drive tumor growth and progression [11]. Indeed, intestinal CSCs, but not intestinal SCs, can regenerate tumors upon transplantation in animals [14]. CSCs are defined by four main characteristics: (i) they can be serially transplanted for multiple generations because of their self-renewal capacity; (ii) CSCs can generate bulk populations of non-tumorigenic cells by asymmetrical division, which is consistent with the hierarchical model of tumor development. Conversely, symmetrical division allows CSC maintenance within the tumor; (iii) CSCs retain their tumorigenic potential when transplanted into animals and (iv) CSCs can be separated from non-SCs using specific surface markers [14, 15]. In the last decades, the concept of CSC hierarchical arrangement has changed our understanding of tumor cell heterogeneity. The current CSC model postulates that CSCs reside at the top of the tumor hierarchy and differentiate unidirectionally into highly proliferative non-CSCs [12].

## **4. Nuclear signaling pathways that control intestinal CSCs**

In this part, we will focus on the major signaling pathways and transcription factors that are involved in the transcriptional regulation of intestinal SC/CSCs (**Figure 2**) and that could consequently be associated with tumor development/progression and/or cancer cell resistance to therapy.

nucleus are the hallmark of the canonical Wnt pathway activity. In the absence of Wnt ligands, β-catenin is phosphorylated, ubiquitinylated and degraded by the proteasomal machinery. Binding of Wnt ligands to their receptors results in the cytoplasmic accumulation of β-catenin that then translocates into the nucleus where it functions as a transcriptional co-activator of Wnt-target genes. The best characterized binding partners of β-catenin in the nucleus are the members of the lymphoid enhancer factor (LEF)/T cell factor (TCF) DNA-binding transcription factors [17]. Some of the downstream targets of the Wnt signaling pathway, such as SOX9 and KLF4/5, are involved in the control of the intestinal CSC phenotype and in CRC develop-

**Figure 2.** Schematic representation of the major signaling pathways involved in cancer stem cell biology. A gradient of BMP and Hh signaling, with relatively high activity in the villus and less activity within the crypt, regulates cell renewal and lineage specification. Wnt and Notch signaling gradients in the opposite direction (highest expression at the crypt

Transcriptional Regulation of the Intestinal Cancer Stem Cell Phenotype

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277

In up to 80% of colorectal carcinomas, mutations in molecules that are part of the Wnt/β-catenin pathway (notably truncating mutations in the *Apc* gene) lead to the formation of constitutive nuclear TCF/β-catenin complexes and to uncontrolled transcription of TCF-4 target genes [18]. In

ment and will be described below (see Sections 4.1.2 and 4.1.3).

base) play an important role in maintaining the stem cell compartment.

#### **4.1. The Wnt pathway and its effectors**

#### *4.1.1. The Wnt pathway*

The Wnt pathway is involved in many biological processes and is essential for epithelial intestinal homeostasis (**Figure 2**) [16]. Accumulation and translocation of β-catenin into the

#### Transcriptional Regulation of the Intestinal Cancer Stem Cell Phenotype http://dx.doi.org/10.5772/intechopen.71859 277

and finally to invasive adenocarcinoma [12]. Studies in humans and in animal models suggest that intestinal tumor development is a process where each successive genetic change confers growth advantage to tumor cells. Collectively, these genetic changes in cancer cells allow tumor progression through different stages [12]. Indeed, CRC development is considered as a paradigm of stepwise tumorigenesis with subsequent histopathological stages that precede invasive neoplastic growth and are associated with a progressively increasing number of specific genetic

276 Gene Expression and Regulation in Mammalian Cells - Transcription Toward the Establishment of Novel Therapeutics

Intriguingly, the biology of intestinal SCs and CRCs is highly interconnected. In many intestinal malignancies, it is assumed that the 'cell of origin' is a SC that acquired the initial mutation(s) necessary for malignant conversion [11]. These genetic alterations promote self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, programmed cell death evasion, limitless replicative potential, sustained angiogenesis, tissue invasion and ultimately metastasis formation [13]. Additionally, heterogeneously differentiated cell types are found in individual CRC specimens, contributing to the idea that such tumors are 'caricatures' of the normal intestinal tissue. This notion is further strengthened by the discovery of SC-like cancer cells that express intestinal SC markers and display multipotency and self-renewal capacities.

It is thought that intestinal CSCs are the cells that drive tumor growth and progression [11]. Indeed, intestinal CSCs, but not intestinal SCs, can regenerate tumors upon transplantation in animals [14]. CSCs are defined by four main characteristics: (i) they can be serially transplanted for multiple generations because of their self-renewal capacity; (ii) CSCs can generate bulk populations of non-tumorigenic cells by asymmetrical division, which is consistent with the hierarchical model of tumor development. Conversely, symmetrical division allows CSC maintenance within the tumor; (iii) CSCs retain their tumorigenic potential when transplanted into animals and (iv) CSCs can be separated from non-SCs using specific surface markers [14, 15]. In the last decades, the concept of CSC hierarchical arrangement has changed our understanding of tumor cell heterogeneity. The current CSC model postulates that CSCs reside at the top of the tumor hierarchy and differentiate unidirectionally into highly proliferative non-CSCs [12].

In this part, we will focus on the major signaling pathways and transcription factors that are involved in the transcriptional regulation of intestinal SC/CSCs (**Figure 2**) and that could consequently be associated with tumor development/progression and/or cancer cell resistance

The Wnt pathway is involved in many biological processes and is essential for epithelial intestinal homeostasis (**Figure 2**) [16]. Accumulation and translocation of β-catenin into the

**4. Nuclear signaling pathways that control intestinal CSCs**

aberrations [11].

to therapy.

*4.1.1. The Wnt pathway*

**4.1. The Wnt pathway and its effectors**

**3.2. Intestinal cancer stem cells**

**Figure 2.** Schematic representation of the major signaling pathways involved in cancer stem cell biology. A gradient of BMP and Hh signaling, with relatively high activity in the villus and less activity within the crypt, regulates cell renewal and lineage specification. Wnt and Notch signaling gradients in the opposite direction (highest expression at the crypt base) play an important role in maintaining the stem cell compartment.

nucleus are the hallmark of the canonical Wnt pathway activity. In the absence of Wnt ligands, β-catenin is phosphorylated, ubiquitinylated and degraded by the proteasomal machinery. Binding of Wnt ligands to their receptors results in the cytoplasmic accumulation of β-catenin that then translocates into the nucleus where it functions as a transcriptional co-activator of Wnt-target genes. The best characterized binding partners of β-catenin in the nucleus are the members of the lymphoid enhancer factor (LEF)/T cell factor (TCF) DNA-binding transcription factors [17]. Some of the downstream targets of the Wnt signaling pathway, such as SOX9 and KLF4/5, are involved in the control of the intestinal CSC phenotype and in CRC development and will be described below (see Sections 4.1.2 and 4.1.3).

In up to 80% of colorectal carcinomas, mutations in molecules that are part of the Wnt/β-catenin pathway (notably truncating mutations in the *Apc* gene) lead to the formation of constitutive nuclear TCF/β-catenin complexes and to uncontrolled transcription of TCF-4 target genes [18]. In the mouse, specific deletion of the *Apc* gene in LGR5+ SCs triggers the formation of many LGR5+ adenomas in the small and large intestine [5]. Similarly, lack of β-catenin repression in intestinal +4 SCs promotes the formation of BMI1+ adenomas [19]. Moreover, loss of APC negative control induces constitutive nuclear β-catenin/TCF complex activation and hyper-proliferation of the SC compartment [19].

