Expression of the hypoxia-inducible monocarboxylate transporter MCT4 is increased in triple negative breast cancer and correlates independently with clinical outcome

Background

¹⁸Fluor-deoxy-glucose PET-scanning of glycolytic metabolism is being used for staging in many tumors however its impact on prognosis has never been studied in breast cancer.

Methods

Glycolytic and hypoxic markers: glucose transporter (GLUT1), carbonic anhydrase IX (CAIX), monocarboxylate transporter 1 and 4 (MCT1, 4), MCT accessory protein basigin and lactate-dehydrogenase A (LDH-A) were assessed by immunohistochemistry in two cohorts of breast cancer comprising 643 node-negative and 127 triple negative breast cancers (TNBC) respectively.

Results

In the 643 node-negative breast tumor cohort with a median follow-up of 124 months, TNBC were the most glycolytic (≈70%), followed by Her-2 (≈50%) and RH-positive cancers (≈30%). Tumoral MCT4 staining (without stromal staining) was a strong independent prognostic factor for metastasis-free survival (HR = 0.47, P = 0.02) and overall-survival (HR = 0.38, P = 0.002). These results were confirmed in the independent cohort of 127 cancer patients.

Conclusion

Glycolytic markers are expressed in all breast tumors with highest expression occurring in TNBC. MCT4, the hypoxia-inducible lactate/H⁺ symporter demonstrated the strongest deleterious impact on survival. We propose that MCT4 serves as a new prognostic factor in node-negative breast cancer and can perhaps act soon as a theranostic factor considering the current pharmacological development of MCT4 inhibitors.     

Statin induces inhibition of triple negative breast cancer (TNBC) cells via PI3K pathway

Primary TNBCs are treated as if they were a single disease entity, yet it is clear they do not behave as a single entity in response to current therapies. Recently, we reported that statins might have a potential benefit for TNBCs associated with ets-1 overexpression. The aim of this study is to investigate the role of PTEN loss in the effects of statin on TNBC cells. In addition, we analyze the relationship between AKT downstream pathways and the effects of statin on TNBC cells. We investigated the effect of a statin on TNBC cells and analyzed the association of PI3K pathways using various TNBC cells in terms of PTEN loss and AKT pathways. Simvastatin treatments resulted in decreased cell viabilities in various TNBC cell lines. Compared with PTEN wild-type TNBC cells, PTEN mutant-type TNBC cells showed a decreased response to simvastatin. Expressions of phosphorylated Akt and total Akt showed an inverse relationship with PTEN expression. The TNBC cell lines, which showed increased expression of p-Akt, appeared to attenuate the expression of p-Akt by PTEN loss in simvastatin-treated TNBC cells. The Akt inhibitor, LY294002, augmented the effect of simvastatin on PTEN wild-type TNBC cells. Simvastatin induces inhibition of TNBC cells via PI3K pathway activation.     

LncRNA POU3F3 promotes proliferation and inhibits apoptosis of cancer cells in triple-negative breast cancer by inactivating caspase 9

It has been reported that lncRNA POU3F3 was upregulated in esophageal squamous-cell carcinomas, indicating its role as an oncogene in this disease. However, the mechanism of its function and its involvement in other malignancies is unknown. In the present study we found that expression levels of lncRNA POU3F3 were higher in tumor tissues than in adjacent healthy tissues of triple negative breast cancer (TNBC) patients and were significantly and inversely correlated with levels of cleaved caspase 9 only in tumor tissues. In addition, plasma levels of lncRNA POU3F3 were higher in TNBC patients than in healthy controls and were significantly and inversely correlated with levels of cleaved caspase 9 only in TNBC patients. In addition, treatment of exogenous Cleaved Caspase-9 significantly attenuated the effects of lncRNA POU3F3 overexpression on cancer cell proliferation and apoptosis. lncRNA POU3F3 may promote proliferation and inhibit apoptosis of cancer cells in triple-negative breast cancer.     

