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. Author manuscript; available in PMC: 2015 Feb 10.
Published in final edited form as: Cancer Cell. 2014 Feb 10;25(2):139–151. doi: 10.1016/j.ccr.2014.01.008

The PRKCI and SOX2 Oncogenes are Co-amplified and Cooperate to Activate Hedgehog Signaling in Lung Squamous Cell Carcinoma

Verline Justilien 1, Michael P Walsh 1, Syed A Ali 1, E Aubrey Thompson 1, Nicole R Murray 1, Alan P Fields 1,*
PMCID: PMC3949484  NIHMSID: NIHMS558180  PMID: 24525231

SUMMARY

We report that two oncogenes co-amplified on chromosome 3q26, PRKCI and SOX2, cooperate to drive a stem-like phenotype in lung squamous cell carcinoma (LSCC). PKCι phosphorylates SOX2, a master transcriptional regulator of stemness, and recruits it to the promoter of Hedgehog Acyl Transferase (HHAT), which catalyzes the rate-limiting step in Hh ligand production. PKCι-mediated SOX2 phosphorylation is required for HHAT promoter occupancy, HHAT expression, and maintenance of a stem-like phenotype. Primary LSCC tumors coordinately overexpress PKCι, SOX2, and HHAT, and require PKCι-SOX2-HHAT signaling to maintain a stem-like phenotype. Thus, PKCι and SOX2 are genetically, biochemically and functionally linked in LSCC, and together they drive tumorigenesis by establishing a cell autonomous Hh signaling axis.

Keywords: Protein Kinase Cι, SOX2, 3q26 amplification, Hedgehog signaling, proliferation, transformed growth, LSCC tumor initiation, clonal expansion

INTRODUCTION

Lung cancer is the major cause of cancer death with a 5 year survival rate of 16% (Siegel et al., 2012). Non-small cell lung cancer (NSCLC) accounts for ~80% of lung cancer cases and is subdivided into adenocarcinoma (LAC), squamous cell carcinoma (LSCC) and large cell carcinoma (LCLC). Distinct histologies, genetic and epigenetic changes, and sites of origin characterize NSCLC subtypes, suggesting they may have unique responses to therapy. Recent therapies targeting pathways active in NSCLC sub-types have resulted in encouraging new treatments for LAC driven by EGFR or EML4-ALK mutations. However, few advances have resulted in better treatment options for LSCC, which accounts for 30% of all lung cancer cases. Thus, there is a need to better understand molecular mechanisms that drive LSCC and translate this knowledge into better intervention strategies.

NSCLC tumors contain stem-like cells responsible for lung tumor initiation, maintenance, relapse and metastasis (Chen et al., 2008; Eramo et al., 2008; Justilien et al., 2012). These cells exhibit resistance to commonly used therapeutic agents (Chen et al., 2008) making them a likely cause of therapeutic failure. Similar cell populations exist in several cancer types (Chen et al., 2012; Driessens et al., 2012; Schepers et al., 2012). Lineage tracing reveals these cells clonally expand to give rise to malignant and non-malignant, differentiated cell types. These highly tumorigenic cells exhibit self-renewal by activating developmental pathways including Wnt, Hedgehog and Notch, and by aberrant expression of stem-related genes such as BMI1 (Siddique and Saleem, 2012), OCT3/4 (Chiou et al., 2010) , SOX2 (Yuan et al., 2010), and NANOG (Chiou et al., 2010) which participate in their maintenance. As tumorigenic drivers, these stem-like cells must be effectively targeted to elicit long-lasting therapeutic responses.

We previously identified PRKCI as an oncogene in LSCC (Regala et al., 2005b). PRKCI is overexpressed in LSCC cells and primary tumors due to tumor specific amplification of a chromosome 3q26 amplicon (Regala et al., 2005b). Tumor PKCι expression is predictive of poor clinical outcome, and PKCι drives LSCC cell invasion and transformed growth in vitro and in vivo (Frederick et al., 2008; Justilien and Fields, 2009; Regala et al., 2008; Regala et al., 2005a). Genetic disruption of Prkci in the LSL-KrasG12D mouse LAC model blocks tumor initiation by inhibiting expansion of putative lung cancer stem cells (Regala et al., 2009). Here, we demonstrate that PRKCI maintains a highly tumorigenic phenotype in lung cancer cells harboring PRKCI amplification, and in LSCC tumors. Our results reveal a genetic, biochemical and functional interaction between PRKCI and SOX2 that coordinately drives growth and maintenance of LSCC stem-like cells.

RESULTS

Lung oncosphere cells exhibit stem-like properties

Highly malignant tumor cells can be enriched in defined medium at low adherence (Eramo et al., 2008; Justilien et al., 2012). These conditions favor growth of highly tumorigenic stem-like cells, while negatively selecting for less tumorigenic differentiated tumor cells. We isolated stem-like cells from five human lung cancer cell lines harboring 3q26 copy number gains (H1299, H1703, ChagoK1, H520 and H1869) using established protocols (Eramo et al., 2008; Justilien et al., 2012). H1299, H1703 and ChagoK1 grew as cell spheres (oncospheres) that exhibit many stem-like properties (Fig. 1A). First, when returned to adherent culture, oncosphere cells redifferentiated and acquired morphology comparable to parental cells (Fig. 1A). Second, oncosphere cultures expressed elevated mRNA for genes associated with a stem-like phenotype including SOX2, OCT3/4, NANOG, ALDHA1, PROM1/CD133, and MMP10 which was lost upon redifferentiation (Fig. 1B). Third, single oncosphere cells clonally expand with high efficiency (H1703 cells: 91/98 cells; 93%; ChagoK1 cells: 41/44 cells, 93%; H1299 cells: 125/138 cells, 91%) (Fig. 1C). Fourth, oncosphere cultures exhibited enhanced soft agar growth, a property lost upon redifferentiation (Fig. 1D). Similar results were obtained in H520 and H1869 LSCC cells (Fig. S1A-D). Finally, oncospheres displayed enhanced tumorigenic potential in vivo. Limiting dilution showed that implantation of as few as 10,000 H1299 oncosphere cells into the lungs of immune-deficient mice resulted in efficient tumor take (5/5), but only occasional tumor take in parental cells (1/5) (Fig. 1E). A similar increase in tumor take was observed in H520 oncospheres when compared to parental H520 cells (Fig. S1E). Oncosphere cells developed larger tumors than parental cells and routinely produced lesions in the contralateral lung, whereas parental cells did not. Oncosphere-derived tumors exhibit morphology similar to parental cell tumors (Fig. 1E, middle panel) and these tumors express SOX2, OCT3/4, NANOG, ALDHA1, CD133, and MMP10 levels similar to parental cells, indicating the ability of oncosphere cells to differentiate in vivo (Fig. S1). Finally, H1299 oncosphere cells efficiently generate tumors of similar morphology through three serial passages in mice (data not shown).

