Therapeutic Target For Triple-negative Breast Cancer Research Articles

Machine learning analysis identifies genes differentiating triple negative breast cancers

Triple negative breast cancer (TNBC) is one of the most aggressive form of breast cancer (BC) with the highest mortality due to high rate of relapse, resistance, and lack of an effective treatment. Various molecular approaches have been used to target TNBC but with little success. Here, using machine learning algorithms, we analyzed the available BC data from the Cancer Genome Atlas Network (TCGA) and have identified two potential genes, TBC1D9 (TBC1 domain family member 9) and MFGE8 (Milk Fat Globule-EGF Factor 8 Protein), that could successfully differentiate TNBC from non-TNBC, irrespective of their heterogeneity. TBC1D9 is under-expressed in TNBC as compared to non-TNBC patients, while MFGE8 is over-expressed. Overexpression of TBC1D9 has a better prognosis whereas overexpression of MFGE8 correlates with a poor prognosis. Protein–protein interaction analysis by affinity purification mass spectrometry (AP-MS) and proximity biotinylation (BioID) experiments identified a role for TBC1D9 in maintaining cellular integrity, whereas MFGE8 would be involved in various tumor survival processes. These promising genes could serve as biomarkers for TNBC and deserve further investigation as they have the potential to be developed as therapeutic targets for TNBC.

Overexpression of IL-8 promotes cell migration via PI3K-Akt signaling pathway and EMT in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a type of malignant and heterogeneous tumor in premenopausal females with ineffective therapeutic targets. IL-8 is one of the earliest discovered chemotaxis cytokines which expression is closely related to the progress of various cancers. Previous studies show that IL-8 determines the prognosis of TNBC patients, nevertheless how IL-8 influence the progress of TNBC is unclear. In our studies, we discovered that overexpression of IL-8 promotes TNBC cells (TNBCs) migration and tumor growth via the PI3K-Akt and MAPK signaling pathway. Cell-cycle of TNBCs arrest at S phase by overexpression of IL-8, however, there is no significant difference on the cell viability and cell apoptosis of TNBCs. Besides, overexpression of IL-8 result in the downregulation of E-cadherin and the upregulation of Cyclin B1 in MDA-MB-231 cells. Taken together, our results suggest that IL-8 plays a crucial role in the progress of TNBC, and it could be a novel therapeutic target of TNBC.

Frequency of MicroRNA Response Elements Identifies Pathologically Relevant Signaling Pathways in Triple-Negative Breast Cancer.

SummaryComplex interactions between mRNAs and microRNAs influence cellular functions. The mRNA-microRNA interactions also determine the post-transcriptional availability of mRNAs and unbound microRNAs. MicroRNAs binds to one or more microRNA response elements (MREs) located on the 3′UTR of mRNAs. In this study, we leveraged MREs and their frequencies in cancer and matched normal tissues to obtain insights into disease-specific interactions between mRNAs and microRNAs. We developed a bioinformatics method “ReMIx” that utilizes RNA sequencing (RNA-Seq) data to quantify MRE frequencies across the transcriptome. We applied ReMIx to triple-negative (TN) breast cancer tumor-normal adjacent pairs and identified MREs specific to TN tumors. ReMIx identified candidate mRNAs and microRNAs in the MAPK signaling cascade. Further analysis of MAPK gene regulatory networks revealed microRNA partners that influence and modulate MAPK signaling. In conclusion, we demonstrate a novel method of using MREs in the identification of functionally relevant mRNA-microRNA interactions in TN breast cancer.