CRC development, thus facilitating transformation and tumor progression [29]. Furthermore, a SOX9 splice variant (MiniSOX9) that contains the HMG domain responsible for binding to DNA but devoid of the trans-activating domain has been discovered [30]. MiniSOX9 inhibits SOX9 activity by a dominant-negative effect *in vitro* and can promote the Wnt/β-catenin pathway, resulting in β-catenin over-activation. In addition, strong MiniSOX9 expression is observed in CRC tumor tissue, while it is undetectable in the adjacent normal tissue [30]. Wild type and many SOX9 mutants regulate tumor proliferation capacity, notably through regulation of the CSC pool. Nevertheless, SOX9 protein level could not be clearly associated with patient prognosis [31].

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KLF4 was originally identified as a gut-enriched transcription factor in the intestine and is expressed in terminally differentiated columnar intestinal epithelial cells [32]. KLF4 regulates intestinal epithelial homeostasis and has a critical role in the development and terminal differentiation of goblet cells [32]. In human HT-29 CRC cells, KLF4 inhibits cell proliferation by blocking progression from the G1 to S phase of the cell cycle through inhibition of cyclin D1

Moreover, mutations in the Wnt/β-catenin pathway are associated with KLF4 downregulation in human CRC cell lines. Indeed, KLF4 is an indirect APC target and is considered to be a repressor of BMI1 transcriptional activity [34, 35]. Furthermore, using a KLF4 inducible system in CRC cell lines, it was demonstrated that KLF4 reduces colony formation, cell migration and invasion [34]. Additionally, KLF4 overexpression in human adenocarcinoma cells leads to reduced [3H]-thymidine uptake, whereas inhibition of KFL4 expression increases DNA synthesis, confirming that KLF4 plays an essential role in colon cell growth arrest [36]. Surprisingly, despite its tumor suppressor activity, KLF4 is overexpressed in colon CSC-enriched spheroids compared with the parental CRC cells from which the spheroids were derived [37]. Moreover, KLF4 knockdown affects the stemness phenotype and decreases the malignant profile of these CSC-enriched spheroid cells, in line with its role in reprogramming murine fibroblasts into stem cells [37, 38]. In agreement with its tumor suppressor activity, KLF4 expression is frequently lost in CRC and its downregulation is strongly associated with tumor development. Moreover, loss of heterozygosity on chromosome 9q31, where the *KLF4* gene is localized, is frequently found in human CRC, and could lead to uncontrolled cell proliferation and to a SC-like phenotype of differentiated cells [33]. Low KLF4 expression levels are also found in colon adenomas and metastases [33]. Lee et al. confirmed that *KLF4* mRNA expression levels are lower in CRC tumor tissue compared with normal tissue [39]. However and surprisingly, they observed that high KLF4 level in normal tissue is correlated with high KLF4 expression in tumors and is associated with poor patient survival [39]. The conflicting results between clinical studies concerning KLF4 prognostic value could be explained by the differential regulation of KLF4 mRNA and protein expression in CRC or by the presence also of KLF4+ stromal cells in the tumor samples. Additional investigations are needed to elucidate these data; nevertheless, KLF4 expression levels in normal and tumor tis-

*4.1.3. Krüppel-like factors (KLF)*

sues are prognostic markers for CRC.

*<IP_ADDRESS>. KLF4*

expression [33].

The Wnt signaling pathway has a role also in human intestinal CSCs. In spheroid cultures of CSCs isolated from biopsies of patients with CRC, Wnt expression is heterogeneous. Injection of Wnthigh cells in mice results in more effective tumor formation compared with Wntlow cells. The heterogeneous Wnt expression pattern is maintained in the tumors and is related to the expression of several intestinal SC markers, such as LGR5 and ASCL2 [20]. Additionally, colonospheres developed from human CSCs show increased β-catenin expression, associated with transcriptional activation of TCF/LEF [21]. Hence, activation of Wnt/β-catenin signaling can convert intestinal SCs into CSCs, which corresponds to the first step of malignant transformation [19].

Several studies tried to correlate β-catenin activation/expression level with the outcome of patients with CRC. For instance, in 2007, Lugli et al. analyzed tissue microarray data on more than 1400 CRC biopsies and found that high level of β-catenin nuclear expression is an independent adverse prognostic factor [22].

#### *4.1.2. SOX9*

The SRY-related high-mobility group box 9 (*SOX9*) gene is a physiological target of the TCF/βcatenin complex that promotes cell proliferation. This key terminal effector of the Wnt pathway is required for +4 SC differentiation into Paneth cells [23]. In the intestinal epithelium, SOX9 expression pattern in the SC compartment almost perfectly overlaps with that of the proliferative marker Ki-67. Interestingly, SOX9 positively regulates its own expression in many cell types and exerts a negative feedback-loop on TCF/β-catenin activity, leading to restriction of intestinal SC proliferation [23, 24].

SOX9-deficient mice exhibit higher cell proliferation, extensive colon hyperplasia with numerous enlarged crypts. However, SOX9 deletion is not sufficient to induce malignancy [25]. Moreover, SOX9 overexpression in human CRC cells results in cell cycle progression and apoptosis bypass, due to increased *BMI1* gene expression [26]. Additionally, in colon epithelial cells, high SOX9 expression is associated with undifferentiated states, SC-like properties and high LGR5 mRNA level *in vitro* [27]. SOX9 has several pro-oncogenic properties, including the ability to promote cell proliferation, to inhibit senescence and to collaborate with other oncogenes in neoplastic transformation [26]. However, recent *in vitro* and *in vivo* studies have described SOX9 tumor suppressor activities in CRC cells. Specifically, SOX9 inhibits β-catenin activity by interacting physically with this protein and removing it from chromatin. It also decreases expression of the c-Myc oncogene, a target of the Wnt/β-catenin pathway [28].

The strong expression of SOX9 in CRC cells due to the constitutive activity of the Wnt pathway can contribute to cancer progression and/or influence tumor differentiation. *SOX9* displays missense or frameshift mutations in almost 10% of CRC [29]. SOX9 mutation rate is higher in more advanced tumors and is correlated with activated KRAS, an oncogene frequently mutated during CRC development, thus facilitating transformation and tumor progression [29]. Furthermore, a SOX9 splice variant (MiniSOX9) that contains the HMG domain responsible for binding to DNA but devoid of the trans-activating domain has been discovered [30]. MiniSOX9 inhibits SOX9 activity by a dominant-negative effect *in vitro* and can promote the Wnt/β-catenin pathway, resulting in β-catenin over-activation. In addition, strong MiniSOX9 expression is observed in CRC tumor tissue, while it is undetectable in the adjacent normal tissue [30]. Wild type and many SOX9 mutants regulate tumor proliferation capacity, notably through regulation of the CSC pool. Nevertheless, SOX9 protein level could not be clearly associated with patient prognosis [31].