Figure 1. Lung cancer oncospheres exhibit stem-like characteristics.

Figure 1

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A) Phase contrast photomicrographs of H1703, ChagoK1 and H1299 parental adherent cells (top panels), oncospheres in low adherence culture (middle panels) and redifferentiated oncosphere cells after return to adherent culture (bottom panels). B) QPCR for putative stem cell markers expressed as fold of parental cells +/−SEM, n = 3; *p<0.05 versus NT adherent cells. Results are representative of five independent experiments. C) Photomicrographs showing clonal expansion of individual cells into oncospheres over a 15 day period. D) Anchorage-independent growth expressed as mean fold-change from parental cells +/− SEM. n = 5, * p<0.05 versus NT adherent cells. Results are representative of five independent experiments. E) Formation of lung orthotopic tumors in immunocompromised mice. 10,000 parental H1299 adherent or oncosphere cells were implanted into the lungs of immune-deficient mice. Lateral and dorsal views of bioluminescent images of a tumor-bearing mouse (a) and corresponding bioluminescence image upon lung dissection (b). H&E of typical parental and oncosphere tumors (c). Oncospheres develop primary tumors at the site of injection (arrows) and multiple lesions to the ipsilateral and contralateral lobes of the lung (arrowheads); parental cells develop either a single, small tumor at the site of injection (shown) or no identifiable tumor. 40X magnification reveals the similar morphology of oncosphere- and parental cell-derived tumors (d). Incidence of tumor formation in limiting dilutions of H1299 oncosphere and parental cells (e). Quantification of tumor growth by IVIS (f); Mean +/−SEM. *p<0.05 n=5, *p<0.05 vs. parental cells. See also Figure S1.

PKCι maintains oncospheres by activating a cell autonomous Hh signaling axis

PKCι is necessary for transformation and expansion of bronchio-alveolar stem cells (BASCs), putative tumor-initiating cells in Kras mediated lung tumorigenesis (Regala et al., 2009). Interestingly, MMP10, a transcriptional target of PKCι in lung cancer cells (Frederick et al., 2008) and transformed BASCs (Regala et al., 2009), is induced in oncospheres (Fig. 1B and Fig. S1), suggesting that PKCι is activated in these cells. Indeed, oncospheres exhibit an increase in T410 PKCι phosphorylation (Fig. S2), an event associated with PKCι activity (Baldwin et al., 2008; Desai et al., 2011; Le Good et al., 1998; Standaert et al., 1999). PKCι RNAi severely impaired soft agar growth and clonal expansion of H1703, ChagoK1 and H1299 oncospheres (Fig. 2A and 2B) indicating that PKCι is required for a stem-like phenotype. Similar results were obtained in H520 and H1869 cells (Fig. S2).

Figure 2. PKCι is required for maintenance of a stem-like phenotype in oncosphere cells.

Figure 2

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A) Effect of RNAi-mediated PKCι knockdown (immunoblot) on anchorage-independent growth of H1703, H1299 and ChagoK1 oncospheres. Results are expressed relative to NT RNAi control cells +/− SEM, n = 5; **p<0.001. B) Oncosphere size expressed as mean diameter in μm +/−SEM. n=98 (H1703), 138 (H1299) and 44 (ChagoK1). C) QPCR for HHAT, GLI1, ADRBK1 and CDK19. Results are expressed as fold NT parental cells +/−SEM, n=3; *p<0.05 compared to NT parental. Results are representative of three independent experiments. D) Effect of SMO inhibitor LDE225 on H1703, H1299 and ChagoK1 oncosphere (onco.) and parental cell (par.) proliferation. Results expressed as % DMSO control +/−SEM; n=6. Results are representative of three independent experiments. See also Figure S2 and Tables S1, S2 and S3.

To identify PKCι-regulated pathways, we conducted total RNA sequencing on NT and PKCι RNAi parental and oncospheres from H1703, ChagoK1 and H1299 cells to identify genes up or down regulated in all three oncosphere lines in a PKCι-dependent manner (Tables S1 and S2). We then used Metacore pathway analysis to determine if PKCι regulates three main pathways associated with stem maintenance, Wnt, Hh and Notch. Results revealed that PKCι significantly regulates Hh (p=0.025) but not Wnt (p=0.091) or Notch (p=0.354), suggesting a role for PKCι-dependent Hh signaling in oncospheres. Hh components Hedgehog Acyl Transferase (HHAT), ADRBK1, CDK19 and GLI1 were identified as PKCι regulated (raw gene counts in Table S3; Hh signaling components listed in Table S4; the entire sequencing dataset is deposited in GEO, accession number GSE48599), and these genes were validated by QPCR in H1703 and H1299 oncospheres (Fig. 2C).

