Regulation of Head and Neck Squamous Cancer Stem Cells by PI3K and SOX2 - PubMed (original) (raw)

. 2016 Sep 15;109(1):djw189.

doi: 10.1093/jnci/djw189. Print 2017 Jan.

Phuong N Le 1, Bettina Miller 1, Brian C Jackson 1, Justin R Eagles 1, Cera Nieto 1, Jihye Kim 1, Binwu Tang 1, Magdalena J Glogowska 1, J Jason Morton 1, Nuria Padilla-Just 1, Karina Gomez 1, Emily Warnock 1, Julie Reisinger 1, John J Arcaroli 1, Wells A Messersmith 1, Lalage M Wakefield 1, Dexiang Gao 1, Aik-Choon Tan 1, Hilary Serracino 1, Vasilis Vasiliou 1, Dennis R Roop 1, Xiao-Jing Wang 1, Antonio Jimeno 1

Affiliations

Regulation of Head and Neck Squamous Cancer Stem Cells by PI3K and SOX2

Stephen B Keysar et al. J Natl Cancer Inst. 2016.

Abstract

Background: We have an incomplete understanding of the differences between cancer stem cells (CSCs) in human papillomavirus-positive (HPV-positive) and -negative (HPV-negative) head and neck squamous cell cancer (HNSCC). The PI3K pathway has the most frequent activating genetic events in HNSCC (especially HPV-positive driven), but the differential signaling between CSCs and non-CSCs is also unknown.

Methods: We addressed these unresolved questions using CSCs identified from 10 HNSCC patient-derived xenografts (PDXs). Sored populations were serially passaged in nude mice to evaluate tumorigenicity and tumor recapitulation. The transcription profile of HNSCC CSCs was characterized by mRNA sequencing, and the susceptibility of CSCs to therapy was investigated using an in vivo model. SOX2 transcriptional activity was used to follow the asymmetric division of PDX-derived CSCs. All statistical tests were two-sided.

Results: CSCs were enriched by high aldehyde dehydrogenase (ALDH) activity and CD44 expression and were similar between HPV-positive and HPV-negative cases (percent tumor formation injecting ≤ 1x10(3) cells: ALDH(+)CD44(high) = 65.8%, ALDH(-)CD44(high) = 33.1%, ALDH(+)CD44(high) = 20.0%; and injecting 1x10(5) cells: ALDH(-)CD44(low) = 4.4%). CSCs were resistant to conventional therapy and had PI3K/mTOR pathway overexpression (GSEA pathway enrichment, P < .001), and PI3K inhibition in vivo decreased their tumorigenicity (40.0%-100.0% across cases). PI3K/mTOR directly regulated SOX2 protein levels, and SOX2 in turn activated ALDH1A1 (P < .001 013C and 067C) expression and ALDH activity (ALDH(+) [%] empty-control vs SOX2, 0.4% ± 0.4% vs 14.5% ± 9.8%, P = .03 for 013C and 1.7% ± 1.3% vs 3.6% ± 3.4%, P = .04 for 067C) in 013C and 067 cells. SOX2 enhanced sphere and tumor growth (spheres/well, 013C P < .001 and 067C P = .04) and therapy resistance. SOX2 expression prompted mesenchymal-to-epithelial transition (MET) by inducing CDH1 (013C P = .002, 067C P = .01), followed by asymmetric division and proliferation, which contributed to tumor formation.

Conclusions: The molecular link between PI3K activation and CSC properties found in this study provides insights into therapeutic strategies for HNSCC. Constitutive expression of SOX2 in HNSCC cells generates a CSC-like population that enables CSC studies.

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Figures

Figure 1.

Figure 1.

Evaluation of tumor recapitulation following serial implantation of cancer stem cells (CSCs) in mice. A) Heterogeneous staining of CD44 cell surface protein and aldehyde dehydrogenase activity (Aldefluor assay) across 10 head and neck squamous cell carcinoma (HNSCC) patient-derived xenograft (PDX) cases. Gated regions are representative of the sorted CSC population for each PDX model defined in Table 1. Diagram depicting the CSC population falling within the overlapping region of the larger aldehyde dehydrogenase (ALDH)+ and CD44high populations. B) Tumor cell composition across PDX models measured as percentages (flow cytometry) for each of the four possible (ALDH-CD44low, ALDH-CD44high, ALDH+CD44low, ALDH+CD44high) tumor populations (average of 2+ sorted tumors). C) Tumors resulting from two consecutive CSC implantations for four HNSCC PDX show tumors retain characteristics of the originating tumor, including morphology (hematoxylin and eosin staining) and epidermal growth factor receptor (EGFR) expression with serial implantation. Scale bars = 100 µm. D) Tumors arising from the first or second/serial CSC implantation consistently cluster with the originating PDX tumor (clustering generated with FPKM values > 1). Tumors arising from CUHN004 and CUHN013 non-CSCs (ALDH-CD44high) were more distantly associated with the original PDX tumor. ALDH = aldehyde dehydrogenase; CSC = cancer stem cell; FPKM = fragments per kilobase of exon per million fragments mapped; HNSCC = head and neck squamous cell carcinoma; HPV-neg. = human papilloma virus–negative; HPV-pos. = human papilloma virus–positive; PDX = patient-derived xenograft.

