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. 2010 Nov 1;10(9):893–902. doi: 10.4161/cbt.10.9.13252

Gli3 mediates cell survival and sensitivity to cyclopamine in pancreatic cancer

Adam Steg 1, Hope M Amm 1, Zdenek Novak 2, Andra R Frost 3, Martin R Johnson 1,4,✉
PMCID: PMC3230294  PMID: 20814245

Abstract

Activation of the hedgehog (HH) pathway plays a critical role in the development and continued growth of pancreatic adenocarcinoma (PA C). Cyclopamine, a HH pathway inhibitor, has been shown to suppress PA C cell proliferation in vitro and in vivo. However, the molecular basis of response to cyclopamine has not been fully elucidated nor have genes that predict sensitivity to this compound been identified. To better understand these features of HH pathway inhibition, we evaluated the biological and molecular effects of cyclopamine in vitro. The viability of nine human PA C cell lines following cyclopamine exposure was determined using MTS assay. Proliferation and induction of apoptosis in treated cells were examined by bromo-deoxyuridine incorporation, caspase activation and mitochondrial membrane potential. Gene expression before and after cyclopamine treatment was determined using Taqman real-time quantitative polymerase chain reaction (RTQ-PCR) and Taqman low-density array (TLDA). Among the cell lines examined, cyclopamine IC50 values ranged from 8.79 to > 30 µM. Response to cyclopamine included reduced cell proliferation and induction of apoptosis with and without mitochondrial membrane depolarization. Regression analysis revealed that GLI3 expression significantly correlated with cyclopamine resistance (r = 0.80; p = 0.0102). Knockdown of GLI3 using siRNAs increased sensitivity to cyclopamine. In addition, GLI3 siRNAs decreased PA C cell viability and reduced expression of genes involved in HH signaling (Patched 1 and GLI1) and cell proliferation, similar to cyclopamine. These effects were not observed in PAC cells with undetectable GLI3 expression. These data suggest that Gli3 mediates cell survival and sensitivity to cyclopamine in pancreatic cancer.

Key words: pancreatic adenocarcinoma, hedgehog, cyclopamine, gli3

Introduction

Pancreatic adenocarcinoma (PAC) is the fourth leading cause of cancer mortality in the United States and is characterized by an unusual resistance to radiation and chemotherapy. Surgical resection remains the most effective treatment for PAC. However, at the time of diagnosis, up to 90% of patients present with advanced (unresectable) disease.1 Chemotherapy agents such as 5-fluorouracil (5-FU) and, more recently, gemcitabine combined with radiation (50–60 Gy) is the current treatment for locally advanced PAC.2–5 Despite highly aggressive therapeutic approaches, the median survival time (6–10 months) for patients with this disease has not appreciably changed in the last 80 years.2,3,6 These dismal statistics indicate a dire need for the development of novel therapeutic targets and treatment strategies.

Recent studies suggest that activation of the hedgehog (HH) signaling pathway is a potential mediator of pancreatic carcinogenesis and sustained tumor growth.7,8 The HH pathway, a critical component of embryogenesis, is comprised of a family of highly regulated proteins that direct cell development and proliferation. Binding of a secreted ligand (Sonic, Indian or Desert HH) to the Patched (Ptch) receptor reverses the inhibition of Ptch on Smoothened (Smo), a transmembrane protein. This results in a signaling cascade, leading to the translocation of the active forms of glioma-associated oncogene (Gli) transcription factors (Gli1, 2 and 3) to the nucleus.9 The Gli homologues have distinct but overlapping functions; Gli1 serves only as a transcriptional activator, whereas Gli2 and Gli3 are capable of both activating and repressing transcription of HH target genes, including PTCH1 and GLI1, the expression of which are frequently measured to evaluate the presence or absence of HH pathway activity.10,11 While a large amount of research has been devoted to studying Gli1 and Gli2, the role of Gli3 in cancer has remained largely unexplored. First identified by Ruppert et al.12 in 1990, studies of GLI3 have focused on the disorders that result from mutations in this gene including the Pallister-Hall and Greig cephalopolysyndactyly syndromes, both of which are characterized by the appearance of extra fingers and toes and abnormal organ development in humans.13,14 Gli3, therefore, plays a critical role in mammalian development and may be involved in cancer formation and maintenance.