Our data suggest that PKCι regulates Hh pathway activity to stimulate oncosphere growth. Consistent with this notion, auranofin (ANF), an anti-arthritic gold salt that selectively inhibits PKCι signaling (Erdogan et al., 2006; Stallings-Mann et al., 2006; Wang et al., 2013), led to dose-dependent inhibition of oncosphere proliferation with IC50s of ~0.5-2.0 μM, and a significant decrease in HHAT and Gli1 expression (Fig. S2). Similarly, the SMO inhibitor LDE225 induced dose-dependent inhibition of oncosphere proliferation with IC50s in the 10-40 nM range, consistent with the sub-μM IC50 for SMO inhibition (Buonamici et al., 2010) (Fig. 2D). Interestingly, parental cells were much less sensitive to LDE225 (IC50s >5 μM; well above the IC50 for SMO inhibition) (Fig 2D), consistent with reports that lung progenitor/stem cells exhibit Hh-dependent proliferation whereas differentiated NSCLC cells do not (Watkins et al., 2003). Interestingly, oncospheres from LAC cell lines (A549, H358 and H1437) are not sensitive to LDE225 (IC50s >10μM) suggesting that Hh signaling is not required for LAC oncospheres (Fig. S2). Furthermore, neither HHAT nor GLI1 are induced in LAC oncospheres, and PKCι KD does not affect expression of these genes (Fig. S2), suggesting that the PKCι-Hh signaling axis is not operative in LAC.

HHAT catalyzes a critical step in Hh ligand processing and can initiate Hh signaling, and GLI1 is a major transcriptional effector of Hh signaling. Therefore, we assessed the effect of genetic inhibition of HHAT and GLI1 on stem-like behavior. HHAT or GLI1 RNAi (Fig. 3A and 3B) significantly inhibited clonal expansion and soft agar growth of H1299 oncospheres (Fig. 3C and 3D). Similar results were observed in H1703, ChagoK1, H520 and H1869 oncospheres (Fig. S3A-D). Results were validated by reconstitution using the small molecule Hh agonist Hh.Ag1.5. PKCι, HHAT and GLI1 RNAi significantly inhibited (Fig. 3E), and Hh.Ag1.5 rescued oncosphere growth in HHAT KD cells, but not PKCι or GLI1 KD cells (Fig. 3E). These results are expected since HHAT acts upstream of ligand, PKCι modulates Hh components both upstream (ie. HHAT) and downstream (ie. GLI1, ADRBK1 and CDK19) of ligand, and GLI1 activates Hh-mediated transcription downstream of ligand. Since PKCι regulates HHAT, an enzyme required for SHH palmitoylation, we assessed the effect of PKCι and HHAT KD on palmitolyated SHH production using the palmitic acid analog ω-alkynyl palmitate (ω-alkC16) as described (Yap et al., 2010). Metabolically labeled FLAG-SHH was immunoprecipitated with anti-FLAG antibody, derivatized with azido-biotin using CLICK chemistry and palmitoylated SHH detected by immunoblot analysis using HRP-Neutra-avidin (Fig. 3F). NT H1299 oncospheres produce more palmitoylated SHH than NT parental cells, and both PKCι and HHAT KD oncospheres exhibit a striking decrease in palmitoylated SHH. Similar results were obtained in H1703 cells (Fig. S3E). Thus, oncospheres utilize a PKCι-dependent cell autonomous Hh signaling pathway to maintain their stem-like phenotype.

Figure 3. Oncosphere growth requires a PKCι-dependent Hh signaling axis.

Figure 3

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A and B) RNAi-mediated knockdown of HHAT and GLI1 in H1299 oncospheres, respectively, expressed as fold of NT RNAi control +/− SEM n=3. *p<0.0005 compared to NT control. C) Effect of HHAT or GLI1 RNAi on clonal expansion expressed as oncosphere diameter in μm +/− SEM, n>15 per RNAi sample; *p<1.0×10−06. Data are representative of three independent experiments. D) Soft agar growth expressed relative to NT RNAi control cells +/− SEM, n = 5; * p<0.003. Data are representative of three independent experiments. E) NT, PKCι, HHAT and GLI1 oncospheres were treated with the indicated amounts of Hh-Ag1.5, a selective Hh agonist, and cell proliferation assessed by MTT at 5 days. Results expressed as % of NT DMSO control +/− SEM, n = 6. F) Detection of palmitoylated SHH in oncospheres. Parental (P) and oncosphere (O) NT, PKCι and HHAT RNAi cultures expressing FLAG-SHH were metabolically labeled with w-alkynyl-palmitate (C16) and palmitoylated SHH (palm-SHH) and total SHH were detected as described in Experimental Procedures. G) Oncosphere growth as lung orthotopic tumors. NT parental and NT, PKCι, HHAT, and GLI1 RNAi oncosphere tumor growth was monitored by bioluminescence detected by IVIS imaging. Data are presented as total flux in photons per second ± SEM; n = 10 per group except for PKCι where n=9; *p<0.05 significantly different than NT RNAi oncosphere tumors. See also Figure S3.

To assess the importance of PKCι-Hh signaling in oncosphere behavior in vivo, we determined the ability of NT, PKCι, HHAT, and GLI1 RNAi oncospheres, and NT parental cells, to initiate lung orthotopic tumors in immune-deficient mice. As expected, H1299 NT RNAi oncospheres exhibited an increased tumor take rate (10/10 vs. 2/10 mice for NT parental cells) and developed significantly larger tumors than NT RNAi parental cells (Fig. 3G). PKCι, HHAT, and GLI1 RNAi oncospheres showed a decreased take rate (1/9, 0/10, 1/10 respectively), and failed to produce large tumors (Fig. 3G). These data indicate that cell autonomous, PKCι-dependent Hh signaling is critical for the tumorigenic growth of oncospheres in vivo.