Figure 2.

Figure 2.

Transcriptome and protein expression profile of head and neck squamous cell carcinoma (HNSCC) cancer stem cells (CSCs). A) Gene set enrichment analysis (GSEA) pathway enrichment in HNSCC CSC populations compared with cells negative for CSC markers and relevant genes statistically significantly upregulated in CSCs across cases (bold = stem cell–related processes; red = phosphoinositide 3-kinase (PI3K)/ mechanistic target of rapamycin (mTOR)–related pathways; blue = transforming growth factor beta–related pathways). Statistical significance was calculated by a two-sided modified Kolmogorov-Smirnov permutation test. B) Immunoblot and reverse transcription polymerase chain reaction analysis of tumor-derived (CUHN013, CUHN022) CSCs and cells negative for CSC markers. C) Co-expression of SOX2 with aldehyde dehydrogenase (ALDH)1A1, markers of PI3K/mTOR pathway activation and E-cadherin in vivo. Scale bars = 100 µm. CSC = cancer stem cell; HNSCC = head and neck squamous cell carcinoma; HPV-neg. = human papilloma virus–negative; HPV-pos. = human papilloma virus–positive; mTOR = mechanistic target of rapamycin; PI3K = phosphoinositide 3-kinase; TGF-β = transforming growth factor beta.

Figure 3.

Figure 3.

Effects of phosphoinositide 3-kinase inhibition on cancer stem cells (CSCs) and SOX2 expression in cell lines. A) Schema for pretreatment of tumors prior to sorting of CSCs and tumor populations. Average tumor growth of pretreated (control, PX-866 and XRT) CSC populations (CUHN014 = 10 aldehyde dehydrogenase [ALDH]+CD44high cells, CUHN022 = 10 000 ALDH+ cells, CUHN013 = 10 000 ALDH+CD44high cells) was substantially decreased with PX-866 or XRT. Numbers represent tumor take rate for implantations in five mice (tumor volume > 500 mm3). B) Expression of ALDH1A1 and (C) SOX2 are upregulated in sphere culture (013C, 067C cells) when compared with cells grown in a monolayer. D) ALDH1A1 and SOX2 protein levels are also increased in sphere culture compared with monolayer cells grown in either RMK media (10% fetal bovine serum) or serum-free CSC media. E) The ALDH+ population is increased in sphere culture. Graphed results are presented as mean ± SD of three independent experiments. Statistical significance was calculated by the two-tailed Student’s t test (*P < .05; †P < .01). ALDH = aldehyde dehydrogenase; CSC = cancer stem cell; PI3K = phosphoinositide 3-kinase.

Figure 4.

Figure 4.

Effects of phosphoinositide 3-kinase (PI3K) inhibition on SOX2 transcriptional activity and the aldehyde dehydrogenase (ALDH)+ population. A) The ALDH+ population is statistically significantly reduced in sphere cultures and (B) monolayer cultures (013C, 067C cells) treated with the PI3K inhibitor ZSTK474. Gated regions are set at 0.1 positive of N,N-diethylaminobenzaldehyde-negative controls. C) PI3K inhibition (ZSTK474) suppresses ALDH1A1 gene expression in 013C cells grown as spheres and (D) patient-derived xenograft tumor-derived cancer stem cells (ALDH+CD44high) grown in sphere culture. E) Immunoblot showing that PI3K pathway inhibition by ZSTK474 decreases ALDH1A1 and SOX2 protein levels within 48 hours in 013C, 067C cells. F) Exogenous expression of SOX2 increases the SORE6-GFP+ population in 013C cells. Gated regions set as 0.5% of control GFP+ cells. G) Sphere culture of 013C-SOX2 cells has increased SORE6-GFP+ cells compared with control cells and monolayer culture. Scale bars = 100 µm. H) PI3K treatment decreased activation of the SORE6 reporter and the ALDH+ population in 013C spheres. Graphed results are presented as mean ± SD of three independent experiments. Statistical significance was calculated by the two-tailed Student’s t test (*P < .05; †P < .01). ALDH = aldehyde dehydrogenase; PI3K = phosphoinositide 3-kinase; SORE6 = SOX2/OCT4 response elements reporter.

Figure 5.

Figure 5.