The clinical impact of the expression of the cancer stem cell marker CD44v9 in early triple negative breast cancer (TNBC).

e12566 Background: The adhesion molecule CD44 is expressed in cancer stem-like cells (CSCs). CD44 variant 9 (CD44v9), a splicing variant of CD44, has emerged as a novel marker of cancer stemness. CD44v9 was reported to be associated with the resistance to chemotherapy in several malignancies. We recently reported that the high expression of CD44v9 is associated with the poor prognosis of the patients with triple negative breast cancer (TNBC), who received neoadjuvant chemotherapy (NAC). In this study, we investigated the clinical impact of the expression of CD44v9 in early TNBC who underwent surgery without NAC. Methods: Among 2257 primary breast cancer patients at clinical stage I-III who underwent surgery without NAC form 2002 to 2015, 201 patients with TNBC were included in this study. The expression of CD44v9 was analyzed immunohistochemistry (IHC). The associations between the CD44v9 expression and the clinicopathological characteristics and the prognosis were investigated. The tumors with the positive staining in more than two third of the cancer cells were regarded to be high expression of CD44v9. Results: CD44v9 high expression was observed in 62 cases (30.8%) and the low expression was in 139 cases (69.2%). Many of the CD44v9 high tumors were low grade than the tumors with low CD44v9. There were no significant associations between the expression levels of CD44v9 and the tumor size, lymph node metastasis and the pathological stage (pStage). There were no differences in the prognosis between the high and low CD44v9 expression in pStage I. However, in the patients with the tumors with pT1c or more (pT2, pT3) and the patients with pStage 2 and more, the high CD44v9 expression was significantly associated with the poor prognosis in terms of distant recurrence-free survival (DRFS) and overall survival (OS). In addition, high CD44v9 was associated with significantly shorter survival after recurrence. Many of these patients received various chemotherapy in the adjuvant setting and after recurrence. Therefore, CD44v9 high expression is considered to be associated with the poor prognosis due to the resistance to various chemotherapeutic agents. Conclusions: The prognosis of the TNBC patients with the high expression of CD44v9 is poor, which might be induced by the resistance to various chemotherapeutic agents. These results suggest that CD44v9 might be a novel therapeutic target for TNBC.

Translational relevance of androgen receptor immunohistochemistry scoring systems for data harmonization in triple negative breast cancer (TNBC).

1078 Background: Androgen receptor (AR) expressing triple negative breast cancer (TNBC) is a sub-set of TNBC with an evolving prognostic and predictive behaviour. AR immunohistochemical threshold for positivity has not been standardized and a wide range of cut-offs have been used across studies ( > 0% to 75%). In this study we explored AR immunohistochemistry thresholds in relation to disease free survival (DFS) and clinical outcomes in non-metastatic TNBC using the Allred and H-Score systems. Increasing interest in AR as a therapeutic target for TNBC and the use of digital tissue image analysis makes it important to standardize AR immunohistochemistry reporting. Methods: 100 FFPE (formalin-fixed paraffin-embedded) tumour blocks were retrieved for non-metastatic TNBCs diagnosed between January 2015 and May 2017 and immunostained using AR441 (IgG1) mouse monoclonal antibody. Clinical follow-up ranged from 59 to 31 months and DFS was calculated. Cut-off scores were explored using Evaluate Cutpoints ( R maxstat package) and X-tile software. The score with maximum split in DFS (based on log-rank statistics and lowest p-value) was chosen as the cut-off. Descriptive and survival statistics was performed. Results: The median age was 51 (SD 11.262; range 28 to 82) years. Using Evaluate Cutpoints ≥3 was found as the threshold for AR by Allred Score. 36% cases were AR positive using Allred score (HR 0.508; CI 0.234 - 1.11; p-value 0.08). Using Evaluate Cutpoints ≥30 was found as the threshold for AR by H-Score (HR 0.624, CI 0.306 - 1.27; p-value 0.19). 35% cases were AR positive using H-Score. X-tile analysis also found the cut-offs as ≥3 and ≥30 for Allred and H-Score respectively (p < 0.05). A significant correlation was seen between the two scoring systems (Pearson Correlation 0.935; p < 0.01). A significantly higher number of grade III TNBCs were AR negative (n = 55/76) compared to grade II (n = 9/24) (p = 0.002). Cut-off for Ki67 was 75 (HR 1.61, CI 0.85-3.04, p-value 0.141) with a significantly higher number of AR negatives in the Ki67≥75 group (21/26; p < 0.05). The overall median DFS was 51.9 months. There was no significant difference in DFS for the AR negative (median: 47.4 months; mean: 39.39 months) and AR positive (Median survival not reached; mean: 41.3 months) groups(p = 0.23). Conclusions: AR immunohistochemistry cut-offs using the Allred (≥3) and H-Score (≥30) are close to the ones used for ER/PR immunohistochemistry as per ASCO/CAP guidelines, making a strong case for universal application of these systems for harmonization of AR data.

