K48-linked deubiquitination of VGLL4 by USP15 enhances the efficacy of tumor immunotherapy in triple-negative breast cancer.

Immunotherapy based on PD-1/PD-L1 antagonists has been demonstrated to be efficacious in inducing tumor remission in patients with triple-negative breast cancer (TNBC). However, tumor immune evasion caused by the PD-1/PD-L1 pathway inhibits the immunotherapeutic effect of PD-1/PD-L1 inhibitors against TNBC. Therefore, identifying potential targets for blocking the PD-1/PD-L1 pathway is a compelling strategy for TNBC treatment. Here, we discovered that VGLL4 could inhibit PD-L1 transcription by suppressing STAT3 activation, thereby enhancing the efficacy of anti-PD-1 antibody immunotherapy in TNBC. Low expression of USP15, a deubiquitinating enzyme of VGLL4, was associated with reduced CD8+ T cell infiltration and poor prognosis in TNBC patients. USP15 was found to inhibit PD-L1 transcription, leading to increased CD8+ T cell infiltration and thus enhancing the efficacy of TNBC immunotherapy. Furthermore, SART3 regulated VGLL4 stability and PD-L1 transcription by influencing the nuclear translocation of USP15. In conclusion, our study provides new insights into the biological regulation of PD-L1, identifies a previously unrecognized regulator of this critical immune checkpoint, and highlights potential therapeutic targets for overcoming immune evasion in TNBC.

miR-186-ANXA9 signaling inhibits tumorigenesis in breast cancer.

Breast cancer (BC) ranks as the highest incidence among cancer types in women all over the world. MicroRNAs (miRNAs) are a class of short endogenous non-coding RNA in cells mostly functioning to silence the target mRNAs. In the current study, a miRNA screening analysis identified miR-186-5p to be downregulated in human breast cancer tumors. Functional studies in vitro demonstrated that overexpression of miR-186-5p inhibited cellular proliferation and induced cell apoptosis in multiple breast cancer cell lines including MDA-MB-231, MCF-7, and BT549 cells. Transplantation of the miR-186-5p-overexpressing MDA-MB-231 cells into nude mice significantly inhibited mammary tumor growth in vivo. Sequence blast analysis predicted annexin A9 (ANXA9) as a target gene of miR-186-5p, which was validated by luciferase reporter assay, QRT-PCR analysis, and western blot. Additional gene expression analysis of clinical tumor samples indicated a negative correlation between miR-186-5p and ANXA9 in human breast cancer. Knockdown of ANXA9 mimicked the phenotype of miR-186-5p overexpression. Reintroduction of ANXA9 back rescued the miR-186-5p-induced cell apoptosis. In addition, miR-186-5p decreased the expression of Bcl-2 and increased the expression of p53, suggesting a mechanism regulating miR-186-5p-induced cellular apoptosis. In summary, our study is the first to demonstrate miR-186-5p-ANXA9 signaling in suppressing human breast cancer. It provided a potential therapeutic target in breast cancer.

DNMT3a-dermatopontin axis suppresses breast cancer malignancy via inactivating YAP.

Breast cancer (BC) is the most common malignant tumor in women worldwide, and its recurrence and metastasis negatively affect patient prognosis. However, the mechanisms underlying its tumorigenesis and progression remain unclear. Recently, the influence of dermatopontin (DPT), which is an extracellular matrix protein, has been proposed in the development of cancer. Here we found that DNMT3a-mediated DPT, promoter hypermethylation results in the downregulation of DPT expression in breast cancer and its low expression correlated with poor prognosis. Notably, DPT directly interacted with YAP to promote YAP Ser127 phosphorylation, and restricted the translocation of endogenous YAP from the cytoplasm to the nucleus, thereby suppressing malignant phenotypes in BC cells. In addition, Ectopic YAP overexpression reversed the inhibitory effects of DPT on BC growth and metastasis. Our study showed the critical role of DPT in regulating BC progression, making it easier to explore the clinical potential of modulating DPT/YAP activity in BC targeted therapies.

E2F1-mediated ectopic expression of PP1A promotes breast cancer progression via activation of YAP1.