The involvement of HH signaling in PAC was first identified when it was found that ectopic expression of Sonic HH in the pancreatic endoderm of transgenic mice resulted in the formation of pancreatic intraepithelial neoplasia (PanIN)-like lesions.7 The histological progression of pancreatic neoplasia in these transgenic mice was accompanied by the induction of HER-2/neu expression and mutations of the proto-oncogene K-ras—genetic alterations that are frequently characterized as early events in PAC tumor etiology.15–17 Immunohistochemical analysis of Sonic HH, Ptch1 and Smo in clinical PAC specimens suggested that expression of these HH signaling components progressively increases from PanIN lesions to adenocarcinomas.7 Studies performed by our laboratory have also demonstrated increased expression of HH signaling components (including Ptch1, Smo and Gli1) in PAC at the mRNA and protein level.18 Taken collectively, these studies suggest that the HH pathway is involved in pancreatic tumor development and remains aberrantly expressed in PAC. The mechanism by which HH signaling contributes to sustained tumor growth has been reported to be either autocrine (within epithelial cancer cells)7,19,20 or paracrine (within the tumor stroma).21

Cyclopamine is a steroidal alkaloid found in the lily plant Veratrum californicum that inactivates HH signaling by antagonizing Smo function.22–24 Cyclopamine has demonstrated significant anti-cancer effects both in vitro and in vivo in models of medulloblastoma, prostate cancer and pancreatic cancer.7,25,26 In addition to the naturally occurring cyclopamine, several other Smo antagonists have been synthesized, including CUR199691, which has been shown to be effective against basal cell carcinoma and medulloblastoma in vivo.27–29 Mice treated with these compounds show little evidence of adverse side effects. These results suggest that inhibition of the HH pathway shows promise as an effective anti-cancer strategy that could be used for future clinical application.

In the current study, we sought to better understand the molecular basis of response to cyclopamine and to identify genes that are associated with this response. To this end, we examined the biological and molecular effects of cyclopamine on human pancreatic cancer cell lines. Differential response to cyclopamine was observed among the cell lines examined and it was this result that ultimately allowed us to identify genes associated with innate sensitivity or resistance to this compound. By comparing gene expression prior to cyclopamine treatment with IC50 values, we found that GLI3 significantly correlated with cyclopamine resistance in vitro. To our knowledge, this is the first study to identify Gli3 as a potential mediator of response to Smo antagonists.

Results

Response to cyclopamine varies among human PAC cell lines.

Initial studies demonstrated that cyclopamine decreased pancreatic cancer cell viability in a dose-dependent manner with variable sensitivity observed among a panel of nine PAC cell lines. As shown in Table 1, HPAF-2 cells (IC50 = 8.79 µM) showed the greatest sensitivity to cyclopamine while S2-013 cells (IC50 = 45.09 µM, approximated from linear regression) demonstrated the least sensitivity to cyclopamine. Tomatidine, an inactive analog of cyclopamine, had no significant effect on the viability of any of the cell lines examined (data not shown).

Table 1.

Response to cyclopamine in human pancreatic cancer cell lines

Cell line IC50 (µM)*
HPAF-2 8.79 ± 0.94
Panc 10.05 11.33 ± 0.41
Panc 8.13 14.49 ± 0.85
Panc 2.03 16.57 ± 0.27
AsPC-1 16.74 ± 1.30
CFPAC-1 19.59 ± 0.32
Panc-1 31.41 ± 0.78†
BxPC-3 36.17 ± 0.31†
S2013 45.09 ± 1.27†
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*

Average of four independent experiments ± SEM.

†

IC50 values > 30 µM were estimated by extending linear regression analysis.

Cyclopamine decreases proliferation and induces apoptosis in PAC cell lines.

To identify potential mechanisms involved in decreased cell viability following cyclopamine treatment, we examined changes in both BrdU incorporation (cell proliferation) and caspase cleavage (apoptosis) in sensitive and resistant PAC cell lines. As shown in Figure 1A, 8 µM cyclopamine (the IC50 of HPAF-2 cells) significantly (p < 0.001) reduced BrdU labeling in HPAF-2 cells by 83% relative to vehicle control. BrdU labeling of Panc-1 cells, treated at the same concentration, was reduced by only 33% (p < 0.001) relative to vehicle control (Fig. 1B). Increasing the concentration of cyclopamine to 30 µM (the IC50 of Panc-1 cells) reduced BrdU-labeling by 54% (p < 0.001) (Fig. 1B). As shown in Figure 1C, cleavage (activation) of initiator caspases-8 and -9 and executioner caspase-3 as well as a decrease in the full-length form of Bid (hallmarks of intrinsic or mitochondrial-mediated apoptosis) were observed in HPAF-2 but not Panc-1 cells treated with 8 µM cyclopamine. Panc-1 cells exposed to 30 µM cyclopamine displayed modest cleavage of caspases-8 and -3, but no cleavage of caspase-9 or a reduction in Bid. These data suggest that cyclopamine decreases PAC cell viability through the mechanisms of both reduced cell proliferation and apoptosis, although the extent to which these biological effects occur is cell-line dependent.

Figure 1.

Figure 1

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Cyclopamine decreases proliferation of and induces apoptosis in PA C cell lines. BrdU incorporation was measured in HPA F-2 (A) and Panc-1 (B) cells exposed to either vehicle alone or cyclopamine. The percentage of BrdU-labeled cells was calculated relative to vehicle control. (C) HPA F-2 and Panc-1 cells were exposed to either vehicle alone or cyclopamine and examined for markers of apoptosis by western blot analysis. β-actin was used as a loading control. Data represent the average of four independent experiments. Error bars represent SD; ***p < 0.001.