PIK3Cδ expression by fibroblasts promotes triple-negative breast cancer progression.

As there is growing evidence for the tumor microenvironment's role in tumorigenesis, we investigated the role of fibroblast-expressed kinases in triple-negative breast cancer (TNBC). Using a high-throughput kinome screen combined with 3D invasion assays, we identified fibroblast-expressed PIK3Cδ (f-PIK3Cδ) as a key regulator of cancer progression. Although PIK3Cδ was expressed in primary fibroblasts derived from TNBC patients, it was barely detectable in breast cancer (BC) cell lines. Genetic and pharmacological gain- and loss-of-function experiments verified the contribution of f-PIK3Cδ in TNBC cell invasion. Integrated secretomics and transcriptomics analyses revealed a paracrine mechanism via which f-PIK3Cδ confers its protumorigenic effects. Inhibition of f-PIK3Cδ promoted the secretion of factors, including PLGF and BDNF, that led to upregulation of NR4A1 in TNBC cells, where it acts as a tumor suppressor. Inhibition of PIK3Cδ in an orthotopic BC mouse model reduced tumor growth only after inoculation with fibroblasts, indicating a role of f-PIK3Cδ in cancer progression. Similar results were observed in the MMTV-PyMT transgenic BC mouse model, along with a decrease in tumor metastasis, emphasizing the potential immune-independent effects of PIK3Cδ inhibition. Finally, analysis of BC patient cohorts and TCGA data sets identified f-PIK3Cδ (protein and mRNA levels) as an independent prognostic factor for overall and disease-free survival, highlighting it as a therapeutic target for TNBC.

Triple Negative Breast Cancer: How Neurokinin-1 Receptor Antagonists Could Be Used as a New Therapeutic Approach.

Breast cancer (BC) is the most frequently diagnosed cancer and the leading cause of cancer death among females. BC cells not showing HER-2/Neu amplification and not expressing estrogen/ progesterone receptors are named triple-negative BC (TNBC) cells. TNBC represents 10-15% of all BC and is associated with an aggressive clinical course. TNBC patient prognosis, survival and response to current therapies are poor and for this reason, it is crucial to search for new therapeutic targets in TNBC to develop new therapeutic strategies. One of these targets is the neurokinin-1 receptor (NK-1R). It is well known that the substance P (SP)/NK-1R system is involved in cancer progression. TNBC cells overexpress the NK-1R and, after binding to this receptor, SP promotes the proliferation/ migration of TNBC cells. Non-peptide NK-1R antagonists (e.g., aprepitant) are known to exert, via the NK-1R, an antitumor action; TNBC cells die by apoptosis. In this review, we update the data on a promising therapeutic innovation: the use of NK-1R antagonists for the treatment of TNBC patients.

Targeted therapy and drug resistance in triple-negative breast cancer: the EGFR axis.

Targeting of estrogen receptor is commonly used as a first-line treatment for hormone-positive breast cancer patients, and is considered as a keystone of systemic cancer therapy. Likewise, HER2-targeted therapy significantly improved the survival of HER2-positive breast cancer patients, indicating that targeted therapy is a powerful therapeutic strategy for breast cancer. However, for triple-negative breast cancer (TNBC), an aggressive breast cancer subtype, there are no clinically approved targeted therapies, and thus, an urgent need to identify potent, highly effective therapeutic targets. In this mini-review, we describe general strategies to inhibit tumor growth by targeted therapies and briefly discuss emerging resistance mechanisms. Particularly, we focus on therapeutic targets for TNBC and discuss combination therapies targeting the epidermal growth factor receptor (EGFR) and associated resistance mechanisms.