Hormone receptor-positive breast cancer is the most common subtype of breast cancer. The protein phosphatase PP1A gene is described as an oncogene in several tumor types; however, the biological function of PP1A in hormone receptor-positive breast cancer remains unclear. The Cancer Genome Atlas data indicates PP1A expression is upregulated in hormone receptor-positive breast cancer tissues than in normal breast tissues. We explored the biological function of PP1A in hormone receptor-positive breast cancer using MTT assays, colony formation assays, and a xenograft mouse model. The results indicated that PP1A promoted hormone receptor-positive breast cancer proliferation, both in vitro and in vivo. Mechanistically, LINC02754 recruited the binding of the transcription factor E2F1 to the PP1A promotor, thereby increasing PP1A expression. The PP1A then interacted with and dephosphorylated YAP1, resulting in YAP1 activation. The dephosphorylated YAP1 moved to the nucleus and increased the expression of the downstream oncogene CTGF, promoting hormone receptor-positive breast cancer progression. Our findings reveal the function of the LINC02754/E2F1/PP1A/YAP1 axis in hormone receptor-positive breast cancer and provide new insight into hormone receptor-positive breast cancer progression.

Circular RNA_0006014 promotes breast cancer progression through sponging miR-885-3p to regulate NTRK2 and PIK3/AKT pathway.

Breast cancer is the most common cancer in women worldwide. Numerous reports have demonstrated that circRNAs play an essential role in regulating the biological characteristics of breast cancer. However, there are currently no reports regarding the role of hsa_circ_0006014 in breast cancer. In this study, qRT-PCR was used to detect the expression of hsa_circ_0006014 and related genes. MTT, colony formation and Transwell assays were used to explore the potential biological functions of hsa_circ_0006014 in breast cancer cells. Western blotting was used to explore the potential molecular mechanisms involving hsa_circ_0006014. In vivo experiments were used to evaluate the influence of hsa_circ_0006014 on animal tumors. In this study, we found higher expression of hsa_circ_0006014 in breast tumor samples than in matched adjacent normal samples, and its expression was positively correlated with histological grade (grade iii). Phenotypically, hsa_circ_0006014 promoted the proliferation of MDA-MB-231 and MCF-7 breast cancer cells. Mechanistically, there were confirmed binding sites between hsa_circ_0006014 and miR-885-3p, and hsa_circ_0006014 promoted breast cancer cell proliferation partially by sponging miR-885-3p and influenced CDK2/CCNE1 and CDK4/6/CCND1. Furthermore, we found that hsa_circ_0006014 regulated NTRK2 through miR-885-3p to modulate the PIK3/AKT signaling pathway. Our results demonstrated that hsa_circ_0006014 promotes breast cancer progression by sponging miR-885-3p to regulate the NTRK2/PIK3CA/AKT axis.

Tumor-Derived circRNAs as Circulating Biomarkers for Breast Cancer.

Early diagnosis is the key to improving the prognosis of breast cancer (BC) patients; however, there are currently no circulating biomarkers that demonstrate good sensitivity and specificity. This study applied circular RNA (circRNA) microarray analysis, screening, and verification in BC plasma samples to identify three tumor-derived differentially expressed circRNAs: hsa_circ_0000091, hsa_circ_0067772, and hsa_circ_0000512. We constructed a diagnostic model using logistic regression analysis in the training set and established an optimal diagnostic model based on the three circRNAs, which showed sensitivity, specificity, and area under the curve (AUC) values of .971, .902, and .974, respectively. We then verified the diagnostic model in the test set which showed satisfactory stability for BC diagnosis. Additionally, the expression of hsa_circ_0000091 in plasma correlated with axillary lymph node (ALN) metastasis, TNM stage, and prognosis of BC patients. Furthermore, hsa_circ_0000091 combined with ultrasound showed predictive ability for ALN metastasis, with an AUC of .808. These findings suggested that the three identified circRNAs can be used as circulating biomarkers for BC diagnosis, with hsa_circ_0000091 potentially representing a prognostic biomarker for BC and novel approach for predicting ALN metastasis.

Circular RNA circRPPH1 promotes breast cancer progression via circRPPH1-miR-512-5p-STAT1 axis.