Induction of mitochondrial membrane depolarization after cyclopamine treatment varies among PAC cell lines.

To determine if cyclopamine induces mitochondrial membrane depolarization (also an indicator of intrinsic apoptosis) in pancreatic cancer cell lines, HPAF-2 and Panc-1 cells were exposed to vehicle alone or cyclopamine (8 and 30 µM, respectively) and mitochondrial membrane potential was determined by °C-1 assay. Cyclopamine significantly reduced the mitochondrial membrane potential of HPAF-2 cells (54%, p < 0.001; Fig. 2A) but not Panc-1 cells (Fig. 2B) compared to vehicle control. These data, in agreement with the western blot analyses, suggest that cyclopamine treatment activates the intrinsic apoptotic pathway in HPAF-2 but not Panc-1 cells. This, in turn, further indicates that there is a molecular basis for the differential response to cyclopamine observed among PAC cell lines.

Figure 2.

Figure 2

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Cyclopamine induces mitochondrial membrane depolarization in HPA F-2 but not Panc-1 cells. Mitochondrial membrane potential was measured in HPA F-2 (A) and Panc-1 (B) cells exposed to either vehicle alone or cyclopamine using a JC-1 assay. The fluorescence ratio (red/green) of cyclopamine-treated cells was compared to that of the vehicletreated cells and calculated as a percent of control. Data represent the average of four independent experiments. Error bars represent SD; n.s., not statistically significant; ***p < 0.001.

Resistance to cyclopamine is associated with expression of GLI3 and knockdown of this gene increases sensitivity to cyclopamine and decreases PAC cell viability.

To identify a molecular basis for differential response to cyclopamine in vitro, a sample of each of the nine PAC cell lines was harvested for RNA extraction prior to cyclopamine treatment. The expression of genes in the HH pathway and downstream of the HH pathway (Sup. Table 1) was then examined in each cell line using TLDA analysis. Gene expression values were subsequently compared with cyclopamine IC50 values to identify genes that may be associated with innate sensitivity or resistance to this compound. We found that resistance to cyclopamine (increasing IC50 values) significantly correlated with increasing mRNA levels of SMO (r = 0.74; p = 0.0239), the target of this compound. Interestingly, we also found that expression of GLI3 significantly correlated with expression of SMO (r = 0.90; p = 0.0021) as well as resistance to cyclopamine (r = 0.80; p = 0.0102).

To further evaluate this association between GLI3 mRNA levels and cyclopamine resistance, we modulated GLI3 expression using two distinct siRNA sequences (GLI3 siRNA1 and 2) and examined the effect of this modulation on response to cyclopamine in vitro. HPAF-2 cells, which have no detectable SMO or GLI3 expression (CT = 40) and are sensitive to cyclopamine and Panc-1 cells, which express both SMO and GLI3 and are more resistant to cyclopamine, were selected for this analysis. As shown in Figure 3A, GLI3 siRNA1 and 2 significantly reduced GLI3 expression by 87 and 92% (p < 0.001), respectively, in comparison to siRNA control. This knockdown significantly increased the sensitivity of Panc-1 cells to cyclopamine (Fig. 3B, right of dashed line). A cyclopamine IC50 value of ∼29 µM was determined for siRNA control-transfected cells whereas GLI3 siRNA1 and 2-transfected cells had cyclopamine IC50 values of ∼13 and 9 µM, respectively. Interestingly, we found that knockdown of GLI3 expression alone (i.e., in the absence of cyclopamine) led to a significant decrease in Panc-1 cell viability in comparison to siRNA control (Fig. 3B, left of dashed line). GLI3 siRNA1 and 2 reduced Panc-1 cell viability by 24 and 34% (p < 0.001), respectively. GLI3 siRNAs had no effect on cyclopamine response or the viability of HPAF-2 cells (Sup. Fig. 1). Similar to that observed with cyclopamine, the reduction in Panc-1 cell viability following GLI3 knockdown was not associated with a significant decrease in mitochondrial membrane potential (Sup. Fig. 2). These data initially suggest that Gli3 is involved in differential response to cyclopamine in PAC cell lines and that Gli3 plays a role in maintaining PAC cell viability.

Figure 3.

Figure 3

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GLI3 knockdown decreases cell viability and increases sensitivity to cyclopamine in Panc-1 cells. (A) GLI3 expression was measured in Panc-1 cells transfected with siRNA control or GLI3 siRNAs using RTQ-PCR. Gene expression was quantified 96 hours post-transfection to determine whether GLI3 mRNA levels would remain knocked down throughout cyclopamine treatment. Gene expression was calculated relative to siRNA control. (B) Panc-1 cells were transfected with siRNA control or GLI3 siRNAs and then exposed to either vehicle alone or increasing concentrations of cyclopamine (8, 15 and 30 µM) for 96 hours. Cell viability was determined using MTS assay and calculated relative to the vehicle + siRNA control group. Data represent the average of four independent experiments. Error bars represent SD; ***p < 0.001; †represents significant (p < 0.001) differences between GLI3 siRNA- and siRNA control-transfected cells among cyclopamine-treated groups.

