SORBS1 suppresses tumor metastasis and improves the sensitivity of cancer to chemotherapy drug

Abstract

Tumor metastasis and invasion are both hallmarks of cancer malignancy and the leading cause of cancer death. Here we show that the adaptor protein SORBS1 (Sorbin and SH3 domain-containing protein 1, also known as CAP/ponsin) is expressed at low levels in clinical cancer samples. In addition, low-level expression of SORBS1 was significantly associated with poor clinical outcomes and the increased tumor cell invasive capacity in breast cancer patients. We demonstrate that depletion of SORBS1 increases protrusions and filopodium-like protrusions (FLPs) formation, as well as the migratory and invasive abilities of cancer cells, via activation of JNK/cJun. Furthermore, silencing of SORBS1 promotes the epithelial-to-mesenchymal transition (EMT) process and attenuates chemical drug sensitivity especially that to cisplatin, by inhibition of p53 in breast cancer cells. Thus, we illustrate that SORBS1 is a potential inhibitor of metastasis in cancer and may be a promising target in chemotherapy.

Keywords: JNK; SORBS1; cisplatin sensitivity; invasion; migration.

Figure 1. SORBS1 is present at a lower level in human breast cancer

(A) Western blots was performed to detect SORBS1 levels in normal human mammary gland epithelial cell line MCF 10A and nine other human breast cancer cell lines. (B) Box plots comparing levels of SORBS1 mRNA in normal human breast tissues and breast carcinomas (left panel) or ductal breast carcinoma tissues (right panel) in published data sets from Oncomine. ***P < 0.001, Student's t-test. (C) mRNA levels of SORBS1 in normal breast tissues vs invasive ductal / lobular breast carcinomas tissues in the case of Curtis Breast, or mRNA levels of SORBS1 in ductal breast carcinomas vs invasive ductal breast carcinoma in Nikolsky Breast in published data sets from Oncomine. *P < 0.05, Student's t-test. (D and E) Kaplan-Meier survival analysis for assessment of overall survival (OS) (D) and distant metastasis-free survival (DMFS) (E) in breast cancer patients with (right panel) or without (left panel) systemic treatment which classified by relative (high or low) tumor SORBS1 level. Survival curves were generated by using the Kaplan-Meier Plotter online tool based on data stratified based on the best cut-off. Curves were compared by hazard ratios (HR) and p values (log rank p).

Figure 2. Loss of SORBS1 increases breast cancer cells migration and invasion properties both in vivo and in vitro

(A) Western blot results to show the knockdown efficiency of SORBS1 gene in MCF10A, HBL100 and MDA-MB-231 cell lines. (B, D and F) Migration transwell assay was done in MCF 10A Control (B), HBL100 Control (D), MDA-MB-231 Control (F) and corresponding shSORBS1 (respectively named as shSORBS1-1 and shSORBS1-2) cell lines, in which cells (B and D: 4 × 10⁴ cells/well, F: 3 × 10⁴ cells/well) were cultured for 24 hours. (C, E and G) Quantitative results are respectively illustrated for panel B, D and F. (H, J and L) Invasion transwell assay was done in MCF 10A control (H), HBL100 Control (J), MDA-MB-231 Control (L) and corresponding shSORBS1 cell lines, in which cells (H and J: 8 × 10⁴ cells/well, L: 6 × 10⁴ cells/well) were cultured for 24 hours. (I, K and M) Quantitative results are respectively illustrated for panel H, J and L. (N) SUM 159 cells were efficiently transfected with a construct encoding SORBS1 or with a control plasmid. (O and P) Transwell assay was performed in SUM159 Control and SUM 159 SORBS1 (OE), in which cells (migration, 3 × 10⁴ cells/well; invasion, 6 × 10⁴ cells/well) were cultured for 24 hours. (P) Quantitative results from panel O. All of images were taken by 10× objective lens. The number of cells was counted from at least four independent microscopic fields. (Q) Lung tissues were photographed, fixed and stained with haematoxylin and eosin (HE). White arrows indicated the lung metastatic lesions. (R) The number of metastatic lesions in each specimen was counted by randomly selected four fields under light microscope. Data are shown as mean ± s.d. (n = 4), **P < 0.01, ***P < 0.001, Student's t-test.