Transcriptional Regulation of the Proteasome: Implications for Cancer Therapy

While proteasome inhibitors are being effectively used in the clinic to treat multiple myeloma and mantle cell lymphoma, the reasons for why this strategy is not generally applicable to other tumor types is poorly understood. A detailed characterization of the cellular events triggered in response to these agents could offer vital clues to target the protein degradation pathway(s) more effectively in a broad range of tumors.Our previous studies have established the transcription factor Nrf1 as a key player in the stress‐response pathway invoked by inhibition of cellular proteasome. Nrf1, by its ability to induce de novo synthesis of proteasome subunit genes in response to proteasome inhibitors, promotes the recovery of proteasome activity, thus mitigating proteotoxic stress and enhancing cellular survival. From a therapeutic stand‐point, this suggests that disabling the Nrf1‐mediated proteasome recovery pathway could potentiate the action of proteasome inhibitor treatments. However, effective means to curb the Nrf1 pathway remain scarce. Here, using an in silico chemical biology approach, we uncovered a class of therapeutic agents that can effectively inhibit the Nrf1 pathway. Mechanistic dissection of this phenomenon provided further novel insight into the way by which Nrf1 maintains its transcriptional activity. Using triple negative breast cancer (TNBC) cell lines and patient‐derived xenograft (PDX) models, we further validated our hypothesis that attenuating the Nrf1 pathway could increase the efficacy of proteasome inhibitor therapeutics. Thus, our study sheds light on the unique biology of Nrf1 along with validating this pathway as a therapeutic target in TNBC. This could serve as a motivation for similar future efforts where Nrf1 pathway inhibitors can be tested as adjuvants for proteasome inhibitor treatments in other cancer types.Support or Funding InformationNIH grant R01GM132396

Assessment of Estrogen Receptor Beta in Patients with Triple Negative Breast Cancer

Introduction: Triple negative breast cancer (TNBC), defined as lacking the expression of estrogen receptor alpha (ERα), progesterone receptor and human epidermal growth factor receptor is an aggressive breast cancer subtype for which there is a need to identify new therapeutic targets. About 50-80% of TNBC express Estrogen Receptor Beta (ERβ). Given its slight differences from ERα in terms of structure, signaling and its anti-proliferative effects on breast cancer cells, ERβ may be a possible therapeutic target. This study sought to assess the presence of ERβ and its correlation with the clinico-pathological features among the Egyptian females with TNBC. Material and Methods: The study was a retrospective analysis. The following data was retrieved from patient’s files and recorded: age, tumor size, lymph nodes metastasis, and stage. Paraffin blocks for patients confirmed by IHC to be TNBC were subjected to immunohistochemical staining for ERβ, CK5/6, and Ki 67. Results: ERβ was positive in 80% of cases (n=32/40). ERβ significantly correlated with Age (rs = -0.473, P = 0.002, n = 40), Tumor size (rs = -0.471, P = 0.007, n = 40), Lymph nodes (rs = -0.365, P = 0.021, n =40), and Stage (rs = -0.468, P = 0.002, n = 40). There was no correlation between ERβ with Ki-67 and CK5/6 a marker of the basal phenotype. All 8 patients without ERβ had an aggressive disease. 4 received neoadjuvant chemotherapy with minimum or no pathologic complete response in the axillary lymph nodes, 2 presented with upfront metastatic disease while the other 2 were cases of local recurrence with a change in the molecular phenotype of the tumor from luminal subtype to TNBC. Conclusion: This study shows ERβ expression occurs in TNBC. It may have the potential to become a therapeutic target for TNBC.

Nippon Shokubai succeeded in improving the performance of the electrolyte for all solid polymer batteries