Cyclopamine and GLI3 knockdown reduce HH transcriptional activity in Panc-1 cells but not HPAF-2 cells.

To determine if the reductions in PAC cell viability observed after cyclopamine treatment alone and GLI3 knockdown alone are similarly mediated through inhibition of the HH pathway, we evaluated gene expression of PTCH1 and GLI1 in treated HPAF-2 and Panc-1 cells. As shown in Figure 4A, cyclopamine (8 µM) did not reduce the expression of PTCH1 and GLI1 in HPAF-2 cells, indicating that this cell line may respond to cyclopamine by a mechanism independent of HH pathway inhibition. Conversely, cyclopamine did reduce the expression of these genes in a dose-dependent manner in Panc-1 cells (Fig. 4B). In addition to PTCH1 and GLI1, cyclopamine also reduced GLI3 gene expression in a dose-dependent manner in Panc-1 cells (Fig. 4B). A maximum decrease of 67% (p < 0.05) was achieved after exposure to 30 µM cyclopamine. Cyclopamine-induced changes in gene expression were found to be time-dependent with maximum decreases in expression resulting after 96 hours of treatment (Sup. Fig. 3). To determine if a second Smo antagonist, CUR199691, has similar molecular effects to cyclopamine, we examined the expression of PTCH1, GLI1 and GLI3 in Panc-1 cells after exposure to this compound. Similar to cyclopamine, CUR199691 reduced the expression of all three genes in a dose-dependent manner (Sup. Fig. 4A). A maximum decrease of 67, 51 and 58% (all p < 0.01) was achieved for PTCH1, GLI1 and GLI3 expression, respectively, after exposure to CUR199691 relative to vehicle control. These changes in gene expression coincided with a dose-dependent reduction in Panc-1 cell viability, similar to that observed with cyclopamine (Sup. Fig. 4B).

Figure 4.

Figure 4

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Cyclopamine and GLI3 knockdown reduce HH transcriptional activity in Panc-1 cells but not HPA F-2 cells. HPA F-2 (A) and Panc-1 (B) cells were exposed to either vehicle alone or cyclopamine for 96 hours and examined for changes in the expression of PTCH1, GLI1 and GLI3 using RTQ-PCR. Gene expression was calculated relative to vehicle control. PTCH1 and GLI1 mRNA levels were quantified in HPA F-2 (C) and Panc-1 (D) cells transfected with either siRNA control or GLI3 siRNAs using RTQ-PCR. Gene expression was calculated relative to siRNA control. Data represent the average of four independent experiments. Error bars represent SD; n.s., not statistically significant; *p < 0.05; **p < 0.01.

The decrease in GLI3 expression after cyclopamine and CUR199691 treatment and the reduction in cell viability observed after GLI3 knockdown led us to examine whether Gli3 could independently regulate HH transcriptional activity (as determined by PTCH1 and GLI1 expression). As expected, PTCH1 and GLI1 mRNA levels were not significantly changed as a result of GLI3 siRNAs in HPAF-2 cells, which do not express GLI3 (Fig. 4C). However, selective knockdown of GLI3 in Panc-1 cells led to a reduction in PTCH1 and GLI1 mRNA levels in comparison to siRNA control. GLI3 siRNA1 reduced PTCH1 and GLI1 expression by 51% (p < 0.01) and 26% (p = 0.0619), respectively and GLI3 siRNA2 reduced PTCH1 and GLI1 expression by 57% (p < 0.01) and 39% (p < 0.01), respectively (Fig. 4D). These data further suggest that Gli3 plays an important role in differential response to cyclopamine in PAC cell lines. Moreover, Gli3 appears to regulate HH transcriptional activity and, by extenstion, mediate response to HH pathway inhibition.

Cyclopamine and GLI3 knockdown similarly affect the expression of genes involved in HH signaling and cell proliferation in Panc-1 cells.

To further examine how Gli3 might contribute to the molecular basis of response to cyclopamine, we quantified the expression of 46 genes involved in HH signaling and cell proliferation (Sup. Table 1) in HPAF-2 and Panc-1 cells after either GLI3 siRNA or cyclopamine treatment using TLDA. As shown in Figure 5A, the expression of GLI3, GLI2, Engrailed 1, E2F1, Cyclin D3, NOTCH2, PTCH1, NOTCH1, GLI1 and Cyclin E1 was significantly (p < 0.05) decreased as a result of both GLI3 knockdown and cyclopamine treatment in Panc-1 cells. None of these genes were significantly changed in HPAF-2 cells following GLI3 siRNA transfection; however, the expression of GLI2, E2F1, Cyclin D3 and Cyclin E1 was significantly reduced after cyclopamine treatment in these cells (Fig. 5B). Whereas GLI3, Engrailed 1, NOTCH2, PTCH1, NOTCH1 and GLI1 were unchanged in HPAF-2 cells exposed to either GLI3 siRNA or cyclopamine, suggesting these genes are involved in a Gli3-mediated response to cyclopamine, a mechanism present in Panc-1 cells.