Breast cancer (BC) is one of the most fatal diseases among women all over the world. Non-coding RNAs including circular RNAs (circRNAs) have been reported to be involved in different aspects during tumorigenesis and progression. In this study, we aimed to explore the biological functions and underlying mechanism of circRPPH1 in BC. Candidate circRNAs were screened in dataset GSE101123 from Gene Expression Omnibus (GEO) database and a differentially expressed circRNA, circRPPH1, was discovered in BC. CircRPPH1 expression was higher in the cancerous tissue compared to paired adjacent tissue. Further in vitro and in vivo experiments indicated that circRPPH1 acted as an oncogene in BC. In addition, circRPPH1 was mainly localized in cytoplasm and played the role of miR-512-5p sponge. By sequestering miR-512-5p from the 3'-UTR of STAT1, circRPPH1 inhibited the suppressive role of miR-512-5p, stabilized STAT1 mRNA in BC and finally affected BC progression. In conclusion, these findings indicated that circRPPH1 acted as an oncogene and regulated BC progression via circRPPH1-miR-512-5p-STAT1 axis, which might provide a potential therapeutic target for BC treatment.

MIR22HG inhibits breast cancer progression by stabilizing LATS2 tumor suppressor.

The long noncoding RNA called MIR22 host gene (MIR22HG) was previously identified as a tumor suppressor in several cancers. However, the biological function of MIR22HG in breast cancer remains unknown. In this study, we aimed to determine the function and molecular mechanism of MIR22HG in breast cancer progression using transcriptomics and biotechnological techniques. Our results showed that MIR22HG expression was lower in the cancerous tissues than in the paired adjacent normal breast tissues. Additionally, MIR22HG was found to be mainly located in the cytoplasm and acted as a miR-629-5p sponge. Notably, MIR22HG stabilized the expression of large tumor suppressor 2 (LATS2), which promoted the LATS2-dependent phosphorylation of YAP1 and suppressed the expression of its downstream target oncogenes, thereby inhibiting the proliferation and migration of breast cancer cells. Therefore, our findings reveal the MIR22HG-dependent inhibition of breast cancer cell proliferation and migration via the miR-629-5p/LATS2 pathway, providing new insights and identifying novel therapeutic targets for breast cancer treatment.

KIF23 promotes triple negative breast cancer through activating epithelial-mesenchymal transition.

BACKGROUND: KIF23 is a member of kinesin family, recent researches indicate KIF23 plays an important role in the proliferation and migration of malignant cancer cells. While the function and specific molecule mechanism of KIF23 in triple negative breast cancer remains unclear. METHODS: QRT-PCR and immunohistochemistry were conducted to analyze expression of KIF23 in triple negative breast cancer tissues and paired paracancer tissues. CCK-8 assay, colony formation assay, wound healing assay and transwell assay were applied for exploring phenotype changing of triple negative breast cancer cell lines MDA-MB-231 and BT549 after siRNA-induced knockdown of KIF23. Several bioinformatic databases were used for predicting miRNAs that combing with KIF23 mRNA and verified by dual luciferase reporter assay. Western blot assay was performed to explore downstream signaling pathway of KIF23. RESULTS: KIF23 was overexpressed in triple negative breast cancer, knockdown of KIF23 by siRNA inhibited proliferation and migration of TNBC cell lines MDA-MB-231 and BT549. Mechanistically, knockdown of KIF23 resulted in the suppression of Epithelial-Mesenchymal Transition. Meanwhile, miR-195-5p was downregulated in TNBC, and dual luciferase reporter assay indicated miR-195-5p could combine with 3'UTR of KIF23 thus promoting degradation of KIF23. CONCLUSIONS: KIF23 is a potential oncogene in triple negative breast cancer, miR-195-5p could combine with 3'UTR of KIF23. Our study reveals a new sight into triple negative breast cancer.

The Role of miR-640: A Potential Suppressor in Breast Cancer via Wnt7b/ß-catenin Signaling Pathway.

In this study, we demonstrated that miR-640 is significantly downregulated in breast cancer (BC) tissues and cell lines. Overexpression of miR-640 inhibited the proliferation and migration of BC in vitro and in vivo, while depletion of miR-640 exhibited the opposite effect. Importantly, miR-640 could directly target Wnt7b, thereby regulating Wnt/ß-catenin signaling pathway in BC. In conclusion, miR-640/Wnt7b suppresses BC cells tumorigenesis via Wnt/ß-catenin signaling pathway, which might be novel targets for BC targeted therapy.

