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. 2020 Jan;14(1):139-158.
doi: 10.1002/1878-0261.12584. Epub 2019 Oct 26.

Distinct roles of PIK3CA in the enrichment and maintenance of cancer stem cells in head and neck squamous cell carcinoma

Xi Chen  1 , Yu Cao  1   2 , Wafik Sedhom  1 , Ling Lu  1 , Yanqiu Liu  1   3 , Haibo Wang  1   4 , Masako Oka  1 , Sophia Bornstein  5 , Sherif Said  6 , John Song  1 , Shi-Long Lu  1   6   7
Affiliations

Distinct roles of PIK3CA in the enrichment and maintenance of cancer stem cells in head and neck squamous cell carcinoma

Xi Chen et al. Mol Oncol. 2020 Jan.

Abstract

Recurrence and metastasis are the major causes of mortality in head and neck squamous cell carcinoma (HNSCC). It is suggested that cancer stem cells (CSCs) play pivotal roles in recurrence and metastasis. Thus, a greater understanding of the mechanisms of CSC regulation may provide opportunities to develop novel therapies for improving survival by controlling recurrence or metastasis. Here, we report that overexpression of the gene encoding the catalytic subunit of PI3K (PIK3CA), the most frequently amplified oncogene in HNSCC, promotes epithelial-to-mesenchymal transition and enriches the CSC population. However, PIK3CA is not required to maintain these traits and inhibition of the phosphatidylinositol 3-kinase (PI3K) signaling pathway paradoxically promotes CSC population. Molecular analysis revealed that overexpression of PIK3CA activates multiple receptor tyrosine kinases (RTKs), in which ephrin receptors (Ephs), tropomyosin receptor kinases (TRK) and mast/stem cell growth factor receptor (c-Kit) contribute to maintain CSC population. Accordingly, simultaneous inhibition of these RTKs using a multi-kinase inhibitor ponatinib has a superior effect at eliminating the CSC population and reduces metastasis of PIK3CA-overexpressing HNSCC cells. Our result suggests that co-targeting of Ephs, TRKs and the c-Kit pathway may be effective at eliminating the PI3K-independent CSC population, thereby providing potential targets for future development of a novel anti-CSC therapeutic approach for HNSCC patients, particularly for patients with PIK3CA amplification.