Figure 5.

Figure 5

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Cyclopamine and GLI3 knockdown similarly affect the expression of genes involved in HH signaling and cell proliferation/differentiation. The expression of 46 genes was measured in HPA F-2 and Panc-1 cells exposed to either GLI3 siRNA or cyclopamine and controls using TLDA. (A) Ten of these 46 genes were significantly (p < 0.05) decreased in Panc-1 cells as a result of either GLI3 siRNA or cyclopamine treatment. (B) This concordance in gene expression changes was not observed in HPA F-2 cells. Gene expression was calculated relative to controls (vehicle or siRNA control). Data represent the average of independent experiments. Error bars represent SD.

Discussion

Initial studies demonstrated that cyclopamine decreased pancreatic cancer cell viability in a dose-dependent manner in vitro. Using BrdU incorporation and caspase cleavage, we found that cyclopamine mediated this effect through both decreased cell proliferation and apoptosis. Response to cyclopamine, however, varied among the PAC cell lines examined with IC50 values differing over 5-fold (from 8.79 to 45.09 µM, Table 1). While these are relatively high concentrations of cyclopamine (>10 µM), previous reports have shown that cyclopamine in excess of 25 µM is necessary to have a significant impact on the viability of many PAC cell lines.19,21 In addition, Panc-1 and BxPC-3 cells (whose IC50s exceeded 30 µM) have previously been identified as cyclopamine-resistant.7,19,43 As shown in Figure 1, decreased cell proliferation and apoptosis were more pronounced in cyclopamine-sensitive cells with activation of apoptotic pathways differing among PAC cell lines exposed to cyclopamine (Fig. 1C). Treated HPAF-2 cells (IC50 = 8 µM) demonstrated caspase-9 cleavage and a reduction in the full-length form of Bid in addition to cleavage of caspases-8 and -3, indicating these cells underwent intrinsic or mitochondrial-mediated apoptosis.44 In contrast, treated Panc-1 cells (IC50 = 30 µM) demonstrated only cleavage of caspases-8 and -3, indicating that these cells underwent apoptosis independent of mitochondrial involvement.44 The significant decrease in mitochondrial membrane potential observed in HPAF-2 cells, but not in Panc-1 cells, further suggests that HPAF-2 cells undergo intrinsic apoptosis as a result of cyclopamine treatment whereas Panc-1 cells do not (Fig. 2). Taken together, these differences in anti-proliferative and apoptotic mechanisms indicate there is an underlying molecular basis for differential response to cyclopamine.

In our study, differential response to cyclopamine among PAC cell lines provided us the opportunity to examine genes associated with innate sensitivity and/or resistance to HH pathway inhibition. By comparing cyclopamine IC50 values with gene expression, we found that SMO and GLI3 expression significantly correlated with increasing resistance to cyclopamine, suggesting that both play a role in mediating response to this compound. This is not surprising for Smo, since it is the target of cyclopamine;24 however, Gli3 has not been previously associated with response to HH pathway inhibition. In order to explore this possibility, GLI3 gene expression was knocked down using siRNAs and the effect on cyclopamine sensitivity was examined (Fig. 3). It was theorized that by decreasing the expression of GLI3, a marker of cyclopamine resistance, sensitivity to this compound would be enhanced. Surprisingly, GLI3 knockdown alone (i.e., in the absence of cyclopamine) significantly reduced Panc-1 cell viability [an effect that, similar to cyclopamine, occurred without mitochondrial membrane depolarization (Sup. Fig. 2)], suggesting that Gli3 may contribute to PAC cell survival. In addition, sensitivity to cyclopamine was, as predicted, significantly increased after GLI3 knockdown, further indicating that GLI3 expression is associated with cyclopamine resistance. How Gli3 potentially mediates this resistance remains to be fully understood. Because active HH signaling promotes the formation of the activator rather than the repressor form of Gli3,9,10 it could be speculated that PAC cells with more Gli3 have more HH pathway activity than PAC cells with little or no Gli3 (such as HPAF-2). This abundance of HH activity could, therefore, overcome or lessen the inhibitory effects of cyclopamine.

