Response to inhibition of smoothened in diverse epithelial cancer cells that lack smoothened or patched 1 mutations

Hedgehog (HH) pathway Smoothened (Smo) inhibitors are active against Gorlin syndrome-associated basal cell carcinoma (BCC) and medulloblastoma where Patched (Ptch) mutations occur. We interrogated 705 epithelial cancer cell lines for growth response to the Smo inhibitor cyclopamine and for expressed HH pathway-regulated species in a linked genetic database. Ptch and Smo mutations that respectively conferred Smo inhibitor response or resistance were undetected. Previous studies revealed HH pathway activation in lung cancers. Therefore, findings were validated using lung cancer cell lines, transgenic and transplantable murine lung cancer models, and human normal-malignant lung tissue arrays in addition to testing other Smo inhibitors. Cyclopamine sensitivity most significantly correlated with high cyclin E (P=0.000009) and low insulin-like growth factor binding protein 6 (IGFBP6) (P=0.000004) levels. Gli family members were associated with response. Cyclopamine resistance occurred with high GILZ (P=0.002) expression. Newer Smo inhibitors exhibited a pattern of sensitivity similar to cyclopamine. Gain of cyclin E or loss of IGFBP6 in lung cancer cells significantly increased Smo inhibitor response. Cyclin E-driven transgenic lung cancers expressed a gene profile implicating HH pathway activation. Cyclopamine treatment significantly reduced proliferation of murine and human lung cancers. Smo inhibition reduced lung cancer formation in a syngeneic mouse model. In human normal-malignant lung tissue arrays cyclin E, IGFBP6, Gli1 and GILZ were each differentially expressed. Together, these findings indicate that Smo inhibitors should be considered in cancers beyond those with activating HH pathway mutations. This includes tumors that express genes indicating basal HH pathway activation.

This study uncovered growth inhibitory responses to Smo inhibition in diverse cancer cells using a robotic-based platform with a genetic database. In this database Ptch1 and Smo sequences were available with information about expression of Response to inhibition of smoothened in diverse epithelial cancer cells that lack smoothened or patched 1 mutations species associated with HH pathway activation. Basal expression of these species in cancer cells was hypothesized to indicate growth dependence of these cells on the HH pathway. It was hypothesized that cancer cells expressing these species would respond to a Smo inhibitor.
Multiple Smo inhibitors were studied in lung cancer because the HH pathway is active in subsets of these cancers. Both murine and human lung cancer cell lines exist. Cyclin E-driven transgenic and transplantable murine lung cancer models that spontaneously activated the HH pathway were available for study as was a paired human normal-malignant lung tissue array with an associated clinical database. The presented findings implicate use of Smo inhibitors for lung and other cancers when a gene profile indicative of HH pathway dependence is expressed in the cancer cells.
Smo inhibitor responses. The HH pathway affects expression of Ptch1, cyclin D1, cyclin E, IGF2, IGFBP6, GILZ, Gli family members, and other species (2)(3)(4)(5). The cor.test function (25) of R (26) compared cyclopamine-dependent growth responses to expressed species. Expression values were from U133 Plus 2.0 Affymetrix arrays and are publically available (27). The data set consisted of 490 samples corresponding to 164 unique cell lines that were typically examined in triplicate. Correlations were done: a) using all samples and b) using samples with a cyclopamine growth inhibitory response ≤0.75 at the 10 µM dosage, corresponding to 110 samples (in triplicate). Cyclopaminemediated growth response was compared with mutation data available from the Sanger database (28).
Ptch1 and smo sequence analyses. Sequencing of the coding regions of Ptch1 was performed as before (29). For murine cell lines, murine-specific primers were used to sequence homologous domains of Ptch1. To assess for Smo mutations in murine and human cell lines, prior mutations in human Smo were searched for (A324T, V404M, D473H, E518K, W535L, and T640A) and corresponding regions of murine Smo were sequenced.
Real-time RT-PCR assays. Total RNA was isolated using the RNeasy kit (Qiagen, Valencia, CA). RT was with the High Capacity cDNA RT kit (Applied Biosystems, Foster City, CA) and a Peltier Thermal Cycler (MJ Research, Waltham, MA). Semi-quantitative real-time RT-PCR assays were with SYBR Green PCR Mastermix (Applied Biosystems) and the 7500 Fast real-time PCR system (Applied Biosystems). Human IGFBP6 and β-actin qPCR assays were performed with TaqMan Universal PCR Master Mix and the manufacturer's described assays Clonal growth assays. Two hundred logarithmically growing ED-1 cells were plated for triplicate replicate clonal growth assays, as before (20). Colonies were stained with Diff Quick (IMEB Inc, San Marcos, CA) and were counted with the Oxford Optronix Col Count counter (Oxford Optronix, Oxford, UK) (31).
