MicroRNA-582-5p promotes triple-negative breast cancer invasion and metastasis by antagonizing CMTM8

ABSTRACT Triple-negative breast cancer (TNBC) commonly have aggressive properties. microRNA-582-5p (miR-582-5p) modulates the progression of several cancers. Yet, the role of miR-582-5p in TNBC progression is undetermined. In the current study, we investigated miR-582-5p expression levels and clinical significance in TNBC. The impact of miR-582-5p modulation on the biological behaviors of TNBC cells were measured. The downstream gene(s) regulated by miR-582-5p in TNBC was explored. We showed that compared to adjacent normal breast tissues, the miR-582-5p level was elevated in TNBC samples. The upregulation of miR-582-5p correlated with lymph node metastasis. Overexpression of miR-582-5p enhanced TNBC cell migration and invasion, whereas knockdown of miR-582-5p had an adverse impact on aggressive phenotype. In vivo xenograft mouse studies demonstrated that miR-582-5p overexpression accelerated TNBC growth and metastasis. Mechanistically, miR-582-5p selectively inhibited CMTM8, leading to a reduction of CMTM8 expression. CMTM8 showed suppressive effects on TNBC cell migration and invasion. Rescue experiments revealed that overexpression of CMTM8 impaired miR-582-5p-induced migration and invasion in TNBC cells. Overall, our data uncover an oncogenic role for miR-582-5p in TNBC metastasis through inhibition of CMTM8. We suggest miR-582-5p as a promising target for managing TNBC.


Introduction
Triple-negative breast cancer (TNBC), which lacks the expression of HER2 or hormone receptors, is a lethal subtype of breast cancer [1]. TNBC accounts for 15-20% of all breast cancers, and commonly has a worse prognosis than other breast cancer types [2,3]. The invasive and metastatic phenotype of cancer cells is responsible for TNBC-related poor prognosis. However, there is no effective systematic therapy available for metastatic TNBC. Therefore, it is important to uncover the molecular mechanisms involved in TNBC metastasis.
CMTM8 is a transmembrane protein ubiquitously expressed in normal tissues [4]. A number of cancers such as liver, lung, colon, rectum, esophagus, stomach, and bladder cancers exhibited downregulation of CMTM8 [5][6][7]. Downregulation of CMTM8 contributes to epithelial-to-mesenchymal transition (EMT) and increases invasive ability in hepatocellular carcinoma cells [6]. Another study has reported that overexpression of CMTM8 suppresses cell proliferation and invasion in bladder cancer cells [7], indicating the involvement of CMTM8 in tumor metastasis.
We hypothesized that miR-582-5p might also play an important role in TNBC progression. Hence, in the present study, we determined the expression of miR-582-5p in TNBC specimens and checked its relationship with clinicopathological parameters of TNBC patients. The ability of miR-582-5p in regulating TNBC cell invasion and metastasis was investigated. Additionally, we identified the direct target gene(s) that mediates the action of miR-582-5p in TNBC.

Patients and tissue specimens
We collected 68 paired TNBC and adjacent normal tissues from TNBC patients who underwent radical mastectomy or breast conserving surgery. Those patients did not receive any anticancer treatment prior to tumor resection. All tissue specimens were frozen immediately in liquid nitrogen and stored at −80°C.

Western blot analysis
Cells were lysed in ice-cold radioimmunoprecipitation assay buffer supplemented with the Protease Inhibitor Cocktail (Sigma-Aldrich). The protein samples were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. After incubation with primary antibodies recognizing CMTM8 and GAPDH (Proteintech, Wuhan, China), the membranes were probed with peroxidaseconjugated secondary antibodies. Signals were developed by enhanced chemiluminescence (Merck Millipore, Darmstadt, Germany).

Cell proliferation
Cells were seeded onto 96-well plates and cultured for 1-3 days. The activity of cells were measured using the MTT assay (Sigma-Aldrich). MTT was used at a concentration of 0.5 mg/mL, and after 4-h incubation at 37°C, dimethyl sulfoxide was then added. Absorbance was measured at 490 nm.

