MicroRNA-145-5p inhibits the tumorigenesis of breast cancer through SENP2-regulated ubiquitination of ERK2

Abstract

Breast carcinoma exhibits the highest incidence among various cancers and is the foremost cause of mortality in women. Increasing evidence shows that SUMOylation of proteins plays a critical role in the progression of breast cancer; however, the role of SENP2 and its molecular mechanism in breast cancer remain underexplored. Here, we discerned that SENP2 promoted the tumorigenesis of breast cancer both in vitro and in vivo. Furthermore, we identified that ERK2 was SUMOylated and that SENP2 played a role by deconjugating ERK2 SUMOylation in breast cancer. SUMOylation of ERK2 promoted its ubiquitin-proteasomal degradation, thus inhibiting the epithelial-to-mesenchymal transition in breast cancer cells. Furthermore, microRNA-145-5p (miR-145-5p) has emerged as a scarce commodity in breast cancer and binds to the 3'-untranslated region of SENP2 mRNA to govern the regulatory dynamics of SENP2 expression. Finally, miR-145-5p inhibits SENP2 transcription, enhances ERK2 SUMOylation, and ultimately suppresses the progression of breast cancer. These revelations suggest evolving ideas for the miR-145-5p-SENP2 axis in therapeutic intervention, thus heralding transformative prospects for the clinical management of breast cancer.

Keywords: Breast cancer; ERK2; MicroRNA-145-5p; SENP2; SUMOylation.

Fig. 1

SENP2 is highly expressed in breast cancer cells and patients. A. The expression levels of SENP2 transcripts in normal breast cell MCF-10 A and different breast cancer cells. The quantification of SENP2 transcript levels was carried out across a range of breast cancer cell lines through real-time PCR. These values were subsequently normalized to those of BT-549 cells (n = 3 biological replicates per group). B. The expression levels of the SENP2 protein in normal breast cell MCF-10 A and different breast cancer cells. Whole cell lysates derived from multiple breast cancer cell lines underwent immunoblot analysis utilizing anti-SENP2 and anti-β-Actin antibodies (left panel). The quantitative analysis of Western blotting results is displayed in the right panel (n = 3 biological replicates per group) C. SENP2 was highly expressed in breast cancer tissue. SENP2 expression levels in breast cancer and normal tissue were analyzed with the GEPIA2 database, and the data did not show statistically significant difference. D. SENP2 was highly expressed in different subtypes of breast cancer tissues. SENP2 expression levels in various subtypes of breast cancer tissues were analyzed with the GEPIA2 database, and the data did not show statistically significant difference. E. SENP2 was highly expressed in breast cancer patients. SENP2 in breast cancer and precancerous tissue from breast cancer patients was analyzed by immunohistochemistry with SENP2 antibody (n = 10 biological replicates per group). Hematoxylin (blue) was applied to visualize the nuclei. The scale bar is 50 μm. F. SENP2 was highly correlated with the progression of breast cancer patients. SENP2 in breast cancer and precancerous tissue chips from breast cancer patients was analyzed by immunohistochemistry with the anti-SENP2 antibody (n = 48 biological replicates per group). The scale bar is 200 μm. G. SENP2 was highly correlated with overall survival in breast cancer patients. The relationship between SENP2 expression and survival rate in breast cancer patients was analyzed by survival analysis with the Kaplan-Meier Plotter database

Fig. 2

SENP2 knockdown inhibits the progression of breast cancer in vitro and in vivo. A. SENP2 knockdown inhibited the proliferation of MCF-7 and MDA-MB-231 cells. Cell growth curves of the SENP2 knockdown and control groups were generated from daily quantification of cell numbers (n = 3 biological repeats/group). B. SENP2 knockdown inhibited the colony formation of MCF-7 and MDA-MB-231 cells. Cells were seeded in 24-well plates for 14 days and stained with 0.1% crystal violet for the colony formation assay (left). The colony formation ability was analyzed (n = 3 biological repeats/group). C. SENP2 knockdown inhibited the migration of MCF-7 and MDA-MB-231 cells. Cell migration was detected by wound healing assay in stably transfected cells (left). The migration rate was measured and analyzed (right, n = 3 biological repeats/group). The scale bar is 200 μm. D. SENP2 knockdown inhibited the invasion of MCF-7 and MDA-MB-231 cells. Cell migration and invasion were analyzed by transwell assay in stably transfected cells (left). The migration and invasion of cells were measured and analyzed (right, n = 5 biological repeats/group). The scale bar is 200 μm. E. SENP2 knockdown inhibited EMT in MCF-7 and MDA-MB-231 cells. The sh-NC and two sh-SENP2 plasmids were packaged and transduced into cells, and the whole cell lysates were detected by Western blotting with the indicated antibodies (left). The results of the quantitative analysis of Western blotting are shown in the right panel (n = 3 biological repeats/group). F-G. SENP2 knockdown inhibited the tumorigenesis of breast cancer in vivo. The tumor volume (F) was measured every four days, and the tumors were harvested and weighed at the end of the experiment (G, n = 5 mice/group). H. SENP2 knockdown inhibited EMT in vivo. The breast tumor extracts from the sh-NC and sh1-SENP2 mice were analyzed by Western blotting with the indicated antibodies (left). The results of the quantitative analysis of Western blotting are shown in the right panel (n = 5 mice with 2 technical repeats/group)

