Triple-negative breast cancer (TNBC) is the most lethal form of breast cancer. TNBC patients have higher rates of metastasis and restricted therapy options. Although chemotherapy is the conventional treatment for TNBC, the frequent occurrence of chemoresistance significantly lowers the efficacy of treatment. Here, we demonstrated that ELK3, an oncogenic transcriptional repressor that is highly expressed in TNBC, determined the chemosensitivity of two representative TNBC cell lines (MDA-MB231 and Hs578T) to cisplatin (CDDP) by regulating mitochondrial dynamics. We observed that the knockdown of ELK3 in MDA-MB231 and Hs578T rendered these cell lines more susceptible to the effects of CDDP. We further demonstrated that the chemosensitivity of TNBC cells was caused by the CDDP-mediated acceleration of mitochondrial fission, excessive mitochondrial reactive oxygen species production, and subsequent DNA damage. In addition, we identified DNM1L, a gene encoding the dynamin-related protein 1 (a major regulator of mitochondrial fission), as a direct downstream target of ELK3. Based on these results, we propose that the suppression of ELK3 expression could be used as a potential therapeutic strategy for overcoming the chemoresistance or inducing the chemosensitivity of TNBC.

Introduction

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer, which lacks the three receptors that are present in more common types of breast cancer; namely, the estrogen receptor, the progesterone receptor, and the human epidermal growth factor receptor type 2. Patients with TNBC have higher rates of metastasis and worse overall survival than those with hormone-receptor-positive breast cancer [1, 2]. Due to a lack of available targeted therapies, chemotherapy is currently the primary systemic treatment option for TNBC patients. Platinum-based chemotherapy, including cisplatin (CDDP), has been widely used as a neoadjuvant treatment for TNBC [3]. Despite the fact that 20% of TNBC patients exhibit a pathologic complete response (pCR) to neoadjuvant chemotherapy, patients without a pCR are more likely to experience early recurrence and metastasis [4]. To achieve the desired therapeutic effect, it is therefore essential to better comprehend the molecular mechanism underlying the response of TNBC to chemotherapy.

Mitochondria are highly dynamic organelles that continuously undergo morphological alterations. These “mitochondrial dynamics” are necessary for mitochondrial quality control and function [5, 6]. Although mitochondria play critical roles in the chemotoxicity of cancer cells, the effect of mitochondrial dynamics on the therapeutic efficacy of chemotherapy is under debate. For instance, mitochondrial fission, followed by the mitophagy of dysfunctional daughter mitochondria, can contribute to cancer cell survival by eliminating irreparable mitochondrial (mt)DNA damage caused by chemotherapy [7]. Conversely, significantly higher levels of mitochondrial fusion have been observed in CDDP-resistant gynecological cancer cells, whereby the fused mitochondria support cancer cell survival by increasing ATP production [8]. Therefore, understanding how mitochondrial dynamics regulate the cellular response to chemotherapy is critical for overcoming chemoresistance in cancer therapy.

The erythroblast transformation specific (ETS)-domain-containing transcription factor, ELK3, belongs to the ternary complex factor subfamily. ELK3 typically functions as a transcriptional repressor. However, phosphorylation of ELK3 transforms it into a transcriptional activator [9, 10]. ELK3 is implicated in various biological processes such as neural cell development, angiogenesis, and tumorigenesis [11,12,13]. We have recently reported that ELK3 activity is also associated with mitochondrial dynamics [14]. ELK3 knockdown in TNBC cells increased the expression of the mitochondrial fission adapter protein, MiD51, and consequently the rate of mitochondrial fission. This led to reactive oxygen species (ROS) accumulation and rendered TNBC cells more sensitive to natural killer cell-mediated cytotoxicity. Considering that mitochondria have a multitude of functions within eukaryotic cells, it is likely that the significance of the ELK3-mediated regulation of mitochondrial dynamics could extend beyond the immune response of cancer cells.

Here, we demonstrated that ELK3-mediated regulation of mitochondrial dynamics affected the chemotherapeutic efficacy of CDDP in TNBC cells. We showed that ELK3-suppressed TNBC cells were more sensitive to CDDP, and that this increase in sensitivity was caused by higher rates of mitochondrial fission, mitochondrial ROS production, and DNA damage. We further identified DNM1L, a gene encoding a major regulator of mitochondrial fission, the dynamin-related protein 1 (DRP1), as a direct downstream target of ELK3.

Results

ELK3 knockdown enhances the CDDP sensitivity of TNBC cells
Previously, we reported that the knockdown of ELK3 with shRNA impaired autophagy and rendered MDA-MB231 cells more sensitive to the anticancer drug, doxorubicin [15]. Furthermore, other groups have reported that ELK3 is linked to platinum drug sensitivity in ovarian and breast cancer [16, 17]. Thus, we investigated whether the ELK3 expression levels in two representative human TNBC cell lines, MDA-MB231, and Hs578T, affected the response of these cells to CDDP. We previously generated the ELK3KD-578T and ELK3KD-231 cell lines, in which ELK3 expression was knocked down, using an ELK3-targeting shRNA [13]. Treatment with various concentrations of CDDP for 48 h reduced the number of ELK3KD-578T and ELK3KD-231 cells to a greater extent than that of control cells (Fig. 1A, B). Additionally, the colony formation rate of ELK3KD-231 cells was significantly lower compared to the control cells upon treatment with CDDP (Supplementary Fig. 1). The anticancer effects of CDDP have been linked to cell cycle arrest because of its ability to crosslink the purine bases of DNA and inhibit DNA repair [18]. We therefore next assessed the effect of CDDP on the cell cycle of ELK3KD-TNBC cells. CDDP treatment halted ~1.5-fold more ELK3KD-231 and ELK3KD-578T cells in G2/M phase than control cells (Fig. 1C). Since the cytotoxic effects of CDDP are also related to the generation of intracellular ROS [19], we evaluated ROS levels in cancer cells following CDDP treatment. We found that the levels of intracellular ROS in ELK3KD-231 cells were much higher than those in control cells, irrespective of CDDP treatment (Fig. 1D). Consistent with the report that the accumulation of intracellular ROS can induce DNA damage and subsequent cell cycle arrest [20, 21], CDDP treatment resulted in a 1.5-fold greater increase in the number of γ-H2AX-positive ELK3KD-TNBC cells than in that of γ-H2AX-positive control cells (Fig. 1E, F). Taken together, these findings imply that ELK3 knockdown rendered TNBC cells more sensitive to CDDP than control cells, by increasing ROS production and subsequently causing DNA damage and cell cycle arrest.

PMID 37422450