Wound-healing assay
TNBC cells were allowed to grow to confluence. We made a 'wound' on the monolayer using a pipette tip. After culturing for 0 and 48 h, the images of the 'wounded' monolayer was taken. We then calculated the percentage of wound closure.

Transwell invasion assay
We used Transwell chambers (8 µm in pore size) to evaluate the invasive ability of cancer cells [14]. Briefly, TNBC cells transfected with indicated constructs were suspended in serum-free media and added onto the upper chamber precoated with Matrigel. The lower chamber was filled with the media containing 10% FBS. After incubation for 48 h, the invaded cells were stained with 0.25% crystal violet and counted.

Animal experiments
Female BALB/c nude mice (4-5 weeks old) were purchased from Hanyu Biomed Company (Beijing, China). They were maintained in a specific pathogen-free animal facility under a condition of a 12-h dark/light cycle, a temperature of 24 ± 1°C, and a humidity of 52 ± 3%. These animals were given free access to standard rodent diet and water. For tumorigenic studies, stably transfected MDA-MB -231 cell lines were subcutaneously injected into nude mice (2 × 10 6 cells/mouse). Tumor growth was measured every week for 4 weeks. For metastatic studies, the MDA-MB-231 cell clones were transfected with firefly luciferase-expressing plasmid and injected into nude mice via the tail vein [21]. Six weeks after cell implantation, mice were injected with D-luciferin. In vivo bioluminescence imaging was then conducted. In each experiment, 3 groups were included. A total of 12 mice were randomly assigned to the 3 groups, and each group had 4 mice. All the procedures were conducted under anesthesia with intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight; Sigma-Aldrich). After the experiments, anesthetized mice were euthanized through cervical dislocation.

Statistics
Results were obtained from at least three independent experiments and are expressed as mean ± standard deviation. GraphPad Prism (version 7.0; GraphPad Software, La Jolla, CA, USA) was used to determine statistical differences. The significance was analyzed using the Student's t test or one-way analysis of variance (ANOVA) followed by the Tukey test. A P-value less than 0.05 was considered statistically significant.

CMTM8 is involved in miR-582-5p-mediated invasiveness of TNBC cells
Next, we asked whether CMTM8 has an impact on TNBC progression. To this end, we overexpressed CMTM8 in MDA-MB-468 and MDA-MB-231  Figure 4(a)). We found that the migratory (Figure 4(b)) and invasive (Figure 4(c)) abilities of TNBC cells were suppressed by enforced expression of CMTM8. Also, we tested whether CMTM8 is involved in the pro-invasive activity of miR-582-5p. To address this, we performed rescue experiments in miR-582-5p-overexpressing TNBC cells. Notably, restoration of CMTM8 expression significantly impaired miR-582-5p-dependent migration (Figure 4(d)) and invasion (Figure 4(e)) in TNBC cells. Taken together, the pro-invasive role of miR-582-5p is causally linked to downregulation of CMTM8.

miR-582-5p enhances TNBC metastasis by targeting CMTM8
We also validated the activity of miR-582-5p in animal models. We injected parental and miR-582-5p-overexpressing MDA-MB-231 cells into the mammary fat pad of SCID mice. The miR-582-5p overexpression group had an increased tumor growth than the control group ( Figure 5  (a-b)). qPCR analysis confirmed the increased expression of miR-582-5p and decreased expression of CMTM8 mRNA in miR-582-5p tumors compared to control tumors ( Figure 5(c)). In addition, miR-582-5p overexpression increased the metastatic burden in the lung (Figure 5(d)). Most importantly, co-expression of CMTM8 significantly reversed the effects of miR-582-5p on metastasis ( Figure 5).