Fig. 3

SENP2 promotes cell proliferation and migration depending on ERK2 in breast cancer cells. A. ERK2, but not ERK1, was significantly decreased in breast cancer tissue from SENP2 knockdown mice. The breast tumor extracts from the sh-NC and sh1-SENP2 mice were analyzed by Western blotting with the indicated antibodies. B. ERK2 was highly expressed in different subtypes of breast cancer tissues. ERK2 expression levels in various subtypes of breast cancer tissues were analyzed with the GEPIA2 database. C. ERK2 was highly expressed in breast cancer patients. ERK2 in breast cancer and precancerous tissue from breast cancer patients was detected by immunohistochemistry with the anti-ERK2 antibody (n = 10 biological repeats/group). Hematoxylin (blue) was applied to show the nuclei. The scale bar is 50 μm. D. ERK2 was highly correlated with distant metastasis-free survival, post-progression survival, and overall survival in breast cancer patients. The relationship between ERK2 expression and survival time in breast cancer patients was analyzed by survival analysis with the Kaplan-Meier Plotter database. E. The overexpression of SENP2 in MDA-MB-231 cells induced proliferation, a phenomenon that was subsequently rescued by the knockdown of ERK2. Growth curves for the specified cell groups were meticulously generated through daily cell number quantification (n = 3 biological replicates per group). F. ERK2 knockdown rescued the colony formation induced by SENP2 overexpression in MDA-MB-231 cells. Cells were seeded for 14 days and stained with 0.1% crystal violet for the colony formation assay (left). The colony formation ability was analyzed (n = 3 biological repeats/group). G. ERK2 knockdown rescued the migration induced by SENP2 overexpression in MDA-MB-231 cells. Cell migration was determined by wound healing assay in stably transfected cells (left). The migration rate was measured and analyzed (right, n = 3 biological repeats/group). The scale bar is 200 μm. H. ERK2 knockdown rescued the migration and invasion that was induced by SENP2 overexpression in MDA-MB-231 cells. Cell migration and invasion were analyzed by transwell assay in stably transfected cells (left). The migration and invasion of cells were measured and analyzed (right, n = 8 biological repeats/group). The scale bar is 200 μm. I. ERK2 knockdown rescued the EMT process promoted by SENP2 overexpression in MDA-MB-231 cells. The whole cell lysates were analyzed by Western blotting with the indicated antibodies

Fig. 4

ERK2 is SUMOylated by SUMO2 and deSUMOylated by SENP2. A. The protein level of ERK2 was decreased in the SENP2-engineered breast cancer cells. The whole cell lysates of SENP2 knockdown MDA-MD-231 (left) and overexpressed MCF7 (middle) cells were analyzed by Western blotting with the indicated antibodies. The quantitative analysis of Western blotting results is displayed in the right panel (n = 3 biological replicates/ group). B. SENP2 interacted with ERK2. The indicated plasmids were transfected into HEK293T cells, and the IP with anti-Flag from whole cell lysates was detected by Western blotting with anti-SENP2 antibody. The whole cell lysates were detected by Western blotting with anti-Flag and anti-SENP2 antibodies (left). The quantitative analysis of Western blotting results is displayed in the right panel (n = 3 biological replicates/ group). C. ERK2 is predominantly modified by exogenous SUMO2 within HEK293T cells. The designated plasmids were transfected into the HEK293T cells, and the IP using anti-Flag from whole cell lysates was detected via Western blotting with anti-HA antibody. The whole cell lysates were detected by Western blotting with anti-HA or anti-Flag antibodies. D-E. ERK2 was mainly modified by endogenous SUMO in MCF7 (D) and MDA-MB-231 (E) cells. The IP with anti-IgG or anti-ERK2 from whole cell lysates was detected by Western blotting with the indicated antibodies. The WCL was detected by Western blotting with the indicated antibodies. F-G. SENP2 knockdown and knockout enhanced the endogenous SUMOylation of ERK2. The IP with anti-ERK2 from whole cell lysates of SENP2 knockdown MDA-MB-231 cells (G) and SENP2 knockout MEF cells (G) was detected by Western blotting with the indicated antibodies. The WCL was detected by Western blotting with the indicated antibodies. H. SENP2 catalyzed the deconjugation of ERK2 SUMOylation within HEK293T cells. The specified plasmids were transfected into HEK293T cells, and the IP with anti-Flag from cell lysates was detected by Western blotting with anti-HA antibody. The WCL was detected through Western blotting with the indicated antibodies. I. ERK2 was co-localized with SUMO2 and SENP2 in the nucleus and cytoplasm. The Flag-ERK2 plasmid was transfected into HEK293T cells, and the cells were harvested for immunofluorescence staining with anti-Flag (red) and anti-SUMO2 (green) or anti-SENP2 (green) antibodies. DAPI (blue) was used to show nuclei, and the colocalization curve was analyzed by ImageJ software. The scale bar is 5 μm. J. K99 and K330 were the primary SUMOylation sites of ERK2. Wild-type (w) or SUMO site mutant Flag-ERK2 and HA-SUMO2 were transiently transfected into HEK293T cells, and the IP with anti-Flag from cell lysates was detected by Western blotting with anti-HA and anti-Flag antibodies. The WCL was detected by Western blotting with anti-HA or anti-Flag antibodies