The Serum Amyloid A (SAA) family of proteins is associated with various pathological conditions, including cancer. However, their role in cancer is incompletely understood. Here, we investigated the role of SAA1 in cell cycle regulation, apoptosis, survival signaling, metabolism, and metastasis in models of triple-negative breast cancer (TNBC), using RNAi. Our data show that in untransformed epithelial cells (MCF12A), the knockdown of SAA1 induces the expression of cell cycle regulators (MCM2, p53), the activation of DNA repair (PARP synthesis), and survival signaling (NFκB). In contrast, knockdown of SAA1 in the TNBC cell line (MDA-MB-231) induced the expression p16 and shifted cells in the cell cycle from the S to G2/M phase, without the activation of DNA repair. Moreover, in SAA1-deficient MDA-MB-231 and HCC70 cells, metabolism (NADH oxidation) continually increased while cell migration (% wound closure and the rate of wound closure) decreased. However, silencing of SAA1 altered epithelial and mesenchymal markers in MCF12A (E-cadherin, Laminin 1β, Vimentin) and MDA-MB-231 (α-Smooth muscle actin) cells, associated with the metastatic program of epithelial-mesenchymal transition. Nonetheless, our data provide evidence that SAA1 could potentially serve as a therapeutic target in TNBC.

Overexpression of IL-8 promotes cell migration via PI3K-Akt signaling pathway and EMT in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a type of malignant and heterogeneous tumor in premenopausal females with ineffective therapeutic targets. IL-8 is one of the earliest discovered chemotaxis cytokines which expression is closely related to the progress of various cancers. Previous studies showed that IL-8 determines the prognosis of TNBC patients, nevertheless how IL-8 influences the progress of TNBC is unclear. In our studies, we discovered that overexpression of IL-8 promotes TNBC cells (TNBCs) migration and tumor growth via the PI3K-Akt and MAPK signaling pathway. Cell-cycle of TNBCs arrest at S phase by overexpression of IL-8, however, there is no significant variation on the cell viability and cell apoptosis of TNBCs. Besides, overexpression of IL-8 result in the downregulation of E-cadherin and the upregulation of Cyclin B1 in MDA-MB-231 cells. Taken together, our results suggest that IL-8 performs a crucial role in the progress of TNBC, and it could be a novel therapeutic target of TNBC.

LncRNA FAM83H-AS1 promotes triple-negative breast cancer progression by regulating the miR-136-5p/metadherin axis.

In this study, we evaluated the function and regulation of the long non-coding RNA (lncRNA) FAM83H-AS1 in triple-negative breast cancer (TNBC). Our data show that the FAM83H-AS1 levels are increased in human TNBC cells and tissues. Proliferation, migration, and invasion of TNBC cells are decreased by FAM83H-AS1 suppression, but increased by FAM83H-AS1 overexpression. Bioinformatics analysis revealed that miR-136-5p is a potential target of FAM83H-AS1. MiR-136-5p expression is decreased in TNBC tissues, and its overexpression suppresses TNBC cell proliferation, migration, and invasion. MiR-136-5p suppression reverses the FAM83H-AS1 silencing-mediated inhibition of TNBC cell proliferation, migration, and invasion, suggesting that FAM83H-AS1 exerts its oncogenic effect by inhibiting miR-136-5p. Our data identify metadherin (MTDH) as the target gene of miR-136-5p, and demonstrate that the MTDH expression is increased in human TNBC tissues, which induces proliferation, migration, and invasion of TNBC cells. Importantly, our in vivo data show that FAM83H-AS1 also promotes tumor growth in TNBC mouse xenografts. Together, our results demonstrate that FAM83H-AS1 functions as an oncogenic lncRNA that regulates miR-136-5p and MTDH expression during TNBC progression, and suggest that targeting the FAM83H-AS1/miR-136-5p/MTDH axis may serve as a novel therapeutic target in TNBC.

Abstract P6-07-04: Mechanosensing calcium channel PIEZO2 accelerates epithelial-mesenchymal transition in triple-negative breast cancer