### *4.1.3. Krüppel-like factors (KLF)*

## *<IP_ADDRESS>. KLF4*

the mouse, specific deletion of the *Apc* gene in LGR5+ SCs triggers the formation of many LGR5+ adenomas in the small and large intestine [5]. Similarly, lack of β-catenin repression in intestinal +4 SCs promotes the formation of BMI1+ adenomas [19]. Moreover, loss of APC negative control induces constitutive nuclear β-catenin/TCF complex activation and hyper-proliferation of the SC

278 Gene Expression and Regulation in Mammalian Cells - Transcription Toward the Establishment of Novel Therapeutics

The Wnt signaling pathway has a role also in human intestinal CSCs. In spheroid cultures of CSCs isolated from biopsies of patients with CRC, Wnt expression is heterogeneous. Injection of Wnthigh cells in mice results in more effective tumor formation compared with Wntlow cells. The heterogeneous Wnt expression pattern is maintained in the tumors and is related to the expression of several intestinal SC markers, such as LGR5 and ASCL2 [20]. Additionally, colonospheres developed from human CSCs show increased β-catenin expression, associated with transcriptional activation of TCF/LEF [21]. Hence, activation of Wnt/β-catenin signaling can convert intestinal SCs into CSCs, which corresponds to the first step of malignant transformation [19].

Several studies tried to correlate β-catenin activation/expression level with the outcome of patients with CRC. For instance, in 2007, Lugli et al. analyzed tissue microarray data on more than 1400 CRC biopsies and found that high level of β-catenin nuclear expression is an independent adverse

The SRY-related high-mobility group box 9 (*SOX9*) gene is a physiological target of the TCF/βcatenin complex that promotes cell proliferation. This key terminal effector of the Wnt pathway is required for +4 SC differentiation into Paneth cells [23]. In the intestinal epithelium, SOX9 expression pattern in the SC compartment almost perfectly overlaps with that of the proliferative marker Ki-67. Interestingly, SOX9 positively regulates its own expression in many cell types and exerts a negative feedback-loop on TCF/β-catenin activity, leading to restriction of

SOX9-deficient mice exhibit higher cell proliferation, extensive colon hyperplasia with numerous enlarged crypts. However, SOX9 deletion is not sufficient to induce malignancy [25]. Moreover, SOX9 overexpression in human CRC cells results in cell cycle progression and apoptosis bypass, due to increased *BMI1* gene expression [26]. Additionally, in colon epithelial cells, high SOX9 expression is associated with undifferentiated states, SC-like properties and high LGR5 mRNA level *in vitro* [27]. SOX9 has several pro-oncogenic properties, including the ability to promote cell proliferation, to inhibit senescence and to collaborate with other oncogenes in neoplastic transformation [26]. However, recent *in vitro* and *in vivo* studies have described SOX9 tumor suppressor activities in CRC cells. Specifically, SOX9 inhibits β-catenin activity by interacting physically with this protein and removing it from chromatin. It also decreases expression of the

The strong expression of SOX9 in CRC cells due to the constitutive activity of the Wnt pathway can contribute to cancer progression and/or influence tumor differentiation. *SOX9* displays missense or frameshift mutations in almost 10% of CRC [29]. SOX9 mutation rate is higher in more advanced tumors and is correlated with activated KRAS, an oncogene frequently mutated during

compartment [19].

prognostic factor [22].

intestinal SC proliferation [23, 24].

c-Myc oncogene, a target of the Wnt/β-catenin pathway [28].

*4.1.2. SOX9*

KLF4 was originally identified as a gut-enriched transcription factor in the intestine and is expressed in terminally differentiated columnar intestinal epithelial cells [32]. KLF4 regulates intestinal epithelial homeostasis and has a critical role in the development and terminal differentiation of goblet cells [32]. In human HT-29 CRC cells, KLF4 inhibits cell proliferation by blocking progression from the G1 to S phase of the cell cycle through inhibition of cyclin D1 expression [33].

Moreover, mutations in the Wnt/β-catenin pathway are associated with KLF4 downregulation in human CRC cell lines. Indeed, KLF4 is an indirect APC target and is considered to be a repressor of BMI1 transcriptional activity [34, 35]. Furthermore, using a KLF4 inducible system in CRC cell lines, it was demonstrated that KLF4 reduces colony formation, cell migration and invasion [34]. Additionally, KLF4 overexpression in human adenocarcinoma cells leads to reduced [3H]-thymidine uptake, whereas inhibition of KFL4 expression increases DNA synthesis, confirming that KLF4 plays an essential role in colon cell growth arrest [36]. Surprisingly, despite its tumor suppressor activity, KLF4 is overexpressed in colon CSC-enriched spheroids compared with the parental CRC cells from which the spheroids were derived [37]. Moreover, KLF4 knockdown affects the stemness phenotype and decreases the malignant profile of these CSC-enriched spheroid cells, in line with its role in reprogramming murine fibroblasts into stem cells [37, 38].

In agreement with its tumor suppressor activity, KLF4 expression is frequently lost in CRC and its downregulation is strongly associated with tumor development. Moreover, loss of heterozygosity on chromosome 9q31, where the *KLF4* gene is localized, is frequently found in human CRC, and could lead to uncontrolled cell proliferation and to a SC-like phenotype of differentiated cells [33]. Low KLF4 expression levels are also found in colon adenomas and metastases [33]. Lee et al. confirmed that *KLF4* mRNA expression levels are lower in CRC tumor tissue compared with normal tissue [39]. However and surprisingly, they observed that high KLF4 level in normal tissue is correlated with high KLF4 expression in tumors and is associated with poor patient survival [39].

The conflicting results between clinical studies concerning KLF4 prognostic value could be explained by the differential regulation of KLF4 mRNA and protein expression in CRC or by the presence also of KLF4+ stromal cells in the tumor samples. Additional investigations are needed to elucidate these data; nevertheless, KLF4 expression levels in normal and tumor tissues are prognostic markers for CRC.

#### *<IP_ADDRESS>. KLF5*

The transcription factor KLF5 can interact with several components of different signaling pathways (e.g., the Wnt, Hippo, TGF-β and Notch signaling cascades) and mediate their activity [40]. In physiological conditions, KLF5 is strongly expressed by intestinal progenitor and stem cells, suggesting a role in cell proliferation control [41].

In CRC, the Notch pathway is strongly activated compared with normal tissue. Moreover, expression analysis of resection biopsies from patients with CRC showed that Notch1 expression level is correlated with poor prognosis and is a good predictive marker of cancer progression [48]. Intriguingly, the expression level of Notch2 is negatively correlated with that of Notch1 in CRC and Notch2 has anti-tumoral properties [48]. These opposite features could be

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BMI1 is a downstream target of Notch signaling and a key component of the Polycomb group [49]. BMI1 is expressed in almost all tissue types and regulates a myriad of cellular processes that are critical for cell growth, cell fate decision, development, senescence, aging, DNA damage repair, apoptosis and SC self-renewal [49, 50]. BMI1 is highly expressed in intestinal SCs and isolated BMI1+ cells can generate epithelial organoids in culture [7]. Additionally, BMI1 loss decreases murine intestinal SC proliferation and promotes their differentiation into goblet cells [49]. BMI1 also contributes to the tumor-initiating and self-renewal abilities of human CRC cells because its downregulation inhibits tumor cell growth and is associated with reduction of tumor-initiating cells [51]. Moreover, BMI1 is involved in intestinal CSC invasion and migration. Indeed, a recent study demonstrated that BMI1 represses E-cadherin expression in colon CSCs, thus promoting metastasis formation via epithelial to mesenchymal transition [50]. Altogether, these data strongly support BMI1 role in the maintenance of the intestinal CSC phenotype. In agreement, clinical studies showed that BMI1 expression is a negative prognostic marker in CRC [52]. BMI1 mRNA and protein are overexpressed in colorectal adenomas and carcinomas compared with normal tissues [53]. A gradient of BMI1 expression has been reported in human colon precancerous and cancerous tissues and is correlated with the cancer stage, suggesting that