To determine if cyclopamine selectively inhibits HH pathway activity, we examined the expression of PTCH1 and GLI1 in treated HPAF-2 and Panc-1 cells (Fig. 4). Interestingly, we found that cyclopamine decreased the expression of PTCH1 and GLI1 in a dose-dependent manner in Panc-1 cells, but had no effect on the expression of these genes in HPAF-2 cells. These data would seem to indicate that the biological effects observed in Panc-1 cells, after cyclopamine treatment, result from selective inhibition of autocrine HH signaling, whereas those observed in HPAF-2 cells (including reduced cell proliferation and intrinsic apoptotic activation) appear to occur through a mechanism unrelated to HH pathway antagonism. A similar conclusion was made in a study performed by Zhang et al. which demonstrated that cyclopamine inhibited the growth of some breast cancer cell lines without antagonizing the HH pathway.45 It therefore becomes important to be able to delineate “on-target” and “off-target” effects of cyclopamine, particularly in cell lines that do not express Smo (e.g., HPAF-2), which, interestingly, tended to be more sensitive to cyclopamine in our study. In cell lines that do express Smo and respond to cyclopamine through HH pathway inhbition, such as Panc-1, the mechanism of this response remains largely undefined; however, it could be suggested that Gli3 plays a role in this phenomenon as well. In our study, we found that the expression of GLI3, like PTCH1 and GLI1, was decreased in a dose- and time-dependent manner after cyclopamine treatment in Panc-1 cells (Fig. 4B and Sup. Fig. 3). CUR199691, a second Smo antagonist, also decreased the expression of PTCH1, GLI1 and GLI3 in a dose-dependent manner in Panc-1 but not HPAF-2 cells (Sup. Fig. 4A, data not shown). Furthermore, knockdown of GLI3 alone led to a reduction in PTCH1 and GLI1 expression (Fig. 4D) and, similar to cyclopamine and CUR199691, these changes in gene expression coincided with a decrease in cell viability (Fig. 3B and Sup. Fig. 4B). These biological and molecular effects were absent in HPAF-2 cells transfected with GLI3 siRNAs (Sup. Fig. 1 and Fig. 4C). Taken together, these data indicate that GLI3 (and SMO) expression may actually be required for inhibition of the HH pathway to occur and that, in cells expressing GLI3, the mechanism of in vitro response to Smo antagonists involves Gli3 inhibition followed by a reduction in autocrine HH transcriptional activity (i.e., a decrease in PTCH1 and GLI1 expression), which ultimately leads to a decrease in cancer cell viability.

To further investigate how Gli3 inhibition potentially contributes to the molecular basis of response to cyclopamine, we examined the expression of 46 genes involved in HH signaling and cell growth and differentiation (Sup. Table 1) in HPAF-2 and Panc-1 cells after either GLI3 siRNA or cyclopamine exposure using TLDA analysis. We found that in Panc-1 cells, both GLI3 siRNA and cyclopamine significantly decreased the expression of 10 out of the 46 genes examined (Fig. 5A). In agreement with the RTQ-PCR results (Fig. 4B and D), GLI3, PTCH1 and GLI1 were among the 10 genes identified. The remaining genes include GLI2 (another member of the Gli family of transcription factors), members of the cell cycle (E2F1, Cyclin D3 and Cyclin E1) and key mediators of cell proliferation/differentiation (Engrailed 1, NOTCH1 and NOTCH2). A decrease in the expression of these genes could certainly contribute to the reduction in Panc-1 cell viability observed after GLI3 knockdown or cyclopamine treatment. These results further support the functional role that Gli3 plays in mediating in vitro response to cyclopamine both in terms of HH pathway antagonism and in terms of cell growth inhibition. It is important to note, however, that these molecular changes do not all appear to be unique to GLI3-expressing cells. As expected, GLI3 siRNA had no effect on gene expression in HPAF-2 cells; however, cyclopamine decreased the expression of GLI2, E2F1, Cyclin D3 and Cyclin E1 in these cells, leaving GLI3, PTCH1, GLI1, Engrailed 1, NOTCH1 and NOTCH2 unaffected (Fig. 5B). It could be speculated that the decrease in GLI2, E2F1, Cyclin D3 and Cyclin E1 expression contributes to the reduced HPAF-2 cell viability/proliferation observed after cyclopamine treatment. In addition, modulation of these genes may be part of the HH-independent mechanism of cyclopamine response in HPAF-2 cells, a mechanism defined by a lack of PTCH1 and GLI1 modulation (Fig. 4A). It is interesting to point out, however, that these genes are also decreased in Panc-1 cells that have been exposed to either GLI3 siRNA or cyclopamine, suggesting their modulation, in additition to the decrease in GLI3, PTCH1, GLI1, Engrailed 1, NOTCH1 and NOTCH2 expression, is part of a HH-dependent (rather than a HH-independent) mechanism in the GLI3-expressing Panc-1 cells. Therefore, another interpretation of this data could be that the gene expression changes in HPAF-2 are part of a non-canonical, but still HH-related mechanism of response to cyclopamine. It could be argued that this mechanism is mediated by Gli2 rather than Gli3 and that inhibition of Gli2 leads to transcriptional changes that do not include the established indicators of HH transcriptional activity, PTCH1 and GLI1. Future studies further examining this distinction between canonical and non-canonical and specific and non-specific effects of cyclopamine are warranted and could ultimately be useful in the design of more selective HH inhibitors.