Abstract Introduction: Breast cancer is the most common female malignancy. Despite improvements in therapeutic options, metastatic disease remains incurable with limited treatments especially in triple-negative breast cancer (TNBC). Clinical outcomes in patients with TNBC continue to be worse as compared to other breast cancer subtypes. Therefore, novel therapeutic targets for TNBC are in urgent need. PIEZO2, which is a transmembrane calcium channel, is known to function as a channel-type mechanosensor in various aspects of mechanotransduction. Recently there have been reports that PIEZO2 may be involved in carcinogenesis and cancer progression. However, the role of PIEZO2 in breast cancer has not been elucidated. We hypothesized that PIEZO2 plays significant roles in progression of TNBC. Methods: To investigate the role of PIEZO2 in human breast cancer patients, The Cancer Genome Atlas (TCGA) breast cancer cohort, in which there are mRNA expression data from RNA-seq and clinical data, was utilized. PIEZO2 expression level was compared among breast cancer subtypes. Patients’ survival was compared based on PIEZO2 expression in each subtype. We used gene set enrichment analysis (GSEA) algorithm to evaluate pathways associated with PIEZO2 expression. To further elucidate the mechanisms of PIEZO2 roles, TNBC cell line, MDA-MB-231 was utilized for in vitro experiments. PIEZO2 expression was downregulated by small interfering RNA (siRNA). Cell viability was quantified by MTT assay. Protein expression was compared by western blot. Results: Breast cancer showed the highest PIEZO2 expression level, among various types of cancers in TCGA. In the breast cancer cohort, PIEZO2 expression level was lower in the TNBC subtype compared to other subtypes (p<0.001). Thus we hypothesized that PIEZO2 has different roles in each subtype. Survival analyses revealed that patients with high expression of PIEZO2 tumors showed significantly worse overall survival (p=0.016) in the TNBC cohort. However, there was no significant difference in the hormone receptor (HR)-positive (p=0.555) and the HER2-positive (p=0.380) cohorts. To explore underlying mechanisms how PIEZO2 high expressing tumors associate with worse prognosis in TNBC, GSEA was conducted. PIEZO2 high expressing tumors enriched epithelial-mesenchymal transition (EMT; NES=1.90, p=0.012) and Angiogenesis (NES=1.91, p<0.001) gene sets, as well as TAZ/YAP gene set (NES=1.57, p=0.028). PIEZO2 knockdown by siRNA significantly suppressed cell viability. This finding was validated by PIEZO inhibitor GSMTx-4 treatment. Further, expression levels of EMT related proteins, such as Vimentin and Snail, were downregulated in the PIEZO2 knockdown MDA-MB-231 cells compared to control cells. Conclusion: We describe a novel signaling pathway mediated by PIEZO2 leading to worse clinical outcomes in patients with TNBC by activation of EMT. Further work is ongoing to validate PIEZO2 as a novel therapeutic target for TNBC. Citation Format: Eriko Katsuta, Marija Vujcic, Shipra Gandhi, Arthur Beyder, Kazuaki Takabe, Mateusz Opyrchal. Mechanosensing calcium channel PIEZO2 accelerates epithelial-mesenchymal transition in triple-negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-07-04.

Abstract P3-06-06: The importance of p300/CBP (CREBBP)-survivin (BIRC5) for cell cycle and apoptosis in triple negative breast cancer

Abstract In breast cancer, both incidence and mortality have been increasing. Triple negative breast cancer (TNBC), one of breast cancer subtypes, is a heterogeneous nature that is negative for estrogen receptor (ER), progesterone receptor (PR) and human epithelial growth factor receptor type 2 (HER2). Due to the lack of expression of therapeutic targets, new therapeutic strategy and elucidation the mechanism of action in TNBC are strongly desired. Histone acetyltransferases E1A binding protein (p300) and CREB binding protein (CBP) are coactivators of a large number of essential transcription factors that contribute to cell survival and proliferation. p300/CBP (CREBBP) interacts with transcription factors and other proteins through histone acetyltransferase (HAT) and regulates several gene expression. Dysregulation of CREBBP HAT activity could contribute to various diseases, including cancer. It is reported that the high expression of p300 correlates with poor prognosis in breast cancers. However, the mechanism of CREBBP HAT activity remains unclear. We, therefore, focused on CREBBP and clarified its important mechanism in TNBC. In breast cancer cell lines, p300 expression was higher than the normal mammary epithelial cell (MCF10A) in mRNA and protein levels and positively correlated with that of CBP. To evaluate the roles of p300 and CBP in cell survival and proliferation, the loss of function studies was conducted. In TNBC cell line (MDA-MB-231), cell survival and proliferation significantly reduced in p300 knockdown cells. The reduction in cell survival and proliferation was also observed using p300 inhibitor (L002). To examine the mechanism of action for the reduction of cell survival and proliferation in p300 inhibition, cell cycle assay and apoptosis assay were performed. We found that the cell-cycle progression was arrested at the cell cycle G2/M phase and apoptosis induction in p300 knockdown cells. These results suggest that cell cycle G2/M phase arrest and the late stage of apoptosis are caused by p300 inhibition and p300 inhibition enhances the anti-tumor effect. To identify a mechanism responsible for the reduction of cell survival and proliferation in p300 inhibition, antibody array was performed. As a result of this assay and TCGA patients cohorts analysis, we identified survivin (BIRC5), a member of the inhibitor of apoptosis (IAP) family, as a functional novel biomarker of TNBC with significantly higher expression. These results suggest that suppression of BIRC5 through CREBBP inhibitor result in G2/M phase cell cycle arrest and apoptotic cell death in vitro and in vivo. In conclusion, CREBBP-BIRC5 transcriptional complex could be a novel therapeutic target for TNBC. Citation Format: Sunao Tanaka, Natsuki Wariishi, Nobuko Kawaguchi-Sakita, Souichi Adachi, Masakazu Toi, Yasuhiko Kamikubo. The importance of p300/CBP (CREBBP)-survivin (BIRC5) for cell cycle and apoptosis in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-06.