Fig. 5

SUMO site mutant ERK2 enhanced the increased cell growth and metastasis induced by wild-type ERK2. A. The decreased expression of ERK2 in breast cancer cells was rescued by MG132 treatment. MDA-MB-231 and MCF-7 cells were treated with MG132, and the WCL were harvested and detected by Western blotting with the indicated antibodies (upper panel). The quantitative analysis of Western blotting results is displayed in the bottom panel (n = 3 biological replicates/ group).B-C. SENP2 accelerated the ubiquitination of the endogenous ERK2 protein. Cells were treated with DMSO or MG132, and the IP with anti-ERK2 from cell lysates of SENP2 knockdown (B) or overexpressing (C) MDA-MB-231 cells were detected by Western blotting with anti-ubiquitin or anti-ERK2 antibodies. The WCL was detected through immunoblotting using the designated antibodies.D-E. SENP2 promoted the proliferation of MDA-MB-231 cells. Cells were treated with DMSO or MG132, and the growth curves of SENP2 knockdown (D) or overexpressing (E) MDA-MB-231 cells were generated from daily quantification of cell numbers (n = 3 biological repeats/group).F. SUMO mutant and shRNA resistant ERK2 enhanced the proliferation of MDA-MB-231 cells. Growth curves of wild-type or SUMO mutant ERK2 cells were generated from daily quantification of cell numbers (n = 3 biological repeats/group).G. SUMO mutant and shRNA resistant ERK2 enhanced the colony formation of MDA-MB-231 cells. Cells were seeded for 14 days and stained with 0.1% crystal violet for the colony formation assay (left). The colony formation ability was analyzed (n = 3 biological repeats/group).H. SUMO mutant and shRNA resistant ERK2 enhanced the migration of MDA-MB-231 cells. Cell migration was detected by wound healing assay in stably transfected cells (left). The migration rate was measured and analyzed (right, n = 4 repeats/group). The scale bar is 200 μm. I. SUMO mutant and shRNA resistant ERK2 enhanced the invasion of MDA-MB-231 cells. Cellular invasiveness was assessed via the transwell assay in cells stably transfected with the construct (left). The invasive cells were measured and analyzed (right, n = 3 biological repeats/group). The scale bar represents 200 μm. J. SUMOylation of ERK2 regulates the EMT process in breast cancer cells. Wild-type or SUMOylation site mutant and shRNA resistant ERK2 was stably expressed in MDA-MB-231 cells, and whole cell lysates were detected by Western blotting with the indicated antibodies