Single peptides and combination modalities for triple negative breast cancer

Unlike other types of breast cancers (BCs), no specific therapeutic targets have been established for triple negative breast cancer (TNBC). Therefore, chemotherapy and radiotherapy are the only available adjuvant therapeutic choices for TNBC. New emerging reports show that TNBC is associated with higher numbers of intratumoral tumor infiltrating lymphocytes. This is indicative of host anti-TNBC immune surveillance and suggesting that immunotherapy can be considered as a therapeutic approach for TNBC management. Recent progress in molecular mechanisms of tumor-immune system interaction and cancer vaccine development studies, fast discoveries and FDA approvals of immune checkpoint inhibitors, chimeric antigen receptor T-cells, and oncolytic virotherapy have significantly attracted attention and research directions toward the immunotherapeutic approach to TNBC. Here in this review different aspects of TNBC immunotherapies including the host immune system-tumor interactions, the tumor microenvironment, the relevant molecular targets for immunotherapy, and clinical trials in the field are discussed.     

miR-483-3p suppresses the proliferation and progression of human triple negative breast cancer cells by targeting the HDAC8>oncogene

Triple negative breast cancer (TNBC) is a heterogeneous subclass of breast cancer (BC) distinguished by lack of hormone receptor expression. It is highly aggressive and difficult to treat with traditional chemotherapeutic regimens. Targeted-therapy using microRNAs (miR) has recently been proposed to improve the treatment of TNBC in the early stages. Here, we explore the roles of miR-483-3p/HDAC8 HDAC8 premiR-vector on tumorigenicity in TNBC patients. Clinical TNBC specimens and three BC cell lines were prepared. miR-483-3p and expression levels were measured using quantitative real-time polymerase chain reaction. Cell cycle progression was assessed by a flow-cytometry method. We also investigated cell proliferation by 3-2, 5-diphenyl tetrazolium bromide assay and colony formation assay. We used a to overexpress miR-483-3p, and a HDAC8-KO-vector for knocking out the endogenous production of HDAC8. Our data showed significant downregulation of miR-483-3p expression in TNBC clinical and cell line samples. The HDAC8 was also upregulated in both tissue specimens and BC cell lines. We found that increased levels of endogenous miR-483-3p affects tumorigenecity of MDA-MB-231. Downregulation of HDAC8 using the KO-vector showed the same pattern. Our results revealed that the miR-483-3p suppresses cellular proliferation and progression in TNBC cell lines via targeting HDAC8. Overall, our outcomes demonstrated the role of miR-483-3p as a tumor suppressor in TNBC and showed the possible mechanism via HDAC8. In addition, targeted treatment of TNBC with miR-483-3p might be considered in the future.     

Activation of phosphatidylinositol 3-kinase by cellular prion protein and its role in cell survival

The cellular prion protein (PrP(C)) is thought to be involved in protection against cell death, however the exact cellular mechanisms involved are still controversial. Herein we present data that strongly indicate a functional link between PrP(C) expression and phosphatidylinositol 3-kinase (PI 3-kinase) activation, a protein kinase that plays a pivotal role in cell survival. Both mouse neuroblastoma N2a cells and immortalized murine hippocampal neuronal cell lines expressing wild-type PrP(C) had significantly higher PI 3-kinase activity levels than their respective controls. Moreover, PI 3-kinase activity was found to be elevated in brain lysates from wild-type mice, as compared to prion protein-knockout mice. Recruitment of PI 3-kinase by PrP(C) was shown to contribute to cellular survival toward oxidative stress by using 3-morpholinosydnonimine (SIN-1) and serum deprivation. Moreover, both PI 3-kinase activation and cytoprotection by PrP(C) appeared to rely on copper binding to the N-terminal octapeptide of PrP(C). Thus, we propose a model in which the interaction of copper(II) with the N-terminal domain of PrP(C) enables transduction of a signal to PI 3-kinase; the latter, in turn, mediates downstream regulation of cell survival.     

Alantolactone promotes ER stress‐mediated apoptosis by inhibition of TrxR1 in triple‐negative breast cancer cell lines and in a mouse model

Triple‐negative breast cancer (TNBC) is a subtype of breast cancer with poor clinical outcome and currently no effective targeted therapies are available. Alantolactone (ATL), a sesquiterpene lactone, has been shown to have potential anti‐tumour activity against various cancer cells. However, the underlying mechanism and therapeutic effect of ATL in the TNBC are largely unknown. In the present study, we found that ATL suppresses TNBC cell viability by reactive oxygen species (ROS) accumulation and subsequent ROS‐dependent endoplasmic reticulum (ER) stress both in vitro and in vivo. Thioredoxin reductase 1 (TrxR1) expression and activity of were significantly up‐regulated in the