Indeed, BMI1 is considered to be a negative CRC prognostic biomarker, and patients with BMI1-positive tumors are at higher risk of disease recurrence and/or metastases compared with those with BMI1-negative tumors. As BMI1 has a role in maintaining the intestinal CSC phenotype, high BMI1 expression could indicate the presence of a large CSC population in the tumor. Consequently, high proportion of CSCs in a tumor could be an indicator of poor prognosis [31,

The Hedgehog (Hh) signaling pathway is a key regulator of intestinal homeostasis. Hh proteins are part of a family of secreted proteins that are involved in the development and maintenance of the gastrointestinal tract [17]. Aberrant activation of the Hh signaling pathway is associated with tumorigenesis in various tissues. The roles of Hh signaling differ at each CRC stage, from adenoma to adenocarcinoma [56]. Moreover, Sonic Hedgehog (SHH), one of the Hh effectors, promotes CRC development, while Indian Hedgehog (IHH) inhibits CRC formation [56].

used to develop a fine prognostic marker of CRC progression and recurrence.

*4.2.2. BMI1*

54, 55].

BMI1 contributes to CRC progression [53].

**4.3. Other signaling pathways**

*4.3.1. The Hedgehog pathway*

Stable KLF5 overexpression in HT-29 CRC cells promotes spheroid formation [40]. Conversely, deletion of the *KLF5* gene in mouse LGR5+ SCs promotes β-catenin nuclear localization and the appearance of abnormal apoptotic cells in the intestinal crypts, due to inhibition of their proliferation and survival capacities [41]. In agreement, KLF5 is required for the tumor-initiating activity of β-catenin during intestinal tumorigenesis in *ApcMin* mice [41]. Inhibition of *KLF5* gene expression in CRC cell lines reduces cell proliferation and transformation as well as anchorage-independent growth [42].

In patients with CRC, intestinal tumor progression is associated with *KLF5* gene upregulation in the primary tumor and also in metastases, compared with healthy tissues [41]. Moreover, comparative genomic hybridization (CGH) array analysis of human CRC samples highlighted the frequent chromosomal amplification of the *KLF5* locus [41]. CRC samples with mutated KRAS also display KLF5 upregulation, associated with increased cell proliferation [42]. As activating KRAS mutations are found in more than 50% of CRC, KLF5 appears to be an important downstream mediator of activated KRAS during CRC development. These findings indicate that KLF5 is a major regulator of intestinal SC proliferation in normal and pathological conditions.

## **4.2. The Notch pathway and BMI1**

#### *4.2.1. The Notch pathway*

The Notch signaling cascade is one of the major pathway involved in intestinal homeostasis and in the direct regulation of cell fate [43]. The initiating step of the Notch signaling cascade is the interaction between one of its five ligands (Delta-like1/3/4, Jagged1/2) and a Notch receptor (Notch1–4). Upon ligand binding, the receptor conformational change through proteolytic cleavage leads to nuclear translocation of cleaved Notch intracellular domain (NICD) and its association with the DNA-binding transcription factor CSL (also called RBP-Jκ). This turns the CSL complex from a transcriptional repressor into a transcriptional activator. The best known targets of the CSL/NICD complex are members of the *HES* gene family and their homologs, the *Hey* (also called HERP) gene family of basic helix-loop-helix transcription factors. This is known as the canonical Notch pathway [17, 43, 44].

In the colon, Notch signaling is an essential gatekeeper of intestinal progenitors and clearly plays an important role in the maintenance of the colon crypt compartment [45] (**Figure 2**). Using small-molecule inhibitors and short hairpin RNA-mediated knock-down, it has been demonstrated that Notch prevents apoptosis of colon cancer-initiating cells (CCICs) and is critical for self-renewal [46]. Moreover, the Notch pathway supports slow-cycling BMI1+ CCICs, by promoting their self-renewal, tumorigenicity and chemoresistance in tumor xenografts [47].

In CRC, the Notch pathway is strongly activated compared with normal tissue. Moreover, expression analysis of resection biopsies from patients with CRC showed that Notch1 expression level is correlated with poor prognosis and is a good predictive marker of cancer progression [48]. Intriguingly, the expression level of Notch2 is negatively correlated with that of Notch1 in CRC and Notch2 has anti-tumoral properties [48]. These opposite features could be used to develop a fine prognostic marker of CRC progression and recurrence.

## *4.2.2. BMI1*

*<IP_ADDRESS>. KLF5*

The transcription factor KLF5 can interact with several components of different signaling pathways (e.g., the Wnt, Hippo, TGF-β and Notch signaling cascades) and mediate their activity [40]. In physiological conditions, KLF5 is strongly expressed by intestinal progenitor

280 Gene Expression and Regulation in Mammalian Cells - Transcription Toward the Establishment of Novel Therapeutics

Stable KLF5 overexpression in HT-29 CRC cells promotes spheroid formation [40]. Conversely, deletion of the *KLF5* gene in mouse LGR5+ SCs promotes β-catenin nuclear localization and the appearance of abnormal apoptotic cells in the intestinal crypts, due to inhibition of their proliferation and survival capacities [41]. In agreement, KLF5 is required for the tumor-initiating activity of β-catenin during intestinal tumorigenesis in *ApcMin* mice [41]. Inhibition of *KLF5* gene expression in CRC cell lines reduces cell proliferation and transformation as well as

In patients with CRC, intestinal tumor progression is associated with *KLF5* gene upregulation in the primary tumor and also in metastases, compared with healthy tissues [41]. Moreover, comparative genomic hybridization (CGH) array analysis of human CRC samples highlighted the frequent chromosomal amplification of the *KLF5* locus [41]. CRC samples with mutated KRAS also display KLF5 upregulation, associated with increased cell proliferation [42]. As activating KRAS mutations are found in more than 50% of CRC, KLF5 appears to be an important downstream mediator of activated KRAS during CRC development. These findings indicate that KLF5 is a major regulator of intestinal SC proliferation in normal and pathological conditions.

The Notch signaling cascade is one of the major pathway involved in intestinal homeostasis and in the direct regulation of cell fate [43]. The initiating step of the Notch signaling cascade is the interaction between one of its five ligands (Delta-like1/3/4, Jagged1/2) and a Notch receptor (Notch1–4). Upon ligand binding, the receptor conformational change through proteolytic cleavage leads to nuclear translocation of cleaved Notch intracellular domain (NICD) and its association with the DNA-binding transcription factor CSL (also called RBP-Jκ). This turns the CSL complex from a transcriptional repressor into a transcriptional activator. The best known targets of the CSL/NICD complex are members of the *HES* gene family and their homologs, the *Hey* (also called HERP) gene family of basic helix-loop-helix transcription fac-

In the colon, Notch signaling is an essential gatekeeper of intestinal progenitors and clearly plays an important role in the maintenance of the colon crypt compartment [45] (**Figure 2**). Using small-molecule inhibitors and short hairpin RNA-mediated knock-down, it has been demonstrated that Notch prevents apoptosis of colon cancer-initiating cells (CCICs) and is critical for self-renewal [46]. Moreover, the Notch pathway supports slow-cycling BMI1+ CCICs, by promoting their self-renewal, tumorigenicity and chemoresistance in tumor xenografts [47].

and stem cells, suggesting a role in cell proliferation control [41].

tors. This is known as the canonical Notch pathway [17, 43, 44].

anchorage-independent growth [42].

