Autophagy regulates cisplatin-induced stemness and chemoresistance via the upregulation of CD44, ABCB1 and ADAM17 in oral squamous cell carcinoma

Abstract

Objective: We inspected the relevance of CD44, ABCB1 and ADAM17 in OSCC stemness and deciphered the role of autophagy/mitophagy in regulating stemness and chemoresistance.

Material and methods: A retrospective analysis of CD44, ABCB1 and ADAM17 with respect to the various clinico-pathological factors and their correlation was analysed in sixty OSCC samples. Furthermore, the stemness and chemoresistance were studied in resistant oral cancer cells using sphere formation assay, flow cytometry and florescence microscopy. The role of autophagy/mitophagy was investigated by transient transfection of siATG14, GFP-LC3, tF-LC3, mKeima-Red-Mito7 and Western blot analysis of autophagic and mitochondrial proteins.

Results: In OSCC, high CD44, ABCB1 and ADAM17 expressions were correlated with higher tumour grades and poor differentiation and show significant correlation in their co-expression. In vitro and OSCC tissue double labelling confirmed that CD44⁺ cells co-expresses ABCB1 and ADAM17. Further, cisplatin (CDDP)-resistant FaDu cells displayed stem-like features and higher CD44, ABCB1 and ADAM17 expression. Higher autophagic flux and mitophagy were observed in resistant FaDu cells as compared to parental cells, and inhibition of autophagy led to the decrease in stemness, restoration of mitochondrial proteins and reduced expression of CD44, ABCB1 and ADAM17.

Conclusion: The CD44⁺ /ABCB1⁺ /ADAM17⁺ expression in OSCC is associated with stemness and chemoresistance. Further, this study highlights the involvement of mitophagy in chemoresistance and autophagic regulation of stemness in OSCC.

Keywords: ABCB1; ADAM17; CD44; autophagy; chemoresistance; stemness.

Figure 1

Expression of CD44, ABCB1 and ADAM17 in normal oral tissue and oral squamous cell carcinoma tissue and their co‐expression. Slide shows representative images of CD44 (A), ABCB1 (B) and ADAM17 (C) staining in normal oral tissue and different grades of OSCC tissue samples. Brown chromogen colour (3,3‐Diaminobenzidine) indicates positive CD44, ABCB1 and ADAM17 staining and the purple colour indicates the nuclear counterstaining by haematoxylin. The square box demonstrates the area of interest shown in larger magnification. Images demonstrate a representative immunofluorescent double labelling of indicated proteins and their cytofluorogram scatter plot depicting the co‐expression (D‐I)

Figure 2

Cisplatin‐resistant cells are endowed with cancer stem‐like features and increased CD44, ABCB1 and ADAM17 expression. The FaDu‐P and FaDu CDDP‐R cells were treated with different concentration of CDDP for 72 h, and the cell viability was measured by MTT dye reduction assay (A). The FaDu P and FaDu CDDP‐R cells were grown in ultralow attachment plate in stem cell media for 10 days, and the number and size of the orosphere produced were quantified (B, C). Expression of β‐catenin in FaDu P and FaDu CDDP‐R cells (D). The corrected total cell fluorescence (CTCF) of β‐catenin expression as analysed by Image J software is depicted (E). Flow cytometric analysis of CD44 expression in FaDu‐P and FaDu‐CDDP‐R cells (F). Flow cytometric analysis of CD44⁺ population with bar diagram represents the comparison between CD44⁺ and CD44⁻ population in FaDu‐P and FaDu CDDP‐R cells (G, I). Immunofluorescence image of indicated protein expression and their quantification in FaDu‐P and FaDu CDDP‐R cells (I‐N). **P value ≤0.01 was considered significant when compared between FaDU‐P and Fadu‐CDDP‐R

Figure 3

Role of autophagy in chemoresistance in FaDu cells. Fadu‐P and Fadu‐CDDP‐R cells were analysed for the autophagy induction in terms of punctate florescence of LC3B‐II expression (A) and quantification of % of LC3B‐II puncta positive cells (B). Autophagic flux analysis in parental and resistant FaDu cells was done after tF‐LC3 transfection, and the numbers of RFP ⁺ GFP ⁺ (yellow) and RFP ⁺ GFP ⁻ (red) puncta per cell were representing autophagosome and autolysosome, respectively, were counted (C and D). Western blot analysis of autophagic protein LC3B, ULK1, Beclin1, ATG5, ATG7, ATG14 and p62 is depicted (E). Western blot image depicts the down regulation of ATG14 with transient transfection of siATG14 in FaDU‐P and FaDu‐CDDP‐R cells (F). Immunofluorescence analysis of LC3B‐II puncta formation in FaDU‐P and FaDu‐CDDP‐R cells in siControl and siATG14 transfected condition were evaluated (G). % of LC3B‐II puncta positive cells in siControl and ATG14‐deficient FaDU‐P and FaDu‐CDDP‐R cells was quantified (H). Densitometry was performed on the original blots; the ratio of protein to actin in control cells was considered as 1. **P value < 0.01 was considered significant when compared between FaDu‐P and FaDu‐CDDP‐R group. ^## P value < 0.01 was considered significant when compared between FaDu‐CDDP‐R and siATG14‐ FaDu‐CDDP‐R

Figure 4

Mitophagy regulates chemoresistance in FaDu cells. Immunofluorescence expression (A) and quantification of outer mitochondrial marker protein TOM20 in FaDU‐P and FaDu‐CDDP‐R (B) were analysed. Western blot analysis of inner mitochondrial marker protein COX‐IV was investigated (C). The mitochondrial delivery to lysosome in order to measure the mitophagic flux (in terms of increased red puncta) was assayed by employing mKeima‐Red‐Mito7 (D and E). Immunofluorescence expression of TOM20 with quantification of % of cells with less/no mitochondrial TOM20 staining in FaDU‐P and FaDu‐CDDP‐R cells after transient transfection of siControl and siATG14 are depicted (F and G). Western blot analysis of COX‐IV expression in ATG14‐deficient FaDU‐P and FaDu‐CDDP‐R cells is represented (H). Densitometry was performed on the original blots, the ratio of protein to actin in control cells was considered as 1. **P value < 0.01 was considered significant when compared between FaDu‐P and FaDu‐CDDP‐R groups. ^## P value < 0.01 was considered significant when compared between FaDu‐CDDP‐R and siATG14‐ FaDu‐CDDP‐R

Figure 5

Autophagy regulates stemness and expression of CD44, ABCB1 and ADAM17 in CDDP‐resistant FaDu cells. After knock down of ATG14, FaDu P and FaDu CDDP‐R cells were grown in ultralow attachment plate in stem cell media for 10 days and the number orospheres produced were quantified (A). Immunofluorescence analysis of β‐catenin, CD44, ABCB1 and ADAM17 in ATG14‐deficient FaDU‐P and FaDu‐CDDP‐R cells and their corresponding quantification was analysed (B‐I). **P value < 0.01 was considered significant when compared between FaDu‐P and FaDu‐CDDP‐R group. ^## P value < 0.01 was considered significant when compared between FaDu‐CDDP‐R and siATG14‐ FaDu‐CDDP‐R