PKCι can phosphorylate Gli1 and activate its transcriptional activity, and Gli1 can regulate PKCι expression, in basal cell carcinoma cells (Atwood et al., 2013). Thus, we assessed Gli1 phosphorylation status in NT and PKCι KD oncospheres (Fig. S3F). We observed a decrease in pSer/Thr Gli1 commensurate with the decrease in total Gli1 in PKCι KD cells. When normalized to total Gli1, no appreciable change in Gli1 phosphorylation was observed, indicating that the PKCι-Hh signaling axis described here is distinct from that reported in basal cell carcinoma (Fig. S3F). Furthermore, neither HHAT nor Gli1 RNAi affected PKCι mRNA abundance in oncospheres indicating that PKCι is not a target of Hh-Gli1 signaling in lung cancer stem cells (Fig S3G).

PKCι regulates SOX2-mediated HHAT expression in oncospheres

PRKCI is amplified as part of a 3q26 amplicon that drives PKCι expression in LSCC tumors, leading us to explore other possible genetic regulators residing on the 3q26 amplicon. SOX2, a SRY-related HMG-box (Sox) transcription factor and master regulator of stem cell maintenance (Sarkar and Hochedlinger, 2013), is consistently induced in our oncosphere cultures (Fig. 1B and Fig.S1). SOX2 is an oncogene in LSCC (Bass et al., 2009), and it can maintain cancer stem cells (Basu-Roy et al., 2012; Ikushima et al., 2011; Tian et al., 2012). SOX2 RNAi (Fig. 4A) significantly inhibited clonal expansion and anchorage-independent growth of H1299 oncospheres (Fig. 4B and 4C), indicating a key role for SOX2 in these cells. Similar results were obtained in H1703, H520 and H1869 oncospheres (Fig. S4).

Figure 4. PKCι regulates HHAT expression through control of SOX2 occupancy of the HHAT promoter.

Figure 4

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A) RNAi-mediated knockdown of SOX2 in H1299 oncospheres. Results expressed as % NT control +/−SEM; n=3, *p<0.05. B) Clonal expansion expressed as oncosphere diameter in μm +/− SEM; n> 22 per RNAi sample, p< 3.0 ×10−14 and are representative of three independent experiments. C) Anchorage-independent growth relative to NT RNAi control cells +/− SEM, n = 5; *p<5.0×10−9. Results are representative of three independent experiments. D) Chromatin immunoprecipitation (ChIP) analysis to assess SOX2 occupancy of the HHAT promoter. Schematic depicts the HHAT promoter region; the position of ChIP probes used are indicated (A and B); Consensus SOX2 binding sites are indicated by vertical slashes. Data presented as % of input; n=3 +/−SEM. *p<0.00002; **p<0.00003. Data are representative of two independent experiments. E) and F) Effect of SOX2 RNAi on HHAT expression in oncosphere cells. Data are expressed relative to NT RNAi control cells +/− SEM; *p<0.0007 and are representative of three independent experiments. G) PRKCI and SOX2 amplification (upper panel) and overexpression (lower panel) in primary LSCC tumors. Red bars, tumors with amplification (upper panel) or overexpression (lower panel); blue bars, tumors with decreased expression; gray bars, tumors with no change in gene copy number (upper panel) or expression (lower panel). H) Analysis of PRKCI, SOX2, HHAT and GLI1 expression in primary LSCC tumors. Primary LSCC tumors were force ranked on PRKCI expression and grouped into top and bottom tertiles corresponding to high and low PRKCI expression, respectively. Box plots denote the expression of PRKCI, SOX2, HHAT and GLI1 in tumors expressing low PRKCI (low) and high PRKCI (high). Bars represent the median, boxes denote the 25 and 75% intervals; whiskers represent the 90% confidence intervals. See also Figure S4, and Tables S4 and S5.

Chromatin immunoprecipitation sequencing (ChIP-Seq) analysis of SOX2 revealed that SOX2 can occupy the HHAT promoter and regulate HHAT expression in glioblastoma cells (Fang et al., 2011). The proximal human HHAT promoter contains 7 potential SOX2 binding motifs (Fig. 4D, inset). Interestingly, SOX2 ChIP assays in NT and PKCι RNAi oncospheres revealed SOX2 occupancy on regions A and B in the HHAT promoter in NT RNAi cells, and a significant decrease in occupancy in PKCι RNAi cells without a change in SOX2 protein abundance (Fig. 4D). Furthermore, SOX2 KD (Fig. 4E) led to a decrease in HHAT mRNA and protein (Fig. 4F). Similar results were obtained in H1703 oncospheres (Fig. S4). Thus, SOX2 regulates HHAT promoter activity and expression by a PKCι-dependent mechanism. Not surprisingly, growth of SOX2 RNAi oncospheres cannot be reconstituted by Hh agonist (Fig. S4E), indicating that SOX2 regulates genes in addition to HHAT that are also required for maintenance of the stem-like phenotype.

