TRIM35 ubiquitination regulates the expression of PKM2 tetramer and dimer and affects the malignant behaviour of breast cancer by regulating the Warburg effect

Abstract

Breast cancer has become the leading cause of death in females. After comprehensive treatment, the lives of patients are still threatened by tumor metastasis and recurrence. Therefore, there is an urgent requirement to find an effective treatment target for breast cancer. Tripartite motif‑containing 35 (TRIM35) is a ubiquitin ligase that has an important role in the recurrence and metastasis of malignant tumors. However, the role of TRIM35 in breast cancer has thus far remained elusive. The expression of TRIM35 was examined in a bioinformatics database and the effects of TRIM35 on the malignant biological behavior of breast cancer were analyzed by Cell Counting Kit‑8, cell migration and invasion assays, flow cytometry and nude mouse xenograft experiments. It was determined that TRIM35 was downregulated in breast cancer tumor tissues and cell lines. Patients with low TRIM35 expression had shorter overall survival. Functional assays revealed that overexpression of TRIM35 inhibited the proliferation, migration and invasion, and promoted apoptosis of breast cancer cells. Furthermore, overexpression of TRIM35 was able to inhibit the Warburg effect in breast cancer cells. Mechanistic analyses indicated that TRIM35 regulates the transition of tetramers and dimers of pyruvate kinase M2 (PKM2) through ubiquitination and thereby affects the Warburg effect. In conclusion, the present results indicated that TRIM35 regulates the tetramer and dimer transition of PKM2 through ubiquitination and affects the malignant biological behavior of breast cancer by modulating the Warburg effect.

Keywords: PKM2; TRIM35; Warburg effect; breast cancer; tumorigenicity; ubiquitination.

Figure 1

Expression profile of TRIM35 in breast cancer and its clinical relevance. (A) mRNA expression of TRIM35 in TCGA database. (B) mRNA expression of TRIM35 in the GEPIA database. (C) mRNA expression of TRIM35 in breast cancer samples. (D) The protein expression of TRIM35 in breast cancer samples. (E) TRIM35 protein expression in breast cancer cell lines. (F) Kaplan-Meier curves indicating the relationship between TRIM35 expression and the prognosis of patients with breast cancer in TCGA database. (G) Kaplan-Meier curves indicating the relationship between the expression of TRIM35 and the prognosis of patients with breast cancer in the GEPIA database. ^*P<0.05. TRIM35, tripartite motif-containing 35; TCGA, The Cancer Genome Atlas; T, tumor sample; N, normal tissue sample; HR, hazard ratio; BRCA, breast cancer.

Figure 2

TRIM35 significantly suppresses cell proliferation, migration and invasion and promotes apoptosis in breast cancer cell lines. (A) Western blot assay indicating the protein expression of TRIM35 in MD-MBA-231 cells. (B) Western blot assay quantifying the protein expression of TRIM35 in MCF-7 cells. (C) CCK-8 assay to detect the proliferation of MD-MBA-231 cells. (D) CCK-8 assay to detect the proliferation of MCF-7 cells. Transwell assays to detect (E) MD-MBA-231 cell migration, (F) MCF-7 cell migration, (G) MD-MBA-231 cell invasion and (H) MCF-7 cell invasion (magnification, ×200). (I) Flow cytometry was used to examine the effects of TRIM35 overexpression on MD-MBA-231 cell apoptosis. (J) Flow cytometry was used to examine the effects of TRIM35 overexpression on MCF-7 cell apoptosis. ^*P<0.05. TRIM35, tripartite motif-containing 35; NC, negative control; Oe, overexpression; CCK-8, Cell Counting Kit-8; PI, propidium iodide.