**4.2. The Notch pathway and BMI1**

*4.2.1. The Notch pathway*

BMI1 is a downstream target of Notch signaling and a key component of the Polycomb group [49]. BMI1 is expressed in almost all tissue types and regulates a myriad of cellular processes that are critical for cell growth, cell fate decision, development, senescence, aging, DNA damage repair, apoptosis and SC self-renewal [49, 50]. BMI1 is highly expressed in intestinal SCs and isolated BMI1+ cells can generate epithelial organoids in culture [7]. Additionally, BMI1 loss decreases murine intestinal SC proliferation and promotes their differentiation into goblet cells [49]. BMI1 also contributes to the tumor-initiating and self-renewal abilities of human CRC cells because its downregulation inhibits tumor cell growth and is associated with reduction of tumor-initiating cells [51]. Moreover, BMI1 is involved in intestinal CSC invasion and migration. Indeed, a recent study demonstrated that BMI1 represses E-cadherin expression in colon CSCs, thus promoting metastasis formation via epithelial to mesenchymal transition [50].

Altogether, these data strongly support BMI1 role in the maintenance of the intestinal CSC phenotype. In agreement, clinical studies showed that BMI1 expression is a negative prognostic marker in CRC [52]. BMI1 mRNA and protein are overexpressed in colorectal adenomas and carcinomas compared with normal tissues [53]. A gradient of BMI1 expression has been reported in human colon precancerous and cancerous tissues and is correlated with the cancer stage, suggesting that BMI1 contributes to CRC progression [53].

Indeed, BMI1 is considered to be a negative CRC prognostic biomarker, and patients with BMI1-positive tumors are at higher risk of disease recurrence and/or metastases compared with those with BMI1-negative tumors. As BMI1 has a role in maintaining the intestinal CSC phenotype, high BMI1 expression could indicate the presence of a large CSC population in the tumor. Consequently, high proportion of CSCs in a tumor could be an indicator of poor prognosis [31, 54, 55].

## **4.3. Other signaling pathways**

## *4.3.1. The Hedgehog pathway*

The Hedgehog (Hh) signaling pathway is a key regulator of intestinal homeostasis. Hh proteins are part of a family of secreted proteins that are involved in the development and maintenance of the gastrointestinal tract [17]. Aberrant activation of the Hh signaling pathway is associated with tumorigenesis in various tissues. The roles of Hh signaling differ at each CRC stage, from adenoma to adenocarcinoma [56]. Moreover, Sonic Hedgehog (SHH), one of the Hh effectors, promotes CRC development, while Indian Hedgehog (IHH) inhibits CRC formation [56].

IHH regulates intestinal SC fates by interfering with the maturation and localization of the underlying stromal cells that in turn generate signaling molecules needed for the maintenance of the intestinal SC niche [56] (**Figure 2**). IHH, expressed by differentiated enterocytes, indirectly inhibits Wnt signaling at the crypt base and reduces the number of proliferating precursor cells [17, 57]. A decrease in Hh signaling is correlated with the expansion of the intestinal SC pool, with blunted enterocyte differentiation and activation of the Wnt pathway. Moreover, *IHH* gene knock-out leads to intestinal SC accumulation [57]. In addition, specific Hh activation in murine stromal cells induces complex transcriptional changes, leading to loss of colon SC-specific gene expression and upregulation of epithelial differentiation markers [58]. Most of the components of the Hh signaling pathway are upregulated (mRNA and protein) in CRC, with the exception of IHH that appears to be downregulated. Overexpression of members of the Hh signaling pathway is associated with poor survival and adverse clinical features [59]. However, in metastatic CRC, treatment with vismodegib, an Hh pathway inhibitor, in combination with standard chemotherapy, does not significantly improve patient survival [60].

*4.3.3. The Hippo pathway*

proliferating and post-mitotic cells [17, 68].

of tumor-initiating cells to adenoma [70].

**4.4. Other intestinal CSC-related transcription factors**

mediated induction of target gene expression [74].

c-Yes and YAP [72].

*4.4.1. PXR*

The Hippo pathway regulates various cellular processes, including cell survival, proliferation and differentiation, but has been involved only recently in SC biology [67]. Yes-associated protein (YAP) and transcriptional co-activator with PDZ binding motif (TAZ) are the central effector molecules of this signaling cascade and are abundantly expressed in the cytoplasm of both

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In cancer, the Hippo pathway inhibits cell proliferation, promotes apoptosis and regulates stem/ progenitor cell expansion. In cancer cells, YAP and TAZ are localized mainly in the nucleus and promotes cell and tumor growth. There is considerable evidence that abnormal Hippo signaling is associated with tumor progression and YAP/TAZ overexpression is frequently observed in CRC [67]. This overexpression could be linked to Wnt/β-catenin over-activation because YAP is a specific target of this pathway. Furthermore, the major components of the Hippo pathway (i.e., MST1/2 and MOBKL1A/B) that control YAP/TAZ activity display low expression levels in colon carcinomas [69]. YAP deletion in *Apcmin* mice prevents polyp formation and blocks the differentiation of *Apc−/−* organoids. Moreover, using a mosaic model of *Yap* and *Apc* gene deletion in intestinal SCs, YAP appears to be dispensable for tumor initiation, but crucial for progression

Hippo pathway dysregulation, leading to loss of YAP repression, has been observed in different cancer types [71]. In patients with CRC, YAP over-activation is closely related to β-catenin over-activation. Moreover, the tyrosine kinase c-Yes is hyper-phosphorylated in 5-fluorouracilresistant cells with CSC features, thus preventing YAP nuclear translocation [72]. Finally, *YES1* and *YAP* levels are correlated with worse prognosis in chemotherapy-treated patients with CRC, suggesting that chemotherapy favors the selection of intestinal CSCs with deregulated

Pregnane X Receptor (PXR, NR1I2), a member of the nuclear receptor superfamily, is highly expressed in the colon. PXR targets are genes that encode phase I and II metabolic enzymes and phase III drug transporters. Members of the nuclear receptor superfamily function as ligand-activated transcription factors and play critical roles in nearly every aspect of development and adult physiology [73]. Interestingly, it has been reported that the Wnt/β-catenin signaling pathway is crucial for PXR activity and notably that β-catenin is required for PXR-

Planque et al. have recently demonstrated that PXR is a potent intestinal CSC phenotype driver by regulating a network of downstream genes involved in self-renewal and chemoresistance [75]. PXR expression is associated with CSC enrichment, after cell sorting of cancer cells using ALDH activity to identify CSCs and after spheroid passaging. In addition, expression of CSC

#### *4.3.2. The BMP pathway*

The BMP pathway regulates many cellular mechanisms, including apoptosis and cell growth, depending on the specific cellular context. BMP ligands are secreted in their active form and homodimerize before binding to their cognate BMP receptors (BMPR). SMAD transcription factors are the main downstream effectors of BMP signaling that plays key roles in adult gut homeostasis, inflammation and cancer.