Hsa_circ_0053063 inhibits breast cancer cell proliferation via hsa_circ_0053063/hsa-miR-330-3p/PDCD4 axis.

Breast cancer (BC) is one of the most common malignancies and its mortality is the highest among females. Circular RNAs (circRNAs), a novel group of non-coding RNAs, play an important regulatory role in angiogenesis and cancer progression. Hsa_circ_0053063 is a circRNA generated from several exons of HADHA. The potential role of hsa_circ_0053063 in BC remains unknown and needs to be explored. Hsa_circ_0053063 was mainly located in the cytoplasm and activated in BC tissues and cell lines. The binding position between hsa_circ_0053063 and miR-330-3p was confirmed by luciferase reporter assay. Moreover, hsa_circ_0053063 inhibited cell viability, proliferation, and progression of BC through the negative regulation of miR-330-3p. Programmed cell death 4 (PDCD4) is a direct target of miR-330-3p. Besides, the over-expression of miR-330-3p promoted cell progression by directly targeting and regulating PDCD4. Mechanistically, hsa_circ_0053063 activated PDCD4 by targeting miR-330-3p to inhibit BC progression. In conclusion, hsa_circ_0053063 inhibits breast cancer cell proliferation via hsa_circ_0053063/hsa-miR-330-3p/PDCD4 axis, which may provide a new therapeutic target for BC patients.

Hsa_circ_0005273 facilitates breast cancer tumorigenesis by regulating YAP1-hippo signaling pathway.

BACKGROUND: Circular RNAs (circRNAs), a novel class of endogenous RNAs, have shown to participate in the development of breast cancer (BC). Hsa_circ_0005273 is a circRNA generated from several exons of PTK2. However, the potential functional role of hsa_circ_0005273 in BC remains largely unknown. Here we aim to evaluate the role of hsa_circ_0005273 in BC. METHODS: The expression level of hsa_circ_0005273 and miR-200a-3p were examined by RT-qPCR in BC tissues and cell lines. The effect of knocking down hsa_circ_0005273 in BC cell lines were evaluated by examinations of cell proliferation, migration and cell cycle. In addition, xenografts experiment in nude mice were performed to evaluate the effect of hsa_circ_0005273 in BC. RNA immunoprecipitation assay, RNA probe pull-down assay, luciferase reporter assay and fluorescence in situ hybridization were conducted to confirm the relationship between hsa_circ_0005273, miR-200a-3p and YAP1. RESULTS: Hsa_circ_0005273 is over-expressed in BC tissues and cell lines, whereas miR-200a-3p expression is repressed. Depletion of hsa_circ_0005273 inhibited the progression of BC cells in vitro and in vivo, while overexpression of hsa_circ_0005273 exhibited the opposite effect. Importantly, hsa_circ_0005273 upregulated YAP1 expression and inactivated Hippo pathway via sponging miR-200a-3p to promote BC progression. CONCLUSIONS: Hsa_circ_0005273 regulates the miR-200a-3p/YAP1 axis and inactivates Hippo signaling pathway to promote BC progression, which may become a potential biomarker and therapeutic target.

Hsa_circ_0091074 regulates TAZ expression via microRNA­1297 in triple negative breast cancer cells.

Triple negative breast cancer (TNBC) has the highest recurrence, metastasis and mortality rate of all breast cancer subtypes, due to its typically more aggressive characteristics and lack of effective targeted treatment options. The Hippo pathway is a signaling cascade composed of a group of conserved kinases, which serves an important role in almost all cancer types. Both circular RNAs (circRNAs) and microRNAs (miRNAs) are types of non­coding RNAs, which influence cancer progression. CircRNAs have been demonstrated to serve as miRNA 'sponges', binding to miRNAs to inhibit their function. In the present study, it was revealed that circular RNA hsa_circ_0091074 binds miR­1297, and that there is an inverse association between the expression levels of the two non­coding RNAs in breast cells, indicating that hsa_circ_0091074 may serve as an endogenous 'sponge' for miR­1297. Subsequently, the potential function and mechanism underlying the involvement of miR­1297 in breast cancer was investigated via MTT, colony formation, wound healing and cell cycle assays. Increased miR­1297 expression resulted in a decrease in the protein levels of critical Hippo pathway transcriptional mediator Transcriptional coactivator with PDZ­binding motif (TAZ), which is a putative target of miR­1297. This was confirmed using dual­luciferase reporter assays, which revealed that miR­1297 targets TAZ by binding its 3'­untranslated region (3'UTR). The current results indicate that miR­1297 serves as a suppressor of breast cancer cell proliferation and invasiveness, and that this can be partially reversed by hsa_circ_0091074, suggesting that the hsa_circ_0091074/miR­1297/TAZ/TEAD4 axis may represent a potential therapeutic target for triple negative breast cancer in the future.