Given the functional link between PRKCI and SOX2 we assessed whether these genes are coordinately amplified and overexpressed in LSCC tumors. Interrogation of a LSCC dataset (178 cases) from The Cancer Genome Atlas (TCGA) revealed that PRKCI and SOX2 are frequently co-amplified and coordinately overexpressed in primary LSCC tumors (Fig. 4G). Analysis also revealed a strong positive correlation between PKCι and SOX2 expression with HHAT and GLI1, two key Hh pathway components regulated by PKCι in oncospheres harboring PRKCI copy number gains (Fig. 4H). SOX2 and HHAT expression also correlated in LSCC tumors, suggesting that SOX2 regulates HHAT expression in vivo (Table S5) and providing evidence for the PKCι-SOX2-HHAT signaling axis in vivo. We next assessed whether co-ordinate PRKCI and SOX2 amplification may drive a “stem-like” genotype in primary LSCC tumors. Airway basal cells exhibit stem-like properties (Hajj et al., 2007; Hong et al., 2004; Rock et al., 2009) and are putative stem or progenitor cells for LSCC (Ooi et al., 2010; Wistuba and Gazdar, 2006). Therefore, we tested for an association between PRKCI and SOX2, and expression of a previously described 13-gene airway basal stem cell signature (Shaykhiev et al., 2013). Analysis revealed a strong positive correlation between PRKCI, SOX2, and expression of 6 of 13 of the airway basal stem cell signature genes (Table S6). Interestingly, this correlation was not observed in primary LAC tumors, consistent with the proposed role of basal cells as putative cells of origin for LSCC, but not LAC (Wistuba and Gazdar, 2006).

Primary LSCC cells require PKCι, SOX2 and HHAT for oncosphere formation and proliferation

To assess whether PKCι-SOX2-Hh signaling regulates behavior of primary LSCC tumors, we established oncosphere cultures from primary LSCCs obtained from patients undergoing tumor resection. Similar to established cell lines, primary LSCC tumor cells readily grow as oncospheres in non-adherent stem cell culture (Fig. 5A). PKCι, SOX2 or HHAT RNAi led to efficient knockdown of their respective target (Fig. 5B). PKCι and SOX2 RNAi cells also exhibited reduced HHAT and GLI1 expression, whereas HHAT KD had no demonstrable effect on PKCι or SOX2 expression (Fig. 5B). PKCι, SOX2 and HHAT RNAi similarly impaired oncosphere proliferation (Fig. 5C). Furthermore, PKCι, SOX2 and HHAT RNAi cells produced small, disorganized oncospheres (Fig. 5D) exhibiting membrane blebbing and intracellular vacuoles indicative of reduced cell viability (Fig. 5E; arrows). Thus, PKCι-SOX2-Hh signaling is important for maintenance and survival of primary human LSCC stem-like cells.

Figure 5. Primary LSCC cells require PKCι, SOX2 and HHAT for oncosphere formation and proliferation.

Figure 5

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A) Phase contrast photomicrographs of oncospheres from three surgically-resected primary LSCC tumors. B) QPCR analysis of PKCι, SOX2, HHAT and GLI1 expressed as % NT RNAi control +/− SEM, n=3, *p<0.02. C) Proliferation of oncospheres by MTT assay expressed as % NT RNAi control +/− SEM; n=3, *p<4.0×10−07. D) Clonal expansion of oncospheres. Results expressed as % NT RNAi control +/− SEM; n=3, *p<0.0002. E) Photomicrographs of NT, PKCι, SOX2 and HHAT KD oncospheres. Arrows show membrane blebbing indicative of cell death. Results are representative of three independent experiments.

SOX2 is a PKCι substrate

Analysis of the SOX2 amino acid sequence indicated that SOX2 may serve as a substrate for atypical PKC. When purified recombinant human PKCι and recombinant human SOX2 were incubated in a kinase reaction, robust incorporation of 32P from ATP into SOX2 was observed that is not observed in the absence of PKCι (Fig. 6A). To identify the site(s) phosphorylated by PKCι, phosphorylated SOX2 was subjected to proteolytic digestion and mass spectrometric (MS) analysis as described in Experimental Procedures. MS analysis recovered peptides encompassing all known phosphorylation sites on SOX2, and revealed a single, previously-uncharacterized phosphorylation site, T118, that conforms to an atypical PKC consensus recognition motif (Fig. 6B).

Figure 6. PKCι directly phosphorylates SOX2 at a unique site, T118, which is required for SOX2 function.

Figure 6

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A) Recombinant human SOX2 was incubated in kinase reaction buffer containing 32P-ATP in the absence or presence of recombinant PKCι. Phosphorylated SOX2 was detected by autoradiography and total SOX2 was detected by immunoblot analysis. B) Schematic of SOX2 protein structure; HMG=high mobility group domain, TAD=transactivation domain, NLS=nuclear localization sequence. Mass Spectrometric analysis revealed a single PKCι-mediated phosphorylation site on SOX2 at T118 which conforms to a consensus atypical PKC phosphorylation site motif (insert). C) Immunoblot analysis of H1299 oncopsheres stably transduced with NT or SOX2 RNAi, and then stably transfected with either empty vector (V) or vector encoding WT-SOX2 (WT), T118A-SOX2 (T118A), or T118D-SOX2 (T118D) mutants. D) QPCR analysis for HHAT and GLI1 expressed relative to NT RNAi control cells +/− SEM; n=3, *p<0.03. E) Clonal expansion expressed relative to NT RNAi control cells +/− SEM; n>20 per cell type, *p<1.0×10−08. F) Soft agar growth expressed relative to NT RNAi control cells +/− SEM; n=5, *p< 4.0×10−05 G) Cellular localization and promoter occupancy of T118-SOX2 phosphorylation mutants. Immunoblot analysis of cytoplasmic and nuclear fractions for FLAG, Lamins A/C and MEK1 (immunoblots). Lamins A/C served as a marker of nuclei and MEK1 as a marker of cytoplasm. HHAT promoter occupancy (bar graph, lower panel) expressed as fold of IgG control +/− SEM; n=3; *p< 0.001. Results in D-G are representative of three independent experiments. See also Figure S5.

