TCRP1 induces tamoxifen resistance by promoting the activation of SGK1 in MCF‑7 cells

Abstract

Tamoxifen is widely used as a highly effective drug for treating estrogen‑receptor (ER) alpha‑positive breast cancer. However, tamoxifen resistance developed during cancer treatment remains a significant challenge. Tongue cancer resistance‑related protein1 (TCRP1), which is recognized as a novel drug target, is related to chemo‑resistance in human cancers, moreover, it is often overexpressed in various cancer cells, such as in lung cancer, breast cancer, and tongue cancer. However, the effects of TCRP1 on tamoxifen‑resistant breast cancer cells and tissues are far from clear. The present study revealed that TCRP1 induced tamoxifen resistance in breast cancer cells. Western blotting, quantitative real‑time polymerase chain reaction (RT‑PCR) and immunohistochemical staining were performed to detect the expression level of TCRP1 in vivo and in vitro between primary breast cancer tissues and tamoxifen‑resistant breast cancer tissues. The data revealed that the expression of TCRP1 was upregulated in the tamoxifen‑resistant breast cancer tissues and human breast cancer cell line, MCF‑7. Further study revealed that knocking down TCRP1 inhibited the growth of MCF‑7 cells with tamoxifen‑resistance (MCF7‑R cells) and induced cell apoptosis. Moreover, TCRP1 promoted serum‑ and glucocorticoid‑inducible kinase 1 (SGK1) activation via phosphorylation of phosphoinositide‑dependent kinase 1 (PDK1) in MCF7‑R cells. In addition, it was also observed that knocking down TCRP1 inhibited tumorigenesis of MCF‑7 cells in nude mice. In conclusion, these data indicated that TCRP1 could induce tamoxifen resistance by regulating the PDK1/SGK1 signaling pathway. Thus, TCRP1 could be explored as a promising candidate for treating tamoxifen‑resistant breast cancer in the future.

Keywords: breast cancer; tamoxifen-resistant; tongue cancer resistance-related protein1; glucocorticoid-inducible kinase 1; MCF-7 cells.

Figure 1.

TCRP1 is overexpressed in tamoxifen-resistant breast cancer tissues compared with primary breast cancer tissues. (A) Total mRNAs were extracted from tamoxifen-resistant breast cancer tissues (n=20) and primary breast cancer tissues (n=20), respectively. TCRP1 expression was analyzed by RT-PCR. (B and C) Pathological sections in tumor tissues were detected by immunohistochemistry with TCRP1 antibody in the tamoxifen-resistant breast cancer tissues and the primary breast cancer tissues. TCRP1, tongue cancer resistance-related protein1.

Figure 2.

TCRP1 induces tamoxifen resistance in MCF7-R cells. (A) MCF-7 and MCF7-R cells were treated by 0 to 2 µM tamoxifen and cultured for 48 h. An MTT assay was performed, and dose-response curves were presented. (B-D) The mRNA and protein expression levels of TCRP1 in MCF-7 and MCF7-R cells were detected by RT-PCR and western blotting. β-Actin served as an internal control. (E and F) MCF7-R cells were transfected with pcDNA3.1-TCRP1 or control plasmid and treated with 0 to 2 µM of tamoxifen for 48 h. MCF7-R cells were transfected with shRNA for TCRP1 or scramble control for 48 h and then treated with 0 to 2 µM tamoxifen for 48 h. The effectiveness of TCRP1 and TCRP1 overexpression were detected by RT-PCR and western blotting. (G and H) Cell viability was assessed by MTT assay. **P<0.01. TCRP1, tongue cancer resistance-related protein1.

Figure 3.

Knockdown of TCRP1 decreases cell proliferation and promotes cell apoptosis in MCF7-R cells. (A) MCF7-R cells were transfected with shRNA for TCRP1 or scramble control and incubated for 0–3 days. (A) Cell viability was detected by MTT assay. (B and C) Clonogenic capacity was assessed by plate cloning assay. (D and E) Cell apoptosis was detected by flow cytometry with Annexin V-FITC/PI dual staining. (F and G) Apoptotic-related proteins were detected by western blotting. **P<0.01. TCRP1, tongue cancer resistance-related protein1.

Figure 4.

TCRP1 phosphorylates PDK1 and further activates PDK1/SGK1 signaling. (A and D) MCF7-R cells were transfected with pcDNA3.1-TCRP1 or control plasmid. MCF7-R cells were transfected with shRNA for TCRP1 or scramble control for 48 h. The levels of p-PDK1 and PDK1 and p-SGK1 and SGK1 were detected by western blotting. (B and E) MCF7-R cells were transfected with PDK1 or control plasmid for 48 h. The levels of p-PDK1 and PDK1 and p-SGK1 and SGK1 were detected by western blotting. (C and F) MCF7-R cells were transfected with a co-transfection system with PDK1 expression vector in combination with the TCRP1 shRNA into MCF7-R cells or transfected with shRNA for TCRP1 alone or control. The expression of p-SGK1 and SGK1 was detected by western blotting. **P<0.01. TCRP1, tongue cancer resistance-related protein1; PDK1, phosphoinositide-dependent kinase 1; SGK1, glucocorticoid-inducible kinase 1.

Figure 5.

TCRP1 induces tamoxifen resistance by activating SGK1. MCF7-R cells were transfected with a co-transfection system with SGK1 expression vector in combination with the TCRP1 shRNA into MCF7-R cells or transfected with shRNA for TCRP1 or control. (A) The effectiveness of SGK1 overexpression was detected by q-PCR. (B and C) The expression levels of p-SGK1 and SGK1 were detected by western blotting. (D) Cell viability was detected by MTT assay. (E and F) Cell apoptosis was detected by flow cytometry with Annexin V-FITC/PI dual staining. **P<0.01. TCRP1, tongue cancer resistance-related protein1; SGK1, glucocorticoid-inducible kinase 1.

Figure 6.

Knockdown of TCRP1 inhibits oncogenesis of MCF7-R cells in nude mice. MCF7-R cells were treated with shRNA for TCRP1 and shRNA control, and then subcutaneously injected into the nude mice. (A and B) Tumor growth was analyzed by measuring the tumor volume. (C) Tumors were excised and weighed. (D) Pathological sections in tumor tissues were subjected to immunohistochemistry with TCRP1, Ki67 and SGK1 antibodies. **P<0.01. TCRP1, tongue cancer resistance-related protein1; SGK1, glucocorticoid-inducible kinase 1.