Long noncoding RNA HOST2, working as a competitive endogenous RNA, promotes STAT3-mediated cell proliferation and migration via decoying of let-7b in triple-negative breast cancer.

BACKGROUND: Human ovarian cancer specific transcript 2 (HOST2) is a long non-coding RNA (lncRNA) reported to be specifically high expressed in human ovarian cancer. However, the mechanism that how HOST2 regulates triple negative breast cancer (TNBC) need to be explored. METHODS: In this study, expression of HOST2 was determined in 40 TNBC patients and matched non-cancerous tissues by qRT-PCR and in situ hybridization (ISH) assay. The biological functions of HOST2 was measured by losing features. The effect of HOST2 on viability, proliferation and migration was evaluated by MTT, colony formation assay, EDU analysis, transwell invasion assay and nude mouse xenograft model. Fluorescence in situ hybridization (FISH), Luciferase report assay, RNA immunoprecipitation (RIP) assay and Western blot were fulfilled to measure molecular mechanisms. RESULTS: The results showed that HOST2 was up-regulated in BC tissues and cell lines. Clinical outcome analysis demonstrated that high expression of HOST2 was associated with poor prognosis of TNBC patients. Functional experiments illustrated that knockdown of HOST2 significantly suppressed TNBC cell proliferation and migration. Western blot assays, qRT-PCR assays, RIP assays and luciferase reporter assays revealed that HOST2 regulated STAT3 via crosstalk with let-7b. Depression of HOST2 suppressed STAT3-mediated proliferation and migration in TNBC cells. HOST2 could function as a decoy of let-7b to depress expression of STAT3. CONCLUSIONS: HOST2 could function as a oncogene and promoted STAT3-mediated proliferation and migration through acting as a competing endogenous RNA, which might act as a potential biomarker for TNBC patients.

A Triple-Regulated Oncolytic Adenovirus Carrying MicroRNA-143 Exhibits Potent Antitumor Efficacy in Colorectal Cancer.

The cancer-targeting gene virotherapy might be a useful strategy for the treatment of cancer, because it could combine the advantages of both gene therapy and virotherapy. This study aimed to construct a triple-regulated oncolytic adenovirus, Ad-RGD-Survivin-ZD55-miR-143, carrying the therapeutic gene miR-143 and evaluate its possible antitumor effect in colorectal cancer. We observed that miR-143 was lowly expressed in patients with colorectal cancer. The upregulation of miR-143 could inhibit cell proliferation and induce cell apoptosis by targeting KRAS in colorectal cancer cells. Then, Ad-RGD-Survivin-ZD55-miR-143 was successfully constructed in this study. Cells infected with Ad-RGD-Survivin-ZD55-miR-143 could inhibit cell proliferation, suppress cell migration and invasion, arrest cells at the G1 phase, and induce cellular apoptosis. At the same time, Ad-RGD-Survivin-ZD55-miR-143 decreased the expression of PARP-1 and KRAS protein in vitro. In a HCT116 xenograft model, intratumoral injection of Ad-RGD-Survivin-ZD55-miR-143 resulted in reduced tumor growth. Furthermore, Ad-RGD-Survivin-ZD55-miR-143 induced apoptosis and decreased the expression level of KRAS in HCT116 xenograft cells. Our results suggested that Ad-RGD-Survivin-ZD55-miR-143 produced a strong antitumor effect by targeting KRAS and that this strategy could broaden the therapeutic options for treating colorectal cancer.