Figure 3. Loss of SORBS1 expression triggers increased protrusion formation and abundant FLPs in 3D culture

(A, C and E) Phase-contrast images of MCF 10A cells (A), HBL100 cells (C) or MDA-MB-231 cells (E) stably expressing control or shSORBS1 and grown as 3D culture (Day 10). (B, D and F) Quantitative results of the length of protrusions were illustrated respectively for panel A, C and E. Results are presented as mean ± s.d. (n = 3), ***P < 0.001, Student's t-test. (G–I) Filopodium like protrusions (FLPs) formation of MCF 10A (G), HBL100 (H), MDA-MB-231 (I) control cells and corresponding two shSORBS1 cell lines. (J) FLPs formation of SUM159 Control cells and forced SORBS1 expression (OE) cells. In FLPs formation assay, 2 × 10³ cells were cultured in 3D matrigel and the location of DAPI (blue) and phalloidin (red) were determined at 24 hours. The white arrows indicated FLPs. The experiment was repeated for three times. Scale bars, 20 μm.

Figure 4. Knockdown of SORBS1 induces epithelial-to-mesenchymal transition in breast cancer cells

(A) By using of quantitative real time PCR (qPCR),mRNA of SORBS1 and EMT markers (E-cadherin, N-cadherin, vimentin, Slug) were detected in MCF 10A control and two shSORBS1 cell lines. Data are shown as mean ± s.d. (n = 3), ***P < 0.001, Student's t-test. (B) The protein levels of SORBS1 and EMT markers (E-cadherin, N-cadherin, vimentin, and Slug) were analyzed by western blot in MCF 10A Control and shSORBS1 cell lines. (C) Immunofluorescence staining images show the protein patterns of EMT markers (E-cadherin, vimentin, Slug) in MCF 10A Control and shSORBS1 cell lines. Scale bar, 20 μm. (D and E) The protein levels of SORBS1 and EMT markers (E-cadherin, N-cadherin, vimentin, Slug, Snail) were detected by western blot in MDA-MB-231 Control or shSORBS1 (D) and HBL100 Control or shSORBS1 (E) cell lines. (F and G) Western blot was performed to analyze the expression of SORBS1 and EMT markers (E-cadherin, N-cadherin, vimentin, Snail) in 4T1 (F) and SUM 159 (G) transiently transfected with 1 μg of control vector or SORBS1 cDNA.

Figure 5. SORBS1 inhibits JNK signaling pathway

(A) JNK, p-JNK, c-Jun, p-c-Jun and SORBS1 were analyzed by the Western blotting assay in MCF 10A Control and MCF 10A shSORBS1 cell lines. (B) P-c-Jun staining were detected by immunofluorescence in MCF10A Control and MCF10A shSORBS1 cells. Scale bar, 20 μm. (C and D) Western blot was performed to detect the levels of JNK, p-JNK, c-Jun, p-c-Jun and SORBS1 in HBL100 Control cells (C), MDA-MB-231 Control cells (D) and respectively stably expressing the shSORBS1 cell lines. (E and F) The levels of JNK, p-JNK, c-Jun, p-c-Jun and SORBS1 in Control and SORBS1 (OE) cells were detected by western blot in 4T1 (E) and SUM159 (F).