Specific inhibition of BMP signaling in intestinal epithelial cell does not lead to initiation of colon tumors *in vivo*, while suppression in mesenchymal myofibroblasts is associated with spontaneous tumor formation. This suggests that inhibition of BMP signaling in the mesenchymal cells surrounding the intestinal epithelium acts as a trigger of gastrointestinal tumorigenesis [61]. Moreover, BMP4 expression is lost in intestinal CSCs, leading to deregulation of the proliferative compartment [62].

Nevertheless, it is still unclear whether BMP limits expansion of intestinal epithelial cells by repressing LGR5+ intestinal SC self-renewal or by inhibiting epithelial cell proliferation. In addition, BMP type Ia receptor (*Bmpr1a*) conditional knock-out in the intestinal epithelium leads to intestine hyperplasia with multiple intestinal polyps due to hyperactive SCs [63]. Moreover, in these mice, the LGR5+ SC pool is enlarged due to increased survival, allowing better intestinal regeneration [63]. Among the BMP family members, BMP2 and BMP4 are specifically involved in intestinal CSC regulation by promoting their differentiation and antagonizing Wnt/β-catenin signaling [64]. Furthermore, a recent study showed that the transcription factor GATA6 is a key regulator of CSC expansion and self-renewal through downregulation of BMP genes [65].

Finally, mutations that affect BMP signaling are frequently observed in patients with juvenile polyposis syndrome that is characterized by non-cancerous polyps, as well as in patients with progressing CRC. Analysis of SMAD4 expression levels in patients with CRC showed that it is downregulated in CRC and associated with poor prognosis [66].

## *4.3.3. The Hippo pathway*

IHH regulates intestinal SC fates by interfering with the maturation and localization of the underlying stromal cells that in turn generate signaling molecules needed for the maintenance of the intestinal SC niche [56] (**Figure 2**). IHH, expressed by differentiated enterocytes, indirectly inhibits Wnt signaling at the crypt base and reduces the number of proliferating precursor cells [17, 57]. A decrease in Hh signaling is correlated with the expansion of the intestinal SC pool, with blunted enterocyte differentiation and activation of the Wnt pathway. Moreover, *IHH* gene knock-out leads to intestinal SC accumulation [57]. In addition, specific Hh activation in murine stromal cells induces complex transcriptional changes, leading to loss of colon SC-specific gene expression and upregulation of epithelial differentiation markers [58]. Most of the components of the Hh signaling pathway are upregulated (mRNA and protein) in CRC, with the exception of IHH that appears to be downregulated. Overexpression of members of the Hh signaling pathway is associated with poor survival and adverse clinical features [59]. However, in metastatic CRC, treatment with vismodegib, an Hh pathway inhibitor, in combination with standard chemotherapy, does not significantly improve patient survival [60].

282 Gene Expression and Regulation in Mammalian Cells - Transcription Toward the Establishment of Novel Therapeutics

The BMP pathway regulates many cellular mechanisms, including apoptosis and cell growth, depending on the specific cellular context. BMP ligands are secreted in their active form and homodimerize before binding to their cognate BMP receptors (BMPR). SMAD transcription factors are the main downstream effectors of BMP signaling that plays key roles in adult gut

Specific inhibition of BMP signaling in intestinal epithelial cell does not lead to initiation of colon tumors *in vivo*, while suppression in mesenchymal myofibroblasts is associated with spontaneous tumor formation. This suggests that inhibition of BMP signaling in the mesenchymal cells surrounding the intestinal epithelium acts as a trigger of gastrointestinal tumorigenesis [61]. Moreover, BMP4 expression is lost in intestinal CSCs, leading to deregulation of the

Nevertheless, it is still unclear whether BMP limits expansion of intestinal epithelial cells by repressing LGR5+ intestinal SC self-renewal or by inhibiting epithelial cell proliferation. In addition, BMP type Ia receptor (*Bmpr1a*) conditional knock-out in the intestinal epithelium leads to intestine hyperplasia with multiple intestinal polyps due to hyperactive SCs [63]. Moreover, in these mice, the LGR5+ SC pool is enlarged due to increased survival, allowing better intestinal regeneration [63]. Among the BMP family members, BMP2 and BMP4 are specifically involved in intestinal CSC regulation by promoting their differentiation and antagonizing Wnt/β-catenin signaling [64]. Furthermore, a recent study showed that the transcription factor GATA6 is a key regulator of CSC expansion and self-renewal through downregulation of BMP genes [65].

Finally, mutations that affect BMP signaling are frequently observed in patients with juvenile polyposis syndrome that is characterized by non-cancerous polyps, as well as in patients with progressing CRC. Analysis of SMAD4 expression levels in patients with CRC showed that it

is downregulated in CRC and associated with poor prognosis [66].

*4.3.2. The BMP pathway*

homeostasis, inflammation and cancer.

proliferative compartment [62].

The Hippo pathway regulates various cellular processes, including cell survival, proliferation and differentiation, but has been involved only recently in SC biology [67]. Yes-associated protein (YAP) and transcriptional co-activator with PDZ binding motif (TAZ) are the central effector molecules of this signaling cascade and are abundantly expressed in the cytoplasm of both proliferating and post-mitotic cells [17, 68].

In cancer, the Hippo pathway inhibits cell proliferation, promotes apoptosis and regulates stem/ progenitor cell expansion. In cancer cells, YAP and TAZ are localized mainly in the nucleus and promotes cell and tumor growth. There is considerable evidence that abnormal Hippo signaling is associated with tumor progression and YAP/TAZ overexpression is frequently observed in CRC [67]. This overexpression could be linked to Wnt/β-catenin over-activation because YAP is a specific target of this pathway. Furthermore, the major components of the Hippo pathway (i.e., MST1/2 and MOBKL1A/B) that control YAP/TAZ activity display low expression levels in colon carcinomas [69]. YAP deletion in *Apcmin* mice prevents polyp formation and blocks the differentiation of *Apc−/−* organoids. Moreover, using a mosaic model of *Yap* and *Apc* gene deletion in intestinal SCs, YAP appears to be dispensable for tumor initiation, but crucial for progression of tumor-initiating cells to adenoma [70].

Hippo pathway dysregulation, leading to loss of YAP repression, has been observed in different cancer types [71]. In patients with CRC, YAP over-activation is closely related to β-catenin over-activation. Moreover, the tyrosine kinase c-Yes is hyper-phosphorylated in 5-fluorouracilresistant cells with CSC features, thus preventing YAP nuclear translocation [72]. Finally, *YES1* and *YAP* levels are correlated with worse prognosis in chemotherapy-treated patients with CRC, suggesting that chemotherapy favors the selection of intestinal CSCs with deregulated c-Yes and YAP [72].

## **4.4. Other intestinal CSC-related transcription factors**

## *4.4.1. PXR*

Pregnane X Receptor (PXR, NR1I2), a member of the nuclear receptor superfamily, is highly expressed in the colon. PXR targets are genes that encode phase I and II metabolic enzymes and phase III drug transporters. Members of the nuclear receptor superfamily function as ligand-activated transcription factors and play critical roles in nearly every aspect of development and adult physiology [73]. Interestingly, it has been reported that the Wnt/β-catenin signaling pathway is crucial for PXR activity and notably that β-catenin is required for PXRmediated induction of target gene expression [74].