Collectively, the data presented in this study demonstrate that cyclopamine reduces PAC cell viability through the mechanisms of both decreased proliferation and apoptosis and that Gli3 plays a critical role in the molecular basis of response to HH pathway antagonism. Moreover, Gli3 appears to mediate PAC cell survival, the importance of which becomes evident when considering that GLI3 gene expression was found by our laboratory to be significantly overexpressed (7.11-fold, p = 0.0011) in clinical PAC specimens in comparison to uninvolved pancreas.18 This suggests that expression of GLI3 is tumor-associated and that targeting this molecule could provide a useful therapeutic strategy for pancreatic cancer. Furthermore, variable expression of GLI3 among newly diagnosed pancreatic cancer patients could ultimately be used to help stratify these patients toward more effective anti-HH therapies.

Materials and Methods

Reagents and cell culture.

Cyclopamine and tomatidine were purchased from Toronto Research Chemicals (North York, Ontario, Canada). CUR199691 was purchased from Genentech Inc., (San Francisco, CA). Human pancreatic adenocarcinoma cell lines (AsPC-1, BxPC-3, CFPAC-1, HPAF-2, Panc-1, Panc 2.03, Panc 8.13 and Panc 10.05) were obtained from the American Tissue Culture Collection. S2-013, a metastatic subclone of the SUIT-2 human pancreatic cancer cell line,30 was kindly provided by Dr. Donald J. Buchsbaum's laboratory. AsPC-1, BxPC-3, Panc 2.03, Panc 8.13 and Panc 10.05 were grown in RPMI 1640 (Cellgro, Herndon, VA); CFPAC-1 was grown in Iscove's MEM (Cellgro); HPAF-2 was grown in Eagle's MEM (Cellgro); Panc-1 and S2-013 were grown in DMEM (Cellgro). All media was supplemented with 10% FBS (Cellgro).

Cell viability assays.

To test for in vitro response to cyclopamine, cells were cultured in triplicate (5,000 cells/well) in 96-well plates for 96 hours in control medium containing 0.5% FBS and vehicle (95% ethanol) alone or in the presence of cyclopamine at concentrations of 1, 2, 3.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5 and 30 µM. Cells were also exposed to tomatidine, a structural analog of cyclopamine that lacks the ability to inhibit HH signaling,31 under the same cell culture conditions. Cell viability was determined by optical density measurements at 490 nm using the CellTiter 96 (Promega, Madison, WI) MTS colorimetric assay. A dose-response curve was then created by comparing drug concentration versus cell viability (relative to vehicle control). For cyclopamine, linear regression analysis was performed to determine the concentration at which 50% inhibition of cell growth (IC50) was achieved. IC50 values were calculated as an average of 4 independent experiments (3 replicates per experiment).

Flow cytometric analysis.

Pancreatic cancer cells were seeded in 6-well plates (150,000 cells/well) and incubated for 96 hours in low-serum medium (0.5% FBS) containing vehicle alone or cyclopamine. To determine the effect of cyclopamine on cell proliferation, cells were incubated in the presence of 0.2 mg/ml 5-bromo-2′deoxyuridine (BrdU) (Calbiochem, San Diego, CA) for 1 hour. Cells were permeabilized, fixed, treated with DNase I (Roche Diagnostics, Penzberg, Germany) and stained with FITC conjugated anti-BrdU (mouse IgG1, Clone B44, BD Biosciences Immunocytometry Systems, San Jose, CA). Labeled cells were quantified by flow cytometry.

Western blot analysis.

Pancreatic cancer cells were seeded in 60 mm cell culture dishes (500,000 cells/dish) and incubated for 96 hours in low-serum medium containing vehicle alone or cyclopamine. Floating and attached cells were subsequently washed in PBS, pH 7.4, lysed, homogenized and centrifuged at 4°C for 10 min at 14,000 rpm to remove insoluble material. The protein concentration of the supernatant was measured by spectrophotometry using the Lowry DC protein assay method (Bio-Rad, Hercules, CA). A total of 25 µg of protein/lane was separated by SDS-polyacrylamide gel electrophoresis. After transfer to PVDF membranes, blots were incubated with a mouse monoclonal antibody to Caspase-8 (BD Pharmingen, San Diego, CA) and rabbit polyclonal antibodies to Caspase-3 (Stressgen, Ann Arbor, MI), Caspase-9 (Cell Signaling Technology, Danvers, MA), Bid (Cell Signaling Technology) and β-actin (Sigma, St. Louis, MO), which was used to monitor equal sample loading. After washing, blots were incubated with goat anti-mouse (for Caspase-8) or goat anti-rabbit (for Caspase-3, Caspase-9, Bid and β-actin) secondary antibodies (Bio-Rad) conjugated with horseradish peroxidase. Visualization was performed by the enhanced chemiluminescence method (Amersham Biosciences, Buckinghamshire, UK).

Mitochondrial membrane potential.