TAK1 signaling regulates p53 through a mechanism involving ribosomal stress

Triple-negative breast cancer (TNBC) is among the most aggressive forms of breast cancer with limited therapeutic options. TAK1 is implicated in aggressive behavior of TNBC, while means are not fully understood. Here, we report that pharmacological blockade of TAK1 signaling hampered ribosome biogenesis (RBG) by reducing expression of RBG regulators such as RRS1, while not changing expression of ribosomal core proteins. Notably, TAK1 blockade upregulated expression of p53 target genes in cell lines carrying wild type (wt) TP53 but not in p53-mutant cells, suggesting involvement of ribosomal stress in the response. Accordingly, p53 activation by blockade of TAK1 was prevented by depletion of ribosomal protein RPL11. Further, siRNA-mediated depletion of TAK1 or RELA resulted in RPL11-dependent activation of p53 signaling. Knockdown of RRS1 was sufficient to disrupt nucleolar structures and resulted in activation of p53. TCGA data showed that TNBCs express high levels of RBG regulators, and elevated RRS1 levels correlate with unfavorable prognosis. Cytotoxicity data showed that TNBC cell lines are more sensitive to TAK1 inhibitor compared to luminal and HER2+ cell lines. These results show that TAK1 regulates p53 activation by controlling RBG factors, and the TAK1-ribosome axis is a potential therapeutic target in TNBC.

RNA-seq analysis identified hormone-related genes associated with prognosis of triple negative breast cancer.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer that currently lacks effective biomarkers and therapeutic targets required to investigate the diagnosis and treatment of TNBC. Here we performed a comprehensive differential analysis of 165 TNBC samples by integrating RNA-seq data of breast tumor tissues and adjacent normal tissues from both our cohort and The Cancer Genome Atlas (TCGA). Pathway enrichment analysis was conducted to evaluate the biological function of TNBC-specific expressed genes. Further multivariate Cox proportional hazard regression was performed to evaluate the effect of these genes on TNBC prognosis. In this report, we identified a total of 148 TNBC-specific expressed genes that were primarily enriched in mammary gland morphogenesis and hormone levels related pathways, suggesting that mammary gland morphogenesis might play a unique role in TNBC patients differing from other breast cancer types. Further survival analysis revealed that nine genes ( FSIP1, ADCY5, FSD1, HMSD, CMTM5, AFF3, CYP2A7, ATP1A2, and C11orf86) were significantly associated with the prognosis of TNBC patients, while three of them ( ADCY5, CYP2A7, and ATP1A2) were involved in the hormone-related pathways. These findings indicated the vital role of the hormone-related genes in TNBC tumorigenesis and may provide some independent prognostic markers as well as novel therapeutic targets for TNBC.

RNA-binding protein NONO contributes to cancer cell growth and confers drug resistance as a theranostic target in TNBC.