Role of phosphoinositide 3-kinase/mechanistic target of rapamycin in the regulation of translation of SOX2 protein and aldehyde dehydrogenase (ALDH)1A1 expression. A) Silencing AKT1 or EIF4E statistically significantly reduces SOX2 protein levels in 013C and 067C cells. B) Treatment of 013C and 067C cells with the EIF4E inhibitor 4EGI-1 decreased SOX2 protein. C) The EIF4E protein physically binds SOX2 mRNA as measured by RNA immunoprecipitation using EIF4E specific antibodies. Western blot analysis showing successful precipitation of EIF4E protein. D) Exogenous expression of SOX2 (013C, 067C cells) increases ALDH1A1 protein. E) ALDH1A1 levels statistically significantly increase within six days following exogenous expression of SOX2. F) SOX2 expression increased the ALDH+ population in both 013C and 067C cells. G and H) Silencing of SOX2 in overexpression cells decreases ALDH1A1 expression and protein levels and (I) the ALDH+ population. J) Exogenous expression of SOX2 activates an ALDH1A1-promoter as measured by a luciferase assay. Chromatin immunoprecipitation using SOX2 antibodies suggests SOX2 associates with the ALDH1A1 promoter in 013C cells. Graphed results are presented as mean ± SD of three or more independent experiments. Statistical significance was calculated by the two-tailed Student’s t test (*P < .05; †P < .01). ALDH = aldehyde dehydrogenase; mTOR = mechanistic target of rapamycin; PI3K = phosphoinositide 3-kinase.

Figure 6.

Figure 6.

Effects of SOX2 expression on cellular morphology and gene expression. A) mRNA-sequencing analysis of 013C cells expressing SOX2 highlights increased expression of stemness pathways, cell-to-cell signaling and growth factor signaling. B) Selected cancer genes upregulated by SOX2 expressing 013C and 067C cells. Red = genes upregulated in both cell lines. C) Exogenous expression of SOX2 in 013C cells suppresses expression of SNAI1 mRNA while (D) increasing CDH1 expression. E) SOX2 expression decreases SNAIL protein levels while increasing E-cadherin in 013C cells. F) E-cadherin expression detected by immunocytochemistry. 067C cells demonstrate “typical” epithelial e-cadherin staining, while increased levels of e-cadherin in SOX2 expression 013C cells appears to be localized in the cytoplasm. This was confirmed by two different antibodies. Scale bars = 10 µm. G) Exogenous expression of SOX2 leads to a more epithelial phenotype in 013C cells. Scale bars = 100 µm. H) SOX2 (red) and E-cadherin (brown) expression are colocalized (red arrow) on the proliferating sphere surface, while SOX2 levels decrease towards the sphere interior (013C-SOX2 cells). Scale bar = 100 µm. I) SOX2 expression statistically significantly decreases invasiveness of 013C, 036C, 067C cells as measured in a matrigel-coated, 8 µm pore chamber assay. Scale bars = 100 µm. Graphed results are presented as mean ± SD of three or more independent experiments. Statistical significance was calculated by the two-tailed Student’s t test (*P < .05; †P < .01).

Figure 7.

Figure 7.

SOX2 expression promotes tumor formation. A) Exogenous expression of SOX2 increased resistance to docetaxel by the MTS assay (013C cells). B) SOX2 expression increases sphere number and sphere size in serum-free low attachment conditions (013C, 067C cells). Scale bar = 400 µm. C) Silencing of SOX2 in tumor- derived CUHN013 cancer stem cells (CSCs) decreases sphere number (sphere initiation) but not sphere size (proliferation) in culture. Graphed results are presented as mean ± SD of three or more independent experiments. Statistical significance was calculated by the two-tailed Student’s t test (*P < .05; †P < .01). D) SOX2 expression increased tumor initiation and growth of relative few (105) 013C cells implanted into five mice compared with parental and empty-vector controls, which had difficulty generating tumors within four months. E) Tumors resulting from SOX2 cells have higher levels of SOX2 and aldehyde dehydrogenase (ALDH)1A1 than control tumors. Scale bars = 100 µm. F) Sorted CUHN013 CSCs readily form spheroids in an anchorage-independent matrigel assay when cultured in serum-free CSC media. Scale bars = 200 µm. G) Single SORE6+ CSCs generate both SORE+ and SORE- daughter cells in anchorage-independent conditions. Three representative time-lapse cases where SORE6-mCherry+ or SORE6-GFP+ CSCs give rise to SORE6- cells. Scale bars = 100 µm. H) Proposed mechanism of the phosphoinositide 3-kinase/mechanistic target of rapamycin role in “stemness” maintenance by regulating SOX2 and ALDH1A1 expression. CSC = cancer stem cell; mTOR = mechanistic target of rapamycin; PI3K = phosphoinositide 3-kinase; SORE6 = SOX2/OCT4 response element.

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