Discussion
TNBC is an aggressive type of breast cancer and lacks effective therapies [1,22]. Identification of key regulators of TNBC progression is of importance for developing novel anticancer strategies. Many miRs have been found to modulate TNBC growth and metastasis [23,24]. For instance, miR-92a-3p overexpression enhances breast cancer cell proliferation and metastasis [23]. Ectopic expression of miR-210-3p stimulates aerobic glycolysis and promotes TNBC cell survival and growth under metabolic stress conditions [24]. In the present study, we show that miR-582-5p plays an oncogenic role in TNBC. Compared to adjacent normal breast tissues, TNBC specimens have greater levels of miR-582-5p. The upregulation of miR-582-5p is associated with lymph node metastasis of TNBC. However, there is no significant association between miR-582-5p expression and tumor size or stage (data not shown). In future work, we will validate the clinical significance of miR-582-5p expression in an independent, larger cohort of breast cancer patients.
Functional studies reveal that miR-582-5p promotes TNBC cell migration and metastasis. These findings suggest miR-582-5p as a novel driver of TNBC metastasis.
Although previous studies have established a link between miR-582-5p and tumor progression [12,14,17,18], its biological effects dramatically vary among different cancer types. For instance, when miR-582-5p is overexpressed, bladder cancer [12] and non-small cell lung cancer [22] had a reduced proliferation and invasion. Conversely, in glioblastoma, miR-582-5p enhances cancer stem cell survival [17]. Under androgen deprived conditions, miR-582-5p augments prostate cancer cell proliferation [18]. Our data also show the oncogenic role of miR-582-5p in TNBC. Depletion of miR-582-5p impairs the migration and invasion of TNBC cells. Moreover, enforced expression of miR-582-5p promotes TNBC cell invasion and metastasis. However, TNBC cell proliferation remains unchanged when miR-582-5p is silenced. Our results support the pro-invasive activity of miR-582-5p in TNBC. NOTCH1, AKT3, and CDK1 [13,15,25] have been proposed as the target genes of miR-582-5p. In hepatocellular carcinoma, miR-582-5p exerts growth-suppressive effects through negative regulation of CDK1 and AKT3 [13]. In prostate cancer, miR-582-5p can inhibit tumor metastasis by targeting several components of TGF-β signaling, including SMAD2, SMAD4, TGFBRI, and TGFBRII [16]. However, miR-582-5p did not affect these gene expression in TNBC cells (data not shown). Most interestingly, we show that miR-582-5p targets CMTM8 mRNA, leading to a reduction in the abundance of CMTM8 mRNA. Luciferase reporter assay confirms that miR-582-5p-mediated repression of CMTM8 is ascribed to the interaction with the target site in the 3′-UTR of CMTM8 mRNA. Furthermore, the expression of miR-582-5p and CMTM8 mRNA shows an opposite trend in TNBC cell lines. In addition to CMTM8, both KLF15 and FOXG1 are also downregulated by miR-582-5p overexpression. The 2 genes seem not to be miR-582-5p targets, as the luciferase reporters carrying their 3′-UTR remained unaffected when miR-582-5p was overexpressed. Collectively, miR-582-5p selectively targets CMTM8 mRNA in TNBC cells. It has been previously documented that CMTM8 expression is reduced in several cancer types such as liver, lung, colon, rectum, esophagus, stomach cancers [5]. However, it is still unclear how CMTM8 is downregulated in malignant tissues. In this study, we demonstrate that miR-582-5p overexpression suppresses CMTM8 expression in TNBC cells, suggesting a mechanism for the downregulation of CMTM8 in cancer. CMTM8 has been reported to inhibit the proliferation and invasion of bladder cancer cells [7]. In agreement with this study, we show that restoration of CMTM8 decreases the migratory and invasive abilities of TNBC cells. Furthermore, enforced expression of CMTM8 impairs miR-582-5p-induced TNBC cell invasion and metastasis. These results suggest that miR-582-5p promotes TNBC metastasis by targeting CMTM8. However, we can not rule out the possibility that other target genes may also mediate the oncogenic activity of miR-582-5p.

Conclusion
We identify miR-582-5p as a novel oncogene in TNBC. MiR-582-5p promotes TNBC invasion and metastasis by negatively regulating CMTM8 expression. We propose miR-582-5p as a potential therapeutic target for TNBC.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Authors' contributions
XZ, XCM, and NZ designed the study, performed the experiments, analyzed the data, and prepared the manuscript. HG, LLW, YTZ, CNS, NH, SCS, and JMZ prepared the materials and collected the data. All authors read and approved the final manuscript.

Consent for publication
Manuscript is approved by all authors for publication.

Data availability statement
The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.