The mitochondrial membrane potential was assessed using JC-1 (5, 5, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolylcarbocyanine iodide), which is a cationic dye that accumulates in the mitochondrial membrane to form aggregates that fluoresce red. When the mitochondrial membrane potential is lost in apoptotic cells, the dye cannot aggregate and remains in its monomeric form, which fluoresces green. Pancreatic cancer cells were seeded in 60 mm cell culture dishes (500,000 cells/dish) and incubated for 96 hours in low-serum medium containing vehicle alone or cyclopamine. Following treatment, floating and attached cells were collected, stained using a JC-1 mitochondrial membrane potential detection kit (Cell Technology, Mountain View, CA) as per manufacturer's instructions and analyzed using a BioTek Synergy HT fluorescent plate reader (BioTek, Winooski, VT). To determine mitochondrial membrane potential changes in vitro, fluorescence ratios were calculated as the red fluorescence value (excitation 485/emission 590) divided by the green fluorescence value (485/528). The fluorescence ratio of cyclopamine-treated cells was then compared to the fluorescence ratio of vehicle-treated cells as a percent of control.

siRNA transfection.

Pancreatic cancer cells were cultured either in triplicate (5,000 cells/well) in 96-well plates or in 60 mm cell culture dishes (500,000 cells/dish) and transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Cells were transfected with a non-targeting control siRNA or GLI3-specific siRNAs (siRNA1, HSS104173, 5′-UGA AUG GAA UGU UUC CGC GAC UGA A-3′; siRNA2, HSS104175, 5′-CCA UUG CAU AUG ACU UCC GCC UUA U-3′) (40 nM, Invitrogen, Carlsbad, CA) for 24 hours prior to treatment with vehicle alone or cyclopamine. Changes in cell viability, mitochondrial membrane potential and gene expression following GLI3 knockdown were determined by MTS assay, JC-1 assay and RTQ-PCR/TLDA, respectively.

RNA extraction.

Total RNA was isolated from pancreatic cancer cells using Trizol reagent (Invitrogen) as per manufacturer's instructions. RNA was then DNase treated and purified using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) as per manufacturer's instructions. RNA was eluted in 50 µL of RNase-free water and stored at −80°C. The concentration of all RNA samples was quantitated using the ribosomal protein, large, P0 (RPLP0) housekeeping gene and linear regression analysis of a standard curve derived from known concentrations of normal pancreas RNA as previously described by our laboratory.32,33

Reverse transcription.

Prior to cDNA synthesis, all RNA samples were diluted to 4 ng/µL using RNase-free water containing 12.5 ng/µL of total yeast RNA (Ambion, Austin, TX) as a carrier. cDNA was prepared using the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA) as per manufacturer's instructions. The resulting cDNA samples were analyzed using RTQ-PCR and/or TLDA.

RTQ-PCR.

Primer and probe sets for GLI1 (Hs00171790_m1), GLI3 (Hs00609233_m1), PTCH1 (Hs00181117_m1) and RPLP0 (Hs99999902_m1) were obtained from Applied Biosystems and used according to manufacturer's instructions. PCR amplification was performed on an ABI Prism 7900HT sequence detection system and gene expression was calculated using the comparative CT method as previously described.33,34 Briefly, this technique uses the formula 2-ΔΔCT to calculate the expression of target genes normalized to a calibrator. The cycling threshold (CT) indicates the cycle number at which the amount of amplified target reaches a fixed threshold. CT values range from 0 to 40 (the latter representing the default upper limit PCR cycle number that defines failure to detect a signal). Vehicle-treated and siRNA control-transfected cells were used as calibrators, for which all gene expression values were assigned a relative value of 1.00.

TLDA.

For each card of the low-density array (Applied Biosystems), there are 8 separate loading ports that feed into 48 separate wells for a total of 384 wells per card. Each 2 µL well contains specific, user-defined primers and probes, capable of detecting a single gene. In this study, the TLDA card was configured into identical 48 gene sets. Genes known to be involved in the HH pathway9,10 and genes influenced by HH signaling, such as those involved in cell differentiation and proliferation,35–42 were selected for examination. Supplemental Table 1 contains a complete listing of all 48 genes, which have been previously described by our laboratory.18 Each set of 48 genes also contains two housekeeping genes, RPLP0 and GAPDH. All samples were loaded onto a TLDA card and PCR amplification was performed on an ABI Prism 7900HT sequence detection system. Gene expression was calculated using the comparative CT method.

Statistical analysis.

Changes in cell viability, BrdU incorporation, mitochondrial membrane potential and gene expression induced by cyclopamine, CUR199691 and/or GLI3 siRNA were assessed by Student's t-test or, in the case of multiple group comparisons, one-way analysis of variance (ANOVA) followed by a Tukey's post-test. Comparisons of gene expression and cyclopamine IC50 values were performed using Pearson correlation analysis. Significance was defined as p < 0.05.

Acknowledgements

The authors thank Amy Petersen, Shibani Mukherjee and Andrea Sadlonova for their technical support.

Footnotes

Financial Support

Supported by P20-CA10195, U10 CA021661 and U10 CA032115.

Supplementary Material

Supplementary Material
cbt1009_0893SD1.pdf (1,020.5KB, pdf)

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Supplementary Materials

Supplementary Material
cbt1009_0893SD1.pdf (1,020.5KB, pdf)

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