To assess the importance of T118 phosphorylation we first introduced a silent mutation into a FLAG-tagged SOX2 cDNA to render it resistant to SOX2 RNAi #2 and then mutagenized T118 to either alanine (T118A) or aspartic acid (T118D) to eliminate or mimic T118 phosphorylation. Immunoblot analysis of H1299 oncospheres stably transfected with WT-SOX2, T118A-SOX2, T118D-SOX2 or empty control vector, followed by SOX2 RNAi revealed efficient loss of endogenous SOX2 and expression of similar levels of WT and T118 SOX2 mutants (Fig. 6C). SOX2 RNAi oncospheres expressing control vector showed reduced HHAT and GLI1 expression as expected (Fig. 6D). In contrast, expression of exogenous WT- or T118D-SOX2 significantly restored HHAT and GLI1 expression in SOX2 RNAi cells, whereas T118A-SOX2 did not (Fig. 6D). As expected, SOX2 RNAi cells expressing empty control vector showed impaired clonal expansion and soft agar growth (Fig. 6E and 6F). Expression of WT- or T118D-SOX2 significantly reconstituted clonal expansion and soft agar growth in SOX2 RNAi oncospheres whereas T118A SOX2 did not (Fig. 6E and 6F). SOX2 ChIP assays revealed reduced SOX2 occupancy of the HHAT promoter in SOX2 RNAi oncospheres expressing empty control vector that is significantly restored by expression of either WT or T118D SOX2, but not T118A SOX2 (Fig. 6G). T118 resides between the HMG domain and a consensus nuclear localization sequence (NLS) in SOX2, suggesting that T118 phosphorylation could affect SOX2 DNA binding and/or nuclear localization. Immunoblot analysis revealed that WT, T118A and T118D SOX2 are predominantly nuclear (Fig. 6G, immunoblots). Next, SOX2 RNAi oncospheres expressing either wild-type, T118A or T118D SOX2, or control empty pCMV vector were transfected with a HHAT promoter luciferase reporter (pGL4-HHAT-luc) and assessed for HHAT promoter activity (Fig. S5). In all four cell lines tested, wild-type and T118D SOX2 stimulated HHAT promoter activity whereas T118A did not. Thus, PKCι-mediated T118 SOX2 phosphorylation regulates SOX2 occupancy and HHAT promoter activity to stimulate HHAT expression, thereby driving a PKCι-Hh signaling axis in LSCC oncospheres.

DISCUSSION

Accumulating evidence supports the existence of tumor-initiating or cancer stem cells in human lung tumors that possess the capacity to self-renew, and differentiate into bulk tumor cells. These cells drive tumor initiation, progression and metastasis, and may contribute to relapse after chemotherapy. The majority of cancer deaths are caused by tumor metastasis and relapse, making these cells an attractive therapeutic target to achieve lasting therapeutic responses. However the molecular mechanisms that drive the enhanced tumorigenic potential and stem-like behavior of tumor-initiating cells are poorly understood. We previously demonstrated that PKCι maintains a transformed phenotype in NSCLC cells, and is critical for tumor initiation in murine lung cancer models (Regala et al., 2009). Prkci is required for tumor initiation and expansion of Kras-transformed bronchio-alveolar stem cells (BASCs), putative lung cancer stem cells in murine LAC models (Regala et al., 2009). Here we show that PKCι plays a vital and previously unrecognized role in maintenance of stem-like cells isolated from human lung cancer cells harboring PRKCI copy number gains, and primary LSCC tumors. We also define a PKCι-SOX2-Hh signaling axis that drives the stem-like phenotype of these cells.

Aberrant Hh signaling has been implicated in the initiation and progression of various cancer subtypes including lung. In basal cell carcinomas and medulloblastomas, Hh signaling is activated by tumor-specific mutations in the Hh pathway components Patched, Smoothened or Suppressor of Fused (Epstein, 2008; Kool et al., 2008). Tumors without activating Hh mutations often utilize paracrine Hh signaling involving Hh ligand produced by tumor-associated stromal cells (Dierks et al., 2007; Tian et al., 2009; Yauch et al., 2008). Finally, some cancer cells exhibit autocrine Hh signaling, in which Hh ligand is produced and utilized by the same or neighboring tumor cells (Bar et al., 2007; Berman et al., 2003; Karhadkar et al., 2004; Stecca et al., 2007; Varnat et al., 2009). Autocrine Hh signaling has been described in SCLC and NSCLC (Park et al., 2011; Rodriguez-Blanco et al., 2013; Watkins et al., 2003), however the factors that regulate and maintain autocrine Hh signaling are largely unknown. We find that PKCι regulates autocrine Hh signaling in oncospheres harboring PRKCI copy number gains, and define a signaling cascade in which PKCι regulates HHAT, which catalyzes the rate-limiting step in Hh ligand processing. Our finding that HHAT KD inhibits clonal expansion which can be reconstituted with Hh agonist, provides compelling evidence for the importance of this autocrine Hh signaling mechanism in LSCC stem cell maintenance. Finally, our finding that HHAT KD oncospheres fail to generate tumors in vivo indicate that autocrine Hh signaling is vital for tumor initiation.

SOX2 is an oncogene in human LSCC (Bass et al., 2009; Hussenet et al., 2010; Yuan et al., 2010). Sox2 overexpression in mouse lung leads to increased lung epithelial cell proliferation and hyperplasia (Tompkins et al., 2011), whereas Sox2 deletion in mouse bronchiolar Clara cells, a potential lung regional stem cell, results in reduced cell proliferation and loss of airway differentiation markers (Tompkins et al., 2009). SOX2 is required for LSCC cell growth in vitro, and SOX2 overexpression in lung epithelial cells induces migration, transformed growth and tumor formation in vivo (Hussenet et al., 2010). Consistent with these findings, we demonstrate that SOX2 maintains the tumorigenic potential of human LSCC oncospheres. We also uncover an unexpected link between SOX2 and Hh signaling that is mediated by PKCι. Our findings are consistent with SOX2-mediated regulation of Shh expression in neural stem cells (NSCs) (Favaro et al., 2009) and provide a potential molecular mechanism for SOX2-mediated Hh signaling in NSC cells.