Keywords: PIK3CA; cancer stem cell; head and neck squamous cell carcinoma; ponatinib; recurrence and metastasis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Overexpression of PIK3CA promotes EMT and enriches CSCs. (A) Western blotting of p110α, total AKT, phospho‐AKT473, E‐cadherin and vimentin in two CUCON and two CU110 cells. GAPDH was used as a loading control. Quantification of western blots by imagej is shown on the right. *P < 0.05 (two‐tailed Student t‐test). (B) qRT‐PCR analysis of transcriptional factors regulating EMT in two CUCON and two CU110 cells. The results are presented as mean of two different experiments with SD (error bars). Each experiment was done in triplicate. *P < 0.05 (two‐tailed Student t‐test). (C) FACS analysis of cell surface marker, CD44 in two CUCON and two CU110 cells. Quantification data are represented as mean ± SD (n = 3). An example of CD44 FACS is shown on the right. *P < 0.05 (two‐tailed Student t‐test). (D) FACS analysis of cell surface marker, CD24 in two CUCON and two CU110 cells. Quantification data are represented as mean ± SD (n = 3). An example of CD44 FACS is shown on the right. *P < 0.05 (two‐tailed Student t‐test). (E) ALDH activity assay using ALDEFLUOR staining in two CUCON cells and two CU110 cells; quantification data are represented as mean ± SD (n = 3). *P ˂ 0.05. An example of ALDH FACS is shown on the right. *P < 0.05 (two‐tailed Student t‐test). (F) SP fraction detected by Hoechst dye‐effluxing assay in two CUCON and two CU110 cells. Right panel: representative FACS plots of CUCON and CU110 cells treated with Hoechst 33342 in the presence and absence of verapamil (ver). The specificity of SP fraction was validated by a verapamil elimination experiment. Left panel: quantification of gated SPs of CUCON and CU110 cells. Quantification data are represented as mean ± SD (n = 3). *P < 0.05 (two‐tailed Student t‐test).
Figure 2
Figure 2
Sphe‐forming is a functional readout of cancer stemness. (A) HNSCC Sphe‐forming ability of two CUCON and two CU110 cells. Left panel: Phase‐contrast images of CUCON and CU110 cells in serum‐free and ultralow attachment culture condition. Right panel: Quantification of Sphe numbers for two CUCON and two CU110 cells. Sphes with diameter ≥ 30 μm were counted. Sphe‐forming capacity is defined as percentage of total number of Sphe formed by CU110 cells in comparison with CUCON cells. Quantification data are represented as mean ± SD (n = 3). *P < 0.05 (two‐tailed Student t‐test). Scale bar: 100 µm. (B) qRT‐PCR for expression of embryonic stem cell genes in the mono‐ or Sphe‐cultured CU110 cells. GAPDH was used as an internal control. The results are presented as mean of two different experiments with SD (error bars). Each experiment was done in triplicate. *P < 0.05 (two‐tailed Student t‐test). (C) Left panel (top): H&E staining for HNSCC Sphe section; left panel (bottom): vimentin‐positive layer was delineated by white dotted lines and hollow center of spheroid was delineated by white dotted lines. Right panel: IF staining on Sphe sections using the antibodies as indicated in figures. Scale bar: 50 µm. (D) In vivo tumorigenicity by subcutaneous injection of 1000 cells isolated from either Sphe‐ or mono‐cultured CU110 cells to the flanks of C57BL6 mice. Left panel: Images of tumors developed from mono (one of three mice) or Sphe‐cultured CU110 cells (three of three mice). The tumor volume is average of tumors developed in mice with SD for each group and is shown on the right. **P ˂ 0.01 (two‐tailed Student t‐test). Scale bar: 2 mm. (E) Correlation between p110α expression and Sphe‐forming ability in human HNSCC cell lines. Left panel: Western blotting and HNSCC Sphe‐forming assay were performed in 17 human head and neck cancer cell lines (UMSCC1/2/10A/10B/22A/22B/47, VU1131/1365, SCC9, Fadu, HN6, Tu167, Cal27, M4C/4E and LNM1). Relative quantification of p110α was done by quantifying band intensity of p110α and GAPDH, and is shown as percentage of GAPDH band intensity. **P ˂ 0.01 (two‐tailed Student t‐test). Right panel: Representative images of Sphe‐forming capacity of HNSCC cell lines in serum‐free and ultralow attachment culture condition. Scale bar: 100 µm.
Figure 3
Figure 3
Knocking down of PIK3CA failed to reverse EMT and reduce CSC population. (A) Western blotting of p110α, E‐cadherin and vimentin in CU110‐2 cells stably transfected with lentiviral‐mediated shRNA (shPIK3CA) or a scrambled control (SCR). GAPDH was used as a loading control. Quantitation of western blots is shown in right. (B) HNSCC Sphe‐forming assay of CU110 cells stably transfected either shPIK3CA or SCR lentivirus. Sphes with diameter ≥ 30 μm were counted; quantification of Sphe is shown on the right, n = 3; error bars indicate SD. *P < 0.05 (two‐tailed Student t‐test). Scale bar: 100 µm. (C) FACS analysis of CD44 in CU110 cells stably transfected either shPIK3CA or SCR lentivirus. Quantification of CD44 population is shown on top. n = 3; error bars indicate SD. (D) FACS analysis of CD24 in CU110 cells stably transfected either shPIK3CA or SCR lentivirus. Quantification of CD24 population is shown on top. n = 3; error bars indicate SD. (E) SP fraction using Hoechst dye‐effluxing analysis in CU110 cells stably transfected either shPIK3CA or SCR lentivirus. Quantification of SP fraction is shown on top. n = 3; error bars indicate SD. *P < 0.05 (two‐tailed Student t‐test). (F) HNSCC Sphe‐forming assay of Fadu or UMSCC47 cell lines stably transfected with either shPIK3CA or SCR. The quantification is shown on the left. Error bars indicate SD. Scale bar: 100 µm.