Normal-malignant lung tissue array. The New Hampshire State Cancer Registry and the Dartmouth-Hitchcock Medical Center Tumor Registry have been described (20). Analyses of normal versus malignant lung tissue arrays were by a pathologist (Vincent A. Memoli) unaware of findings from the associated clinical database. Signed consents were obtained; studies were reviewed and approved by Dartmouth's Institutional Review Board (IRB) for human subjects. A paired normal-malignant lung tissue microarray was constructed (20).
Statistical analysis. Results were expressed as means ± standard deviations. Results of all independent experiments were pooled to assess for statistical significance. Z-test and two-sided t-tests were used for statistical analyses with significance considered for values of P<0.05.
Cyclopamine treatments of transgenic mice. Three 9-month-old female mice expressing transgenic wild-type human cyclin E were each treated daily (intraperitoneal) for 5 consecutive days with cyclopamine (40 mg/kg) or vehicle (45% hydroxypropylß-cyclodextrin, HBS) with a total of six mice studied. Mice were examined using an Institutional Animal Care and Use Committee (IACUC)-approved protocol. Tissues were formalinfixed, paraffin-embedded and sectioned for histopathology (20). Hematoxylin and eosin staining as well as Ki-67, cyclin D1, and cyclin E immunostaining were performed as before (16,19). Analyses were by a pathologist (Vincent A. Memoli) unaware whether harvested tissues were from cyclopamine or vehicletreated mice.
Murine syngeneic lung cancer transplantation assays. ED-1 cells were transduced with a GFP expression vector and transductants were sorted (15,16) with 8x10 5 cells injected into tail veins of each female FVB mouse (8-week old). Ten mice per arm were used and replicate experiments were performed. To investigate cyclopamine antineoplastic effects, 10 mice were each intraperitoneally treated daily for 14 days with cyclopamine (40 mg/kg); 10 additional mice were each treated with vehicle (45% HBS). Treatments began 2 weeks after tail-vein injections because lung tumors formed by then (data not shown). Ten syngeneic FVB mice were each injected with ED-1 cells that were treated with either MK-4101 (10 µM) or vehicle as part of an IACUC-approved protocol. Mice were sacrificed 28 days post-tail vein injections. Replicate experiments were performed. Lung tissues were formalin-fixed, paraffin-embedded and sectioned for histopathology (16,19,20). A rabbit polyclonal anti-GFP antibody (product ab290) (Abcam, Cambridge, MA) was used to identify lung tumors. Hematoxylin counterstaining was used.
Twenty lines were of SCLC and 107 were of NSCLC origins. Subsets of SCLC (Fig. 1B) and NSCLC (Fig. 1C) lines responded to cyclopamine with substantial growth inhibition. NSCLC responses occurred independently of mutations for Ptch1 or Smo (Table IA). These responses were also independent of ras, epidermal growth factor receptor (EGFR), or p53 mutations (Table IB). K-RAS mutations in NSCLC are associated with resistance to EGFR-tyrosine kinase inhibitors (TKIs) (32)(33)(34)(35). Growth inhibition in the most cyclopamine-responsive NSCLC cells lines (NCI-H1703 and NCI-H2122) were independently confirmed in Fig. 2.
Studies were next conducted in lung cancer because an autocrine HH pathway is reported in lung cancer (36). HH signaling is important for growth of NSCLC and SCLC (6,15). Other reasons for studying lung cancer included the finding that increased cyclin E expression was associated with cyclopamine response (Fig. 1A) and cyclin E transgenic mice recapitulated key features of human lung cancer biology (16,30).