Phosphorylation can regulate Sox2 function in mouse embryonic stem cells (Jeong et al., 2010) and phosphoproteome analysis has identified Ser249, Ser250, and Ser251 as potential SOX2 phosphorylation sites (Van Hoof et al., 2009). However, the potential role of SOX2 phosphorylation in human cancer cells has not been investigated. We find that PKCι phosphorylates SOX2 at on a previously uncharacterized site T118. T118 phosphorylation regulates SOX2 binding to, and activity of the HHAT promoter. Our MS analysis, which interrogated all known SOX2 phosphorylation sites, detected pT118 but no other sites in LSCC oncospheres (data not shown). These data indicate that T118 is a major SOX2 phosphorylation site, though we cannot exclude the presence of other low level phosphorylation sites. Regardless, our functional data demonstrate the importance of T118 phosphorylation in SOX2 function and stem-like behavior.

Chromosome 3q26 amplification is one of the most frequent chromosomal alterations in human cancer and is found in a majority of LSCC (Balsara et al., 1997; Brass et al., 1996), serous ovarian (Sonoda et al., 1997; Sugita et al., 2000), cervical (Sugita et al., 2000), head and neck (Snaddon et al., 2001) , oral (Lin et al., 2005) and esophageal tumors (Imoto et al., 2001). Interestingly, a major susceptibility locus for chemically-induced mouse LSCC is syntenic with human 3q26 and includes Prkci and Sox2, suggesting a role for coordinate genetic alteration of Prkci and Sox2 in mouse LSCC (Wang et al., 2004). In this regard, SOX2 overexpression is only weakly transforming (Hussenet et al., 2010), and PKCι overexpression is not sufficient alone to cause cellular transformation (Fields laboratory, unpublished observation) suggesting that these oncogenic factors may act in concert to exhibit full tumorigenic potential. Consistent with this idea, PRKCI and SOX2 are frequently co-amplified and coordinately overexpressed in LSCC tumors, and these tumors appear to exhibit activate PKCι-SOX2-Hh signaling. Based on our data we propose that in LSCC tumors, and possibly other lung cancer subtypes harboring 3q26 amplification, PRKCI and SOX2 are co-oncogenic drivers activated through a single genetic alteration, that cooperatively drive tumorigenesis by maintaining a stem-like phenotype through activation of a PKCι-SOX2-Hh signaling axis. Interestingly, whereas each of the cell lines analyzed here harbor PRKCI gene copy number gain and exhibit PKCι-dependent Hh signaling, not all of these cell lines are classified as LSCC. Three cell lines are classified histologically as LSCC (H1703, H520 and H1869), whereas ChagoK1 and H1299 cells are classified as bronchiogenic carcinoma and LCLC, respectively, suggesting that PKCι-SOX2-HHAT signaling may be operative in lung tumor types other than LSCC that harbor PRKCI gene copy number gain. Given the prevalence of chromosome 3q26 amplification in human tumors, further studies are warranted to determine whether the PKCι-SOX2-Hh signaling axis elucidated here maintains a stem-like phenotype in other major tumor types harboring these genetic alteration.

Finally, our results have broad implications for development of new therapeutic strategies to target LSCC stem-like cells. SMO inhibitors are in clinical development, but their utility is limited in tumors that acquire Hh mutations that confer resistance. PKCι, which regulates the Hh pathway both upstream and downstream of SMO, may serve as an alternative therapeutic strategy for modulating Hh signaling in tumors harboring such mutations. Our data also provide a rationale for combined use of PKCι and Hh inhibitors for treatment of LSCC tumors. Finally, selective HHAT inhibitors have recently been developed (Petrova et al., 2013). Such inhibitors may exhibit particular potency in tumors harboring chromosome 3q26 amplification and commensurate activation of the autocrine PKCι-SOX2-HHAT signaling axis described here.

EXPERIMENTAL PROCEDURES

Cell lines, reagents and antibodies

Antibodies used: PKCι (BD Transduction Laboratories, San Jose, CA); β-actin, SOX2, GLI1, Lamin A/C, MEK1, phospho-aPKC Thr 410 (Cell Signaling, Danvers, MA); HHAT (Abgent San Diego, CA), phosphoserine/threonine (AbCAM, Cambridge, MA), SHH (Santa Cruz, Dallas, TX), FLAG (Sigma, St. Louis, MO). Auranofin (Prometheus Pharmaceuticals), LDE225 and Hh-Ag1.5 (Cellagen, San Diego, CA) were diluted in dimethyl sulfate (Me2SO). Cells were treated at concentrations indicated in figures, and control cells received an equal volume of Me2SO at 0.1% v/v. H1703, H1299, H520, H1869 and ChagoK1 lung carcinoma cell lines were obtained from American Type Culture Collection (Manassas, VA) and maintained in low passage culture as recommended. H1299 firefly luciferase cells were established by transducing cells with retroviral firefly luciferase.

Enrichment, clonal expansion and redifferentiation of lung cancer cell exhibiting a stem-like phenotype

Oncospheres were enriched from H1703, H1299, H520, H1869 and ChagoK1 cells and from primary human LSCC tumors essentially as described previously (Eramo et al., 2008; Justilien et al., 2012). Human tumor tissues were obtained under informed consent and their collection was approved by the Mayo Clinic Institutional Review Board. Experimental details are provided in Supplemental Experimental Procedures.