Planque et al. have recently demonstrated that PXR is a potent intestinal CSC phenotype driver by regulating a network of downstream genes involved in self-renewal and chemoresistance [75]. PXR expression is associated with CSC enrichment, after cell sorting of cancer cells using ALDH activity to identify CSCs and after spheroid passaging. In addition, expression of CSC markers and self-renewal are increased in CRC cells with enhanced PXR transcriptional activity [75]. PXR expression in intestinal CSCs is also associated with tumor aggressiveness and chemoresistance [76]. Specifically, PXR increases the oxaliplatin efflux capacity of cancer cells, thus reducing the cell drug concentration and preventing its effects on cell proliferation and apoptosis [76]. Another study demonstrated that PXR is a master regulator of chemoresistance by regulating genes involved in drug resistance, such as cytochrome P450, multidrug resistance 1 and multidrug resistance-associated protein 2 [77]. Furthermore, PXR is associated with poor survival, particularly after drug treatment. Indeed, in patients with CRC, it allows clonal selection after treatment, leading to the emergence of resistant and more aggressive clones with molecular signatures of poor prognosis [75, 77].

**5. Conclusion**

Cancer management is one of the major issues in our society and therefore, much research is focused on improving our understanding of cancer development and progression. Here, we presented an overview of the transcriptional dysregulation that affect intestinal epithelium homeostasis and that can lead to tumor initiation and development. In the last decade, considerable progress has been made in understanding the molecular and cellular mechanisms linked to CRC development/progression and a major breakthrough was the identification of cells with CSC properties. Studies in mouse models have shown that CRC development is mainly supported by intestinal CSCs that can self-renew and generate tumor cell heterogeneity even after *in vitro* or *in vivo* passaging. However, CSCs do not cycle as fast as cancer cells. This means that the current therapies that target cycling cancer cells are not efficient against the relatively quiescent CSCs.

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CSC fate and properties are regulated through a wide transcriptional network controlled by

**Figure 3.** Transcriptional landscape associated with the CSC phenotype in CRC. Schematic representation of the positive (arrows) and negative (bar-ended arrows) regulations between transcription factors and signaling pathways and

associated with the CSC phenotype within the tumor. ISEM = intestinal subepithelial myofibroblasts.

signaling cascades that often crosstalk and regulate each other (**Figure 3**).

#### *4.4.2. HOPX*

The homeodomain-only protein homeobox (HOPX) is strongly expressed in normal colorectal mucosa, and is considered a marker of the +4 SC population in the intestine [78]. Conversely, HOPX-β (an isoform of HOPX) represses conversion to the CBC phenotype in +4 SCs in physiological contexts in mice [78].

HOPX shows tumor suppressor functions in CRC by regulating cell proliferation and inhibiting angiogenesis [79]. Microarray data analysis revealed that, in CRC samples, HOPX downregulates oncoproteins, such as c-FOS and EGR-1. Moreover, EphA2 (which increases tumor invasion and survival) is overexpressed in patients with *HOPX* gene hypermethylation. In addition, HOPX-β promoter is frequently hypermethylated in CRC cell lines and tissues. This methylation results in the downregulation of HOPX mRNA and protein levels. Importantly, in patients with stage III CRC, HOPX-β promoter hypermethylation is associated with worse prognosis [79]. Moreover, in patients with CRC, *HOPX* gene hypermethylation is accompanied by increased expression of Cyr61/CCN1, a critical downstream member of the Hh signaling pathway that affects the pro-angiogenic tumor microenvironment [80].

#### *4.4.3. Sp1*

Specificity protein 1 (Sp1) is a transcription factor ubiquitously expressed in mammalian cells that recruits the basal transcription machinery. Sp1 is active in all cell types, but it is also tightly regulated because Sp1 activity can alter the expression of genes involved in cell cycle and growth (including many tumor suppressor genes and oncogenes) in response to signaling pathways and specific cellular conditions [81].

Interestingly, Sp1 levels are higher in colon CSCs than in the parental tumor cells [82]. Moreover, siRNA-mediated *SP1* silencing suppresses the specific features of CSCs derived from CRC cells and promotes apoptosis of colon CSCs *in vitro* [82]. *SP1* silencing also decreases the expression of several CSC markers. Hence, colon CSC self-renewal ability, drug resistance and metastasis potential could be partially related to high Sp1 expression. In agreement, Sp1 overexpression correlates with tumor stage and poor prognosis [81].

## **5. Conclusion**

markers and self-renewal are increased in CRC cells with enhanced PXR transcriptional activity [75]. PXR expression in intestinal CSCs is also associated with tumor aggressiveness and chemoresistance [76]. Specifically, PXR increases the oxaliplatin efflux capacity of cancer cells, thus reducing the cell drug concentration and preventing its effects on cell proliferation and apoptosis [76]. Another study demonstrated that PXR is a master regulator of chemoresistance by regulating genes involved in drug resistance, such as cytochrome P450, multidrug resistance 1 and multidrug resistance-associated protein 2 [77]. Furthermore, PXR is associated with poor survival, particularly after drug treatment. Indeed, in patients with CRC, it allows clonal selection after treatment, leading to the emergence of resistant and more aggressive clones with molecular

284 Gene Expression and Regulation in Mammalian Cells - Transcription Toward the Establishment of Novel Therapeutics

The homeodomain-only protein homeobox (HOPX) is strongly expressed in normal colorectal mucosa, and is considered a marker of the +4 SC population in the intestine [78]. Conversely, HOPX-β (an isoform of HOPX) represses conversion to the CBC phenotype in +4 SCs in

HOPX shows tumor suppressor functions in CRC by regulating cell proliferation and inhibiting angiogenesis [79]. Microarray data analysis revealed that, in CRC samples, HOPX downregulates oncoproteins, such as c-FOS and EGR-1. Moreover, EphA2 (which increases tumor invasion and survival) is overexpressed in patients with *HOPX* gene hypermethylation. In addition, HOPX-β promoter is frequently hypermethylated in CRC cell lines and tissues. This methylation results in the downregulation of HOPX mRNA and protein levels. Importantly, in patients with stage III CRC, HOPX-β promoter hypermethylation is associated with worse prognosis [79]. Moreover, in patients with CRC, *HOPX* gene hypermethylation is accompanied by increased expression of Cyr61/CCN1, a critical downstream member of the Hh signaling pathway that affects the pro-angiogenic tumor

Specificity protein 1 (Sp1) is a transcription factor ubiquitously expressed in mammalian cells that recruits the basal transcription machinery. Sp1 is active in all cell types, but it is also tightly regulated because Sp1 activity can alter the expression of genes involved in cell cycle and growth (including many tumor suppressor genes and oncogenes) in response to signal-

Interestingly, Sp1 levels are higher in colon CSCs than in the parental tumor cells [82]. Moreover, siRNA-mediated *SP1* silencing suppresses the specific features of CSCs derived from CRC cells and promotes apoptosis of colon CSCs *in vitro* [82]. *SP1* silencing also decreases the expression of several CSC markers. Hence, colon CSC self-renewal ability, drug resistance and metastasis potential could be partially related to high Sp1 expression. In agreement, Sp1

signatures of poor prognosis [75, 77].

physiological contexts in mice [78].

microenvironment [80].

ing pathways and specific cellular conditions [81].

overexpression correlates with tumor stage and poor prognosis [81].