Breast cancer (BC) is one of the most common cancers in women. TNBC (Triple-negative breast cancer) has limited treatment options and still lacks viable molecular targets, leading to poor outcomes. Recently, RNA-binding proteins (RBPs) have been shown to play crucial roles in human cancers, including BC, by modulating a number of oncogenic phenotypes. This suggests that RBPs represent potential molecular targets for BC therapy.Methods: We employed genomic data to identify RBPs specifically expressed in TNBC. NONO was silenced in TNBC cell lines to examine cell growth, colony formation, invasion, and migration. Gene expression profiles in NONO-silenced cells were generated and analyzed. A high-throughput screening for NONO-targeted drugs was performed using an FDA-approved library.Results: We found that the NONO RBP is highly expressed in TNBC and is associated with poor patient outcomes. NONO binds to STAT3 mRNA, increasing STAT3 mRNA levels in TNBC. Surprisingly, NONO directly interacts with STAT3 protein increasing its stability and transcriptional activity, thus contributing to its oncogenic function. Importantly, high-throughput drug screening revealed that auranofin is a potential NONO inhibitor and inhibits cell growth in TNBC.Conclusions: NONO is an RBP upstream regulator of both STAT3 RNA and protein levels and function. It represents an important and clinically relevant promoter of growth and resistance of TNBCs. NONO is also therefore a potential therapeutic target in TNBC.

MiR-518a-3p Suppresses Triple-Negative Breast Cancer Invasion and Migration Through Regulation of TMEM2.

MicroRNAs (miRNAs) are emerging as critical mediators in tumors, including triple-negative breast cancer (TNBC). The role of miR-518a-3p in TNBC was investigated to identify potential therapeutic target. Data from KM Plotter database (www.kmplot.com) showed that high miR-518a-3p expression was significantly associated with overall survival of patients with TNBC (p = 0.04). The expression of miR-518a-3p was dysregulated in TNBC cells. Functional assays revealed that over-expression of miR-518a-3p inhibited cell invasion and migration of TNBC. Additionally, miR-518a-3p could target TMEM2 (transmembrane protein 2), and decreased protein and mRNA expression of TMEM2 in TNBC cells. Knockdown of TMEM2 suppressed cell invasion and migration through inhibiting phospho (p)-JAK1 (Janus kinase 1) and p-STAT (signal transducer and activator of transcription protein) 1/2. Moreover, over-expression of TMEM2 counteracted the suppressive effect of miR-518a-3p on TNBC invasion and migration through promoting the levels of p-JAK1 and p-STAT1/2. In conclusion, miR-518a-3p negatively regulates the JAK/STAT pathway via targeting TMEM2 and suppresses invasion and migration in TNBC, suggesting that miR-518a-3p may be a potential therapeutic target in TNBC.

Structural analysis of human SEPHS2 protein, a selenocysteine machinery component, over-expressed in triple negative breast cancer

Selenophosphate synthetase 2 (SEPHS2) synthesizes selenide and ATP into selenophosphate, the selenium donor for selenocysteine (Sec), which is cotranslationally incorporated into selenoproteins. The action and regulatory mechanisms of SEPHS2 as well as its role in carcinogenesis (especially breast cancer) remain ambiguous and need further clarification. Therefore, lacking an experimentally determined structure for SEPHS2, we first analyzed the physicochemical properties of its sequence, modeled its three-dimensional structure and studied its conformational behavior to identify the key residues (named HUB nodes) responsible for protein stability and to clarify the molecular mechanisms by which it induced its function. Bioinformatics analysis evidenced higher amplification frequencies of SEPHS2 in breast cancer than in other cancer types. Therefore, because triple negative breast cancer (TNBC) is biologically the most aggressive breast cancer subtype and its treatment represents a challenge due to the absence of well-defined molecular targets, we evaluated SEPHS2 expression in two TNBC cell lines and patient samples. We demonstrated mRNA and protein overexpression to be correlated with aggressiveness and malignant tumor grade, suggesting that this protein could potentially be considered a prognostic marker and/or therapeutic target for TNBC.