Lentiviral RNAi constructs, cell transduction, plasmids, transfections and immunoblot analysis

Lentiviral vectors containing short hairpin RNAi against human PKCι, HHAT, GLI1 and SOX2 were obtained from Sigma-Aldrich (St. Louis, MO) and packaged into recombinant lentiviruses as described previously (Frederick et al., 2008). The human SOX2 cDNA was cloned into a FLAG mammalian expression vector (QIAGEN, Valencia, CA, Cat# DAM-105) and rendered RNAi-resistant by silent mutation to disrupt the SOX2 RNAi #2 target site as described previously (Frederick et al., 2008). GLI1 was immunoprecipated from cell lysates using mouse anti-GLI1 and protein A/G Agarose beads (Santa Cruz Biotechnologies) according to manufacturer’s procedure. Phospho(S/T)-GLI1 was detected in GLI1 immunoprecipitates by immunoblot analysis using phospho-S/T antibody. Detailed protocols are provided in Supplemental Experimental Procedures.

Anchorage-independent growth and orthotopic implantation studies

Anchorage-independent growth was assessed as described previously (Justilien et al., 2012). All animal procedures were approved by the Mayo Clinic IACUC. Experimental details are provided in Supplemental Experimental Procedures.

Metabolic labeling and detection of palmitoylated SHH

Metabolic labeling and detection of palmitoylated SHH was performed essentially as described (Yap et al., 2010). Experimental details are provided in Supplemental Experimental Procedures.

Deep sequence analysis of lung cancer cell transcriptomes

Deep sequencing analysis was performed essentially as described previously (Kalari et al., 2012). Experimental details are provided in Supplemental Experimental Procedures.

Analysis of TCGA gene expression data

Data from TCGA was analyzed using cBIO (http://www.cbioportal.org/public-portal/) software to correlate gene amplifications and gene expression in 178 human LSCC and 129 LAC tumors. Gene count (RPKM) values for these tumors were rank ordered according to PRKCI expression and the top and bottom third were compared for differential expression of HHAT, SOX2, GLI1, and the 13 gene airway basal stem cell gene signature (Shaykhiev et al., 2013).

In vitro PKCι kinase assays

PKCι in vitro kinase assays were performed as described previously (Justilien et al., 2011). Details are provided in Supplemental Experimental Procedures.

Chromatin immunoprecipitation and analysis

Chromatin immunoprecipitation assays (ChIPs) were performed to assess SOX2 occupancy on the HHAT promoter as described in Supplemental Experimental Procedures.

HHAT promoter cloning and luciferase assays

A ~1 kB fragment of the promixal 5’ promoter region of human HHAT was cloned by PCR from genomic DNA from H1703 cells. The promoter was purified, sequenced to validate a match with the Pub-Med database (NM_001170564), and TA cloned into pGL4-luciferase reporter plasmid using standard techniques. Luciferase assays were conducted as described in Supplemental Experimental Procedures.

Mass spectrometry analysis of SOX2 phosphorylation

PKCι phosphorylated SOX2 or SOX2 immunoprecipitated from H1703 cells was isolated and submitted to the Mayo Clinic Cancer Center Protein Chemistry and Proteomics Shared Resource for proteolytic cleavage and phosphorylation site analysis by mass spectrometry. Details are provided in Supplemental Experimental Procedures.

Accession Number

All sequencing data are deposited in GEO (accession number GSE48599).

Supplementary Material

01
02
03
04

HIGHLIGHTS.

  • PRKCI and SOX2 are co-amplified and coordinately overexpressed in LSCC tumors.

  • PKCι transcriptionally regulates expression of Hedgehog Acyl Transferase (HHAT).

  • PKCι directly phosphorylates and recruits SOX2 to the HHAT promoter.

  • PKCι and SOX2 activate autocrine Hh signaling to maintain LSCC stem-like cells.

SIGNIFICANCE.

Lung cancer is the leading cause of cancer deaths worldwide. LSCC represents 30% of lung cancer diagnoses, and is characterized by poor therapeutic response, a high relapse rate and poor prognosis. Here we identify a genetic, biochemical and functional link between two oncogenes on chromosome 3q26, PRKCI and SOX2, which are co-amplified and overexpressed in a majority of LSCC tumors. Our data indicate that chromosome 3q26 copy number gains serve to genetically activate PRKCI and SOX2 which together establish a PKCι-SOX2-HHAT signaling axis that drives a stem-like phenotype. Our results provide a compelling rationale for use of PKCι inhibitors currently in clinical development to target LSCC.

ACKNOWLEDGEMENTS

We thank Dr. Robert Bergen and Benjamin Madden of the Mayo Clinic Proteomics Research Center for performing mass spectrometric analysis of SOX2 phosphorylation, Dr. John Odell for consenting patients and obtaining fresh primary lung tumor tissue, Dr. Al Copland for the retroviral firefly luciferase construct, Ms. Capella Weems and Dr. Lee Jamieson for technical assistance, Ms. Brandy Edenfield for immunohistochemical analysis, the Mayo Clinic Florida Biospecimen Acquisition and Processing laboratory for procurement of primary lung tumor tissues, and the Mayo Clinic Advanced Genomic Technology Center for RNA-seq and data analysis. We also acknowledge Dr. Howard Crawford, and members of the Fields laboratory, for critical feedback on the manuscript. This work was supported by grants from National Institutes of Health/National Cancer Institute (R01 CA081436-16 and R21 CA151250-02), the V Foundation for Cancer Research, the James and Esther King Biomedical Research Program (1KG-05-33971) and the Mayo Clinic Center for Individualized Medicine (CIM) to APF; and a National Institutes of Health Research Supplement to Promote Diversity in Health-related Research Award from the National Cancer Institute (VJ). APF is the Monica Flynn Jacoby Professor of Cancer Research, an endowment fund that provides partial support for the investigator’s research program.

Footnotes

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Supplementary Materials

01
NIHMS558180-supplement-01.docx (1.3MB, docx)
02
NIHMS558180-supplement-02.xlsx (45.3KB, xlsx)
03
NIHMS558180-supplement-03.xlsx (25.2KB, xlsx)
04
NIHMS558180-supplement-04.xlsx (13.5KB, xlsx)

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