VGLL4 interacts with STAT3 to function as a tumor suppressor in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive malignancy with a poor prognosis, and there are no effective molecular-targeted drugs for TNBC patients in clinical practice. The JAK-STAT pathway is implicated in tumorigenesis and the progression of various cancers. In this study, the results demonstrated that VGLL4 is expressed at low levels in both TNBC specimens and cell lines and that VGLL4 expression is negatively correlated with Ki67 expression and tumor size in TNBC patients. VGLL4 knockdown can promote the growth of TNBC cells, while VGLL4 overexpression significantly suppresses the growth of TNBC cells in vitro. More importantly, VGLL4 significantly inhibits tumor progression in a nude mouse model. In addition, VGLL4 is a direct target of miR-454, and the upregulation of miR-454 decreases VGLL4 expression and promotes the cell growth of TNBC cells. Furthermore, we also demonstrated that VGLL4 interacts with STAT3, the core component of the JAK-STAT pathway, leading to the inactivation of STAT3 and the inhibition of STAT3 downstream transcription. Collectively, these findings indicate that VGLL4 expression is negatively associated with poor prognosis in TNBC patients. High expression of miR-454 may be one of the causes of the downregulation of VGLL4 in TNBC, and VGLL4 acts as a tumor suppressor in TNBC by interacting with STAT3 and subsequently suppresses the STAT3 signaling axis, providing potential biomarkers and therapeutic approaches for this fatal disease.

MicroRNA-301b promotes cell proliferation and apoptosis resistance in triple-negative breast cancer by targeting CYLD.

Aberrant expression of microRNAs (miRNAs) plays important roles in carcinogenesis and tumor progression. However, the expression and biological role of miR-301b in triple-negative breast cancer (TNBC) remains unclear. Here we aimed to evaluate the roles and mechanisms of miR-301b in TNBC cells. miR-301b expression was assessed in TNBC specimens and cell lines by quantitative Real-Time PCR (qRT-PCR). TNBC cells were transfected with miR-301b mimics, inhibitors or Cylindromatosis (CYLD) small interfering RNA (siRNA) using Lipofectamine 2000. The functional roles of miR-301b were determined by cell proliferation, colony formation, and apoptosis assays. Western blots and qRT-PCR were used to measure the expression of mRNAs and proteins in the cells. We found that miR-301b was upregulated in TNBC specimens and cell lines. Overexpression of miR-301b promoted cell proliferation in TNBC cells, while inhibited the apoptosis induced by 5-FU. CYLD was downregulated by miR-301b at both mRNA and protein levels in TNBC cells. Dual-luciferase report assay confirmed that miR-301b downregulated CYLD by direct interaction with the 3'-untranslated region(3'-UTR) of CYLD mRNA. NF-κB activation was mechanistically associated with miR-301b-mediated downregulation of CYLD. However, inhibition of miR-301b reversed all the effects of miR-301b. In conclusion, miR-301b plays an oncogenic role in TNBC possibly by downregulating CYLD and subsequently activating NF-κB p65, and this may provide a novel therapeutic approach for TNBC. [BMB Reports 2018; 51(11): 602-607].

VGLL4 is a transcriptional cofactor acting as a novel tumor suppressor via interacting with TEADs.

Vestigial Like Family Member 4 (VGLL4) is a transcriptional cofactor of VGLL family, which includes VGLL1-4. Unlike other members of VGLL family, VGLL4 was described as a novel tumor suppressor containing two TDU motifs. VGLL4 executes its biological function through two TDU domains via interacting with TEA domain (TEAD) transcription factors. Lower expression of VGLL4 usually indicates poor survival in many cancers, such as lung cancer, gastric cancer, breast cancer, colorectal cancer, bladder cancer, pancreatic adenocarcinoma and esophageal squamous cancer. In cancer cells, the expression of VGLL4 is lower than that of normal tissues, moreover, expression level of VGLL4 is positively related to survival rate. VGLL4 is found to play an important role in several signal pathways, mainly acts as a tumor suppressor interacting with TEADs. In Hippo signaling pathway, VGLL4 competes with YAP in binding to TEADs and inhibits the downstream of YAP. In Wnt/ß-catenin signaling pathway, VGLL4 negatively regulates Wnt/ß-catenin signaling pathway via inhibiting ß-catenin and TCF (T-cell factor). VGLL4 can also suppress epithelial-mesenchymal transition (EMT) and contribute to apoptosis signaling pathway.