Figure 4
Figure 4
Knocking down of key components in PI3K pathway promotes CSC population. (A) Western blotting of p110α and p85α in two CUCON and two CU110 cells. GAPDH was used as a loading control. Quantitation of western blots is shown on the right. *P < 0.05 (two‐tailed Student t‐test). (B) HNSCC Sphe‐forming assay of CU110 cells stably transfected with either shPIK3R1 or SCR lentivirus. The quantification is shown on top. Sphes with diameter ≥ 30 μm were counted. n = 3; error bars indicate SD. *P < 0.05 (two‐tailed Student t‐test). Scale bar: 100 µm. (C) FACS analysis of CD44 population in CU110 cells stably transfected with either shPIK3R1 or SCR lentivirus. Error bars indicate SD. n = 3. (D) FACS analysis of CD24 population in CU110 cells stably transfected with either shPIK3R1 or SCR lentivirus. Error bars indicate SD. n = 3. (E) SP fraction using Hoechst dye‐effluxing analysis in CU110 cells stably transfected either shPIK3R1 or SCR lentivirus. Quantification of SP fraction is shown on top. Error bars indicate SD. n = 3. *P < 0.05 (two‐tailed Student t‐test). (F) HNSCC Sphe‐forming assay of CU110 cells stably transfected with shAKT1, shAKT2 or SCR lentivirus. The quantification is shown on top. Sphes with diameter ≥ 30 μm were counted. n = 3; error bars indicate SD. *P < 0.05 (two‐tailed Student t‐test). Scale bar: 100 µm. (G) FACS analysis of CD44 population in CU110 cells stably transfected with shAKT1, shAKT2 or SCR lentivirus. Error bars indicate SD. n = 3. (H) SP fraction using Hoechst dye‐effluxing analysis in CU110 cells stably transfected shAKT1, shAKT2 or SCR lentivirus. Quantification of SP fraction is shown on top. Error bars indicate SD. n = 3. *P < 0.05 (two‐tailed Student t‐test).
Figure 5
Figure 5
Identifying multiple RTK pathways effectively eliminates CSC populations. (A) RTK protein array in the PIK3CA‐overexpressing cells (CU110) and control cells (CUCON) using the PathScan RTK Signaling Antibody Array Assay Kit. Top panel: quantification, lower panel: chemiluminescent images. (B) Screening for pharmaceutical inhibitors effectively reducing CSC population in CU110 cells using HNSCC Sphe‐forming assay. The quantification is shown on the left. Error bars indicate SD. n = 3. *P < 0.05 (two‐tailed Student t‐test). Scale bar: 100 µm.
Figure 6
Figure 6
Targeting multiple RTK pathways effectively eliminates CSC populations with inhibiting Ephs, TRKs, and c‐Kit the most prominent. (A) FACS analysis of CD44 (left) or ALDH (right) in CU110 cells treated with LDN211904. Error bars indicate SD. n = 3. *P < 0.05 (two‐tailed Student t‐test). (B) FACS analysis of ALDH in CU110 cells treated with GNF5837. Error bars indicate SD. n = 3. *P < 0.05 (two‐tailed Student t‐test). (C) FACS analysis of CD44 (left) or SP fraction (right) in CU110 cells treated with imatinib. Error bars indicate SD. n = 3. *P < 0.05 (two‐tailed Student t‐test). (D) Effect of pharmaceutical inhibitors (as indicated in the figure) on reducing CSC population in human HNSCC cell lines: Fadu and UMSCC47, using Sphe‐forming assay. The quantification is shown on right. n = 3; error bars indicate SD. *P < 0.05 (two‐tailed Student t‐test). Scale bar: 100 µm.
Figure 7
Figure 7
Ponatinib, a multi‐kinase inhibitor targeting Ephs, TRKs and c‐Kit, effectively eliminates CSC population in HNSCC. (A) HNSCC Sphe‐forming assay of CU110 cells treated with ponatinib. The quantification is shown on top. Sphes with diameter ≥ 30 μm were counted. n = 3; error bars indicate SD. *P < 0.05 (two‐tailed Student t‐test). Scale bar: 100 µm. (B) FACS analysis of ALDH in CU110 cells treated with ponatinib. The quantification is shown on top. n = 3; error bars indicate SD. *P < 0.05 (two‐tailed Student t‐test). (C) FACS analysis of SP fraction in CU110 treated with ponatinib (left). The quantification is shown on top. n = 3; error bars indicate SD. *P < 0.05 (two‐tailed Student t‐test). (D) Left: Gross pictures of lung tissues harvested from mice received tail vein injection of CU110 cells treated with DMSO, PX866 or ponatinib. Red arrow indicates metastatic foci on the surface of the lung. Right: Relevant quantification of the metastatic foci on the surface of lung. n = 4; error bars indicate SD. *P < 0.05 (two‐tailed Student t‐test). Scale bar: 100 µm. (E) Effect of ponatinib on reducing CSC population in human HNSCC cell lines: Fadu and UMSCC47, using Sphe‐forming assay. The quantification is shown on the right. n = 3; error bars indicate SD. *P < 0.05 (two‐tailed Student t‐test). Scale bar: 100 µm.
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