Figure 3

TRIM35 impedes the energy metabolism in breast cancer cells. Examination of the effects of TRIM35 overexpression on (A) glucose uptake, (B) pyruvate, (C) lactate and (D) ATP in MD-MBA-231 cells. Examination of the effects of TRIM35 overexpression on (E) glucose uptake, (F) pyruvate, (G) lactate and (H) ATP in MCF-7 cells. ^*P<0.05. TRIM35, tripartite motif-containing 35; NC, negative control; Oe, overexpression.

Figure 4

TRIM35 overexpression inhibits breast cancer cell proliferation in vivo. (A) Representative images of excised xenograft tumors grown on nude mice from MDA-MB-231 cells, which had increased expression of TRIM35. (B) Xenograft tumor volume in each group derived from MDA-MB-231 cells with increased expression of TRIM35. (C) Tumor weight in each group derived from MDA-MB-231 cells with increased expression of TRIM35. (D) Western blot analysis revealed the expression of EMT components, including proliferation- and apoptosis-related indicators in MDA-MB-231 cells with increased expression of TRIM35. (E) Representative images of excised xenograft tumors grown on nude mice from MCF-7 cells with increased expression of TRIM35. (F) Tumor volume in each group derived from MCF-7 cells with increased expression of TRIM35. (G) Tumor weight in each group derived from MCF-7 cells with increased expression of TRIM35. (H) Western blot analysis revealed the expression of EMT components, including proliferation- and apoptosis-related indicators in MCF-7 cells with increased the expression of TRIM35. ^*P<0.05. TRIM35, tripartite motif-containing 35; NC, negative control; Oe, overexpression; EMT, epithelial-to-mesenchymal transition.

Figure 5

TRIM35 combines with PKM2. (A) Co-IP experiments indicated that TRIM35 interacted with PKM2 in the MDA-MB-231 and MCF-7 cell lines. (B) The Co-IP experiments indicated that TRIM35 interacted with PKM2 in the MDA-MB-231 and MCF-7 cell lines. (C-F) TRIM35 was overexpressed or knocked down. (C) mRNA expression of PKM2 in MDA-MB-231 cells. (D) mRNA expression of PKM2 in MCF-7 cells. (E) Protein expression of TRIM35 and PKM2 in MDA-MB-231 cells. (F) Protein expression of TRIM35 and PKM2 in MCF-7 cells. ^*P<0.05; ns, no significance; TRIM35, tripartite motif-containing 35; IB, immunoblot; IP, immunoprecipitation; NC, negative control; Oe, overexpression; sh, short hairpin RNA; PKM2, pyruvate kinase M2.

Figure 6

TRIM35 regulates the tetramer-dimer transition of PKM2 through ubiquitination, thereby regulating the Warburg effect. (A) The levels of PKM2-ub were examined by western blot with anti-ubiquitin antibody in MDA-MB-231 cells. (B) The levels of PKM2-ub were examined by western blot with anti-ubiquitin antibody in MCF-7 cells. (C) Western blot analysis of the protein expression levels of monomeric, dimeric and tetrameric PKM2 in MDA-MB-231 cells. (D) Relative expression of tetrameric PKM2 in MDA-MB-231 cells. (E) Relative expression of dimeric PKM2 in MDA-MB-231 cells. (F) Proportion of PKM2 tetramers and PKM2 dimers in MDA-MB-231 cells. (G) Western blot analysis of the protein expression levels of monomeric, dimeric and tetrameric PKM2 in MCF-7 cells. (H) Relative expression of tetrameric PKM2 in MCF-7 cells. (I) Relative expression of dimeric PKM2 in MCF-7 cells. (J) Proportion of PKM2 tetramers and PKM2 dimers in MCF-7 cells. (K) Schematic illustration of TRIM35 regulating the tetramer-dimer transition of PKM2 through ubiquitination, thereby regulating the Warburg effect. ^*P<0.05. TRIM35, tripartite motif-containing 35; IP, immunoprecipitation; NC, negative control; Oe, overexpression; PKM2, pyruvate kinase M2; PKM2-ub, ubiquitinated PKM2 protein; WB, western blot.