*4.4.3. Sp1*

*4.4.2. HOPX*

Cancer management is one of the major issues in our society and therefore, much research is focused on improving our understanding of cancer development and progression. Here, we presented an overview of the transcriptional dysregulation that affect intestinal epithelium homeostasis and that can lead to tumor initiation and development. In the last decade, considerable progress has been made in understanding the molecular and cellular mechanisms linked to CRC development/progression and a major breakthrough was the identification of cells with CSC properties. Studies in mouse models have shown that CRC development is mainly supported by intestinal CSCs that can self-renew and generate tumor cell heterogeneity even after *in vitro* or *in vivo* passaging. However, CSCs do not cycle as fast as cancer cells. This means that the current therapies that target cycling cancer cells are not efficient against the relatively quiescent CSCs.

CSC fate and properties are regulated through a wide transcriptional network controlled by signaling cascades that often crosstalk and regulate each other (**Figure 3**).

**Figure 3.** Transcriptional landscape associated with the CSC phenotype in CRC. Schematic representation of the positive (arrows) and negative (bar-ended arrows) regulations between transcription factors and signaling pathways and associated with the CSC phenotype within the tumor. ISEM = intestinal subepithelial myofibroblasts.

In this review, we focused on some of these transcription factors and major signaling pathways involved in the regulation of the intestinal CSC phenotype and in CRC development. The basis of CRC development is the over-activation of the Wnt/β-catenin signaling cascade. Then, the disruption of other signaling pathways potentiates the oncogenic process by maintaining or even amplifying these alterations. Similarly, mutations or altered expression of different transcription factors also contribute to the oncogenic network. All these mechanisms concur to promote tumor growth and aggressiveness due to CSC enrichment. Moreover, some of these pathways and transcription factors might confer chemoresistance to the CSC population and are involved in CRC relapse (**Table 1**). Therefore, they are considered poor prognostic markers. Consequently, effective CRC therapies should target not only the highly proliferative cancer cells but also colon CSCs, or sensitize them to therapies. These different signaling pathways and their downstream

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effectors could represent biomarkers of CRC progression and therapeutic targets.

**Abbreviations**

ACF Aberrant crypt foci

ALDH Aldehyde dehydrogenase

APC Adenomatous polyposis coli

BMP4 Bone morphogenetic protein 4

CBC Crypt-based columnar

CRC Colorectal cancer CSCs Cancer stem cells

CCICs Colon cancer-initiating cells

BMPR Bone morphogenetic protein receptor

CGH Comparative growth hybridization

Cyr61 Cysteine-rich angiogenic inducer 61

EGR1 Early growth factor response 1

ASCL2 Achaete-scute family bHLH transcription factor 2 BMI1 B lymphoma Mo-MLV insertion region 1 homolog

To conclude, these data do not give the solution on how to cure CRC, but help understanding why its management is not simple. Several topics presented in this review are field of active research. Indeed, there are multiple and complex interactions between key signaling pathways known to control SC behavior. The knowledge on the transcriptional networks that control intestinal CSCs is not complete yet, and some findings are controversial. A better characterization and comprehension of these regulatory mechanisms, notably through network analysis, are needed to identify new therapeutic targets in order to improve patient care.

**Table 1.** Phenotypic outcomes associated with the different transcription factors in normal and tumoral intestinal epithelium.

In this review, we focused on some of these transcription factors and major signaling pathways involved in the regulation of the intestinal CSC phenotype and in CRC development. The basis of CRC development is the over-activation of the Wnt/β-catenin signaling cascade. Then, the disruption of other signaling pathways potentiates the oncogenic process by maintaining or even amplifying these alterations. Similarly, mutations or altered expression of different transcription factors also contribute to the oncogenic network. All these mechanisms concur to promote tumor growth and aggressiveness due to CSC enrichment. Moreover, some of these pathways and transcription factors might confer chemoresistance to the CSC population and are involved in CRC relapse (**Table 1**). Therefore, they are considered poor prognostic markers. Consequently, effective CRC therapies should target not only the highly proliferative cancer cells but also colon CSCs, or sensitize them to therapies. These different signaling pathways and their downstream effectors could represent biomarkers of CRC progression and therapeutic targets.

To conclude, these data do not give the solution on how to cure CRC, but help understanding why its management is not simple. Several topics presented in this review are field of active research. Indeed, there are multiple and complex interactions between key signaling pathways known to control SC behavior. The knowledge on the transcriptional networks that control intestinal CSCs is not complete yet, and some findings are controversial. A better characterization and comprehension of these regulatory mechanisms, notably through network analysis, are needed to identify new therapeutic targets in order to improve patient care.

## **Abbreviations**

**Transcription factors**

SOX9 Differentiation of

KLF4 Differentiation of

KLF5 Promotion of cell

BMI1 Promotion of SC

IHH Differentiation of

SHH Promotion of cell

SMAD Differentiation of

YAP/TAZ Promotion of cell

PXR Increase of

Paneth cells Promotion of SC proliferation

Goblet cells

proliferation

proliferation and renewal Prevention of senescence DNA damage repair

enterocyte cells Inhibition of cell proliferation

proliferation

enterocyte cells Inhibition of Lgr5+ SC expansion

proliferation

repair

HOPX Maintenance of +4 SC identity

SP1 Cell cycle and

epithelium.

cholesterol uptake Promotion of intestinal epithelial wound healing and

growth control

**Physiological roles Status in CRC CRC-associated**

286 Gene Expression and Regulation in Mammalian Cells - Transcription Toward the Establishment of Novel Therapeutics

**phenotype**

Apoptosis bypassing undifferentiated

Overexpression Cell cycle progression

Low expression Increased DNA

Overexpression Promotion of cell

Overexpression Tumor initiation

Low expression Expansion of the

Overexpression Promotion of CRC

Low expression Promotion of cell

Overexpression CSC renewal ability

**Table 1.** Phenotypic outcomes associated with the different transcription factors in normal and tumoral intestinal

Overexpression

and over-activation cells

CSC pool Promotion of cell proliferation

development

CSC self-renewal Drug resistance

proliferation Promotion of angiogenesis

Drug resistance Metastasis potential

Low expression Poor [61, 66]

Tumor progression Poor [67, 70, 72]

state

synthesis Uncontrolled cell proliferation CSC-like phenotype

proliferation Increase of cell survival capacities

Self-renewal of CRC

Promotion of cell invasion and migration

**Prognosis References**

No correlation [23–27, 31]

Poor [32, 33, 36, 39]

Poor [31, 49–51,

Poor [56, 59]

Poor [73, 75, 77]

Poor [78, 79]

Poor [81, 82]

53–55]

[17, 56, 57]

No correlation [41]

**Author details**

**References**

274-284

Antoine Gleizes1,2,3,4, Vincent Cavaillès1,2,3,4\* and Marion Lapierre1,2,3,4

4 Institut Régional du Cancer de Montpellier, Montpellier, France

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3 Université de Montpellier, Montpellier, France

## **Author details**

EphA2 Ephrin receptor A2

HH Hedgehog

GATA6 GATA binding protein 6

HMG High-mobility group

IHH Indian hedgehog

KLF Kruppel-like factor

LEF Lymphoid enhancer factor

MYC Myelocytomatosis oncogene NICD Notch intracellular domain

PXR Pregnane X receptor

NOG Noggin

SC Stem cells

SHH Sonic hedgehog

SMAD Mother against Dpp