Fig. 6

miR-145-5p is a direct upstream regulator of SENP2 in breast cancer cells. A. miR-145-5p is predicted to be an upstream regulator of SENP2. The potential upstream microRNA that regulates SENP2 transcription was predicted by miRDB, miRBase, miRcode, and RNA22. B. The miR-145-5p mimic inhibited the promoter activity of wild-type but not mutant SENP2. HEK293T cells were transiently transfected with the wild-type mutant SENP2 promoter, and relative luciferase activity was detected after treatment with the miR-145-5p mimic or control (n = 3 biological repeats/group). C. miR-145-5p mimic inhibited the transcription of SENP2. MDA-MB-231 cells were treated with miR-145-5p mimic or control, and the expression level of SENP2 transcripts was calculated by real-time PCR and normalized to the control (n = 3 biological repeats/group). D. Inhibition of miR-145-5p resulted in the upregulation of SENP2 transcription. MDA-MB-231 cells were treated with either a miR-145-5p inhibitor or a control, and the transcription level of SENP2 were determined by real-time PCR and normalized to the control group (n = 3 biological repeats/group). E. The miR-145-5p mimic inhibited the protein levels of SENP2 and ERK2. MDA-MB-231 cells were treated with miR-145-5p mimic or control, and the protein expression levels of SENP2 and ERK2 were measured by Western blotting with the indicated antibodies (left). The quantitative analysis of Western blotting results is displayed in the right panel (n = 3 biological replicates/group). F. The miR-145-5p inhibitor promoted the protein expression of SENP2 and ERK2. MDA-MB-231 cells were treated with miR-145-5p inhibitor or control, and the protein expression levels of SENP2 and ERK2 were measured by Western blotting with the indicated antibodies (left). The quantitative analysis of Western blotting results is displayed in the right panel (n = 3 biological replicates/group). G-H. The miR-145-5p mimic inhibited the level of SENP2 and ERK2 protein in a concentration (G) and time-dependent (H) manner. MDA-MB-231 cells were treated with miR-145-5p mimic for different concentrations and times, and the protein expression levels of SENP2 and ERK2 were measured by IB with the indicated antibodies (left). The quantitative analysis of Western blotting results is displayed in the right panel (n = 3 biological replicates/group). I. MiR-145-5p enhanced the SUMOylation level of ERK2. The specified plasmids were transiently transfected into HEK293T cells, followed by IP using anti-Flag antibodies from cellular lysates, and subsequent Western blotting was performed using anti-HA antibodies. The WCL was detected by Western blotting with anti-HA and anti-Flag antibodies

Fig. 7

miR-145-5p acts as a tumor suppressor in breast cancer cells. A. Quantification of miR-145-5p transcript abundance across normal breast cancer cell MCF-10 A and diverse breast cancer cell lines. The transcript abundance of miR-145-5p in distinct breast cancer cell lines was assessed via real-time PCR and subsequently standardized to the expression levels in MDA-MB-231 cells (n = 3 biological repeats/group). B. miR-145-5p was poorly expressed in breast cancer tissue. miR-145-5p expression levels in various cancer tissues and normal tissues were analyzed with the Tumor-miRNA-Pathway database. C. miR-145-5p was poorly expressed in breast cancer patients. miR-145-5p in breast cancer and precancerous tissue from TNBC patients was analyzed by immunofluorescence staining with the miR-145-5p probe. DAPI (blue) was applied to label the nuclei. The scale bar is 50 μm. D. miR-145-5p was positively correlated with the overall survival of breast cancer patients. The relationship between miR-145-5p expression and survival rate in breast cancer patients was analyzed by survival analysis with the Kaplan-Meier Plotter database. E. The miR-145-5p mimic inhibited the proliferation of MDA-MB-231 cells. MDA-MB-231 cells were treated with miR-145-5p mimic or control, and growth curves of cells were calculated from daily quantification of cell numbers (n = 3 biological repeats/group). F. The miR-145-5p inhibitor promoted the proliferation of MDA-MB-231 cells. MDA-MB-231 cells were treated with miR-145-5p inhibitor or control, and cell growth curves were calculated from daily quantification of cell numbers (n = 3 biological repeats/group). G. miR-145-5p inhibited the migration of MDA-MB-231 cells. Cell migration was detected by wound healing assay in control or miR-145-5p mimic treated cells (left). The migration rate was calculated and analyzed (right, n = 3 biological repeats/group). The scale bar is 200 μm. H. The miR-145-5p inhibitor promoted the migration of MDA-MB-231 cells. Cell migration was detected by wound healing assay in control or miR-145-5p inhibitor-treated cells (left). The migration rate was calculated and analyzed (right, n = 3 biological repeats/group). The scale bar is 200 μm. I. SENP2 promoted the proliferation of MDA-MB-231 cells that were inhibited by the miR-145-5p mimic. SENP2 stably transfected MDA-MB-231 cells were treated with miR-145-5p mimic or control, and growth curves of cells were generated from daily quantification of cell numbers (n = 3 biological repeats/group). J. SENP2 promoted the migration of MDA-MB-231 cells that were inhibited by the miR-145-5p mimic. Cell migration was determined by wound healing assay in control or miR-145-5p inhibitor-treated cells (left). The migration rate was calculated and analyzed (right, n = 3 biological repeats/group). The scale bar is 200 μm

Fig. 8

Schematic diagram of SENP2-deconjugated SUMOylation of ERK2 in miR-145-5p inhibited tumorigenesis of breast cancer. Firstly, miR-145-5p targets and binds to the 3’-UTR of SENP2 mRNA, resulting in the suppression of SENP2 expression; secondly, reduced SENP2 levels lead to the accumulation of SUMOylated ERK2, and SUMOylation of ERK2 favors its ubiquitination and proteasomal degradation; finally, degradation of ERK2 weakens the signaling pathways that promote EMT and tumorigenic processes in breast cancer cells