Figure 6. Inhibition of JNK activity decreases migration, invasion and FLPs formation in SORBS1 knockdown cells

(A, D and G) Transwell assay were conducted in MCF 10A Control (A), MDA-MB-231 Control (D), HBL100 Control cells (G) and corresponding stably expressing shSORBS1 cells which were treated with DMSO or sp600125 (10 μM) for 24 hours. Migration assay: MCF 10A and HBL100 (4 × 10⁴ cells/well), MDA-MB-231 (3 × 10⁴ cells/well). Invasion assay: MCF 10A and HBL100 (8 × 10⁴ cells/well), MDA-MB-231 (6 × 10⁴ cells). Images were taken with a 10× objective lens. (B and C) Quantitative results are illustrated for migration and invasion in panel A. (E and F) Quantitative results from panel D. (H and I) Quantification of transwell migration and invasion assay from panel G. (J and K) Transwell assay were conducted in SUM159 Control and SORBS1 overexpressed (OE) cells, in which cells (migration, 3 × 10⁴ cells/well; invasion, 6 × 10⁴ cells/well) were cultured and treated with DMSO or sp600125 (10 μM) for 24 hours. (K) Quantification of transwell migration and invasion assay from panel J. The numbers of cells were counted from at least four independent microscopic fields. Data are shown as mean ± s.d. (n = 3), **P < 0.01, ***P < 0.001, Student's t-test. (L–O) FLPs formation of MCF10A shSORBS1 (L), HBL100 shSORBS1 (M), MDA-MB-231 shSORBS1 (N), SUM159 SORBS1 (OE) (O) and corresponding control cell lines. 3 × 10³ cells/well were seeded in 3D matrigel and treated with DMSO or sp600125 (10 μM) for 24 hours. The location of DAPI (blue) and phalloidin (red) were determined. The experiment was repeated for three times. Scale bars, 20 μm.

Figure 7. SORBS1 promotes cisplatin-related drug sensitivity in breast cancer cells

(A–C) flow cytometry (FCM) was performed to analyze apoptosis in MCF 10A control (A), HBL100 control (B), MDA-MB-231 control (C) and respectively shSORBS1 cell lines. Cells were treated with 10 ug/ml cisplatin for 24 hours and data are shown as mean ± s.d. (n = 3). **P < 0.01, ***P < 0.001, Student's t-test. (D) SUM159 cells were transiently transfected with 1 μg of vector (Control) or human SORBS1 (OE). After 24 hours, cells were treated with 10 ug/ml cisplatin for another 24 hours. The rate of apoptosis was analyzed by FCM. Data are shown as mean ± s.d. (n = 3). **P < 0.01, Student's t-test. (E–G, I) Western blot was conducted to detect the expression of apoptosis related proteins in MCF 10A shSORBS1 (E), MDA-MB-231 shSORBS1 (F), HBL100 shSORBS1 (G), SUM159 SORBS1 (OE) (I) and respectively control cell lines treated with 10 ug/ml cisplatin for 24 hours. (H) Immunofluorescence staining was performed to detect the protein patterns of cleaved-caspase3 in HBL100 Control and shSORBS1 cell lines treated with 10 ug/ml cisplatin for 24 hours. Images were taken with a 40× objective lens. (J) IC50 values were detected by MTT in MCF7-Adr (anti Adriamycin), Skov3-DDP (anti cisplatin) and SGC7901-DDP (anti cisplatin) and respectively parent cell lines. The IC50values were calculated. The experiment was repeated for three times. (K) qPCR and western were performed to analyze the mRNA and protein levels of SORBS1 in MCF7, Skov3, SGC7901 and corresponding chemical drug resistance cell lines. Data are shown as mean ± s.d. (n = 3), ***P < 0.001, Student's t-test. (L) Kaplan-Meier survival analysis for assessment of overall survival (OS) analysis based tumor SORBS1 level in 69 breast cancer patients with systemic cisplatin chemotherapy (left panel) and 176 chemotherapy treated lung cancer patients (right panel). Survival curves were generated by using the Kaplan-Meier Plotter online tool based on data stratified based on the best cut-off. Curves were compared by hazard ratios (HR) and p values (log rank p).