Gain or loss of expression was achieved for the most significantly associated candidate regulators of cyclopamine response (cyclin E and IGFBP6) or resistance (GILZ). Gli1 is an established HH pathway regulator in lung cancer (15). To determine whether cyclin E affected cyclopamine response, cyclin E was overexpressed in murine immortalized C-10 lung epithelial cells ( Fig. 2A, left panel shows a 2.26-fold increase in cyclin E protein versus actin expression) and this significantly (P<0.01) increased cyclopamine-mediated growth inhibition versus the inactive isomer tomatidine ( Fig. 2A, right panel). Similar results were obtained using a second Smo inhibitor, SANT-1 (data not shown), which was consistent with prior studies (15). Two shRNAs (Fig. 2B, left panel) were selected to reduce IGFBP6 expression independently in ED-1 lung cancer cells derived from a transgenic mouse that expressed wild-type cyclin E (30). Individual knock-down with these shRNAs significantly (P<0.01) increased Smo-mediated growth inhibition in this murine lung cancer cell line versus an inactive shRNA control (Fig. 2B, right panel). Thus, gain of cyclin E or loss of IGFBP6 expression regulated response to a Smo inhibitor. In contrast, two different shRNAs independently repressed GILZ expression (Fig. 2C, left panel), but these did not appreciably affect cyclopamine-mediated growth suppression at 72 h (Fig. 2C, right panel). Findings were confirmed in human NSCLC cells engineered with IGFBP6 knock-down ( Fig. 2D and E).
Lung cancers arising in transgenic mice from increased cyclin E expression in the lung exhibit high levels of Gli1 (16,30). These transgenic lung cancers are also shown to express low levels of IGFBP6 and GILZ versus the adjacent normal lung  (Fig. 3A). Whether cell lines derived from these murine lung cancers responded to a Smo inhibitor was independently studied in ED-1 and ED-2 lung cancer cells derived respectively from transgenic mice whose lung tumors expressed wild-type (ED-1) or degradation-resistant (ED-2) cyclin E species (30).
Effects of Smo inhibition were explored by targeting Smo in ED-1 cells. Gli1, Gli2, Gli3, and Smo were independently targeted using two different siRNAs engineered to repress each of these species. Independent knock-down of Gli1, Gli2, and Smo each significantly inhibited ED-1 cell growth (as did cyclopamine treatment), but Gli3 knock-down did not appreciably affect growth (Fig. 3B, left and middle panels). As expected, the Smo agonist SAG augmented Gli1 expression; cyclopamine co-treatment prevented this (Fig. 3B, right panel). Compensatory changes in expressed HH pathway members were found after these different knock-downs in Fig. 3C and D. Smo knock-down substantially decreased lung cancer cell growth.
Cyclopamine treatment also significantly (P<0.01) decreased ED-1 clonal growth while increasing apoptosis (Fig. 4A). ED-1 and ED-2 cells expressed multiple HH pathway components as did the examined murine and human immortalized pulmonary epithelial or cancer cell lines (Fig. 4B). Genomic DNA from each of these cell lines was sequenced for the entire coding region of Ptch1 and for Smo mutations in regions that conferred Smo inhibitor resistance (A324T, V404M, D473H, E518K, W535L, and T640A). No mutations were detected (data not shown). Human lung cancer cell lines studied were those in which responses to Smo inhibition were already reported (15,36). To establish whether the HH pathway was functional in ED-1 cells, these cells were treated with recombinant sHH at different dosages in low serum (0.5%) containing medium. HH pathway activity in these cells was confirmed; sHH treatment significantly (P<0.01) increased mRNA expression for Gli1 and Ptch (Fig. 4C). Although ED-1 cells arose from transgenic lung cancers that activated the HH pathway (16), these cells had relatively low basal Gli1 and Gli2 mRNA levels (Fig. 4B) and low basal Gli1-reporter activity (Fig. 4D). This is consistent with differences reported between ex vivo versus in vivo HH pathway dependence of lung cancer cells (37). It was therefore not unexpected to detect prominent Gli1 immunostaining of lung tumors arising from ED-1 cells after their tail-vein injections into syngeneic FVB mice (data not shown).
To explore independently Smo inhibitor effects on lung cancer cells, ED-1 cells were engineered to have Smo knockdown. This was done using two independent shRNAs versus an inactive control shRNA (Fig. 4E). This conferred significant repression (Smo1 shRNA P=0.002 and Smo2 shRNA P=0.001) of transplanted lung cancers (Fig. 4F). Ex vivo treatment of ED-1 cells with another Smo inhibitor MK-4101 significantly reduced ED-1 colony formation (P<0.01, data not shown) and tumorigenicity (P=0.009) after tail-vein injections into syngeneic mice of these versus control cells (Fig. 4G).
Cyclopamine responses were examined in cyclin E transgenic mice because their lung cancers expressed a gene profile that was indicative of dependence on HH signaling. Cyclopaminetreatment decreased immunohistochemical expression of the proliferation markers Ki-67, cyclin D1 and cyclin E in the neoplastic versus normal lungs of treated versus age and sexmatched control mice (Fig. 5A, left panel). This result suggested that Smo inhibition would also reduce lung tumor formation in This significantly (P=0.00002) reduced lung cancers in cyclopamine versus vehicle-treated mice (Fig. 5A, right panel).
To learn whether a profile indicative of HH pathway dependence was expressed in human lung cancers, a normal-malignant lung tissue array (20) was studied. Immunohistochemical expression profiles revealed significant differences in the malignant versus normal lung for cyclin E (P<0.01), IGFBP6 (P<0.01), and GILZ (P<0.01) in Fig. 5B. Lung cancers with high cyclin E levels expressed significantly (P<0.01) higher levels of Gli1 than did cases with reduced cyclin E levels (Fig. 5B). Cases with high cyclin E and low IGFBP6 levels had increased (P<0.0001) Gli1 immunohistochemical expression. Representative results are presented (Fig. 5C).

Discussion
Smo inhibitors are active against Gorlin syndrome-associated BCC or medulloblastoma where Ptch mutations occur (9)(10)(11)(12)(13)  Smo inhibitor growth response was most significantly associated with high cyclin E (P= 0.000009), low IGFBP6 (P=0.000004), and high Gli1 (P=0.04) levels; high GILZ levels were associated with reduced response (P= 0.002) in Fig. 1. This profile implicated a basal dependence on the HH pathway for the growth of these cancer cells. This possibility was validated in murine and human lung cancer cell lines and transgenic as well as transplantable murine lung cancer models in Figs. 1-5. Functional consequences were shown using several pharmacological and genetic Smo inhibitors.
Differential expression of the same species was observed in a malignant versus normal human lung tissue array (Fig. 5). A profile indicative of Smo inhibitor response was also observed in murine lung cancers (Fig. 3). While high IGFBP6 levels were present in some lung tumors, it is notable that lung cancers with both high cyclin E and reduced IGFBP6 expression significantly (P<0.0001) increased Gli1 expression. This pattern was one indicating response to a Smo inhibitor. This points out the potential clinical need to discern profiles of HH pathway-regulated species in human tumors to learn which are likely to be responsive to Several Smo inhibitors conferred similar effects. Stromal effects are engaged to confer some of these anti-neoplastic effects. This could account for differences between in vitro and in vivo effects of Smo inhibition (37,38). The findings presented in Fig. 1  Only some cancers express a gene profile indicating possible HH pathway dependence. Notably, autocrine HH pathway signaling occurs in lung cancers (36). The findings reported here are consistent with this prior study.
These findings have implications for combination cancer therapy. Responses to Smo inhibition occurred whether or not RAS or p53 (or Ptch1 or Smo) mutations were present in cancer cells (Table I). In clinical lung cancers, K-RAS mutations confer resistance to EGFR-TKIs (32,34,35). Yet, clinical trials revealed activity against lung cancers having K-RAS mutations when an EGFR-TKI was combined with a rexinoid (33). This clinical activity was associated with reduced cyclin D1 expression in posttreatment lung cancer biopsies (33). In the present study, in vivo responses to Smo inhibition were linked to cyclin D1 repression (Fig. 5). Adding a Smo inhibitor to a regimen that targets cyclin D1 for repression might enhance clinical anti-tumor activity. Smo responses were also associated with cyclin E expression (Figs. 1 and 5). Targeting the cyclin E-cdk2 complex exerted anti-tumor responses despite presence of K-RAS mutations by inducing anaphase catastrophe (19,39). Combining a cdk2 antagonist with a Smo inhibitor might augment anti-neoplastic activity.
High Smo inhibitor dosages were associated with these anti-neoplastic effects. A similar dose-response relationship for HH inhibition was observed in different tumor contexts and this might depend on expressed drug transporters (40,41). It is notable that the findings displayed in Fig. 3B (right panel) using a Smo agonist argue against off-target effects of cyclopamine. The reduction of lung tumors after Smo inhibition in the transplantation model reported here is notable since these lung cancers did not have Ptch1 or Smo mutations. Smo inhibitors might treat or prevent other cancers that lack these mutations.
Taken together, findings presented here indicate a Smo inhibitor should be considered in cancers that lack Smo or Ptch1 mutations. This is especially the case when the tumors express a gene profile indicating basal activation of the HH pathway. This could implicate a dependence on the HH pathway for growth or survival of the same tumors. Future clinical research should explore the translational consequences of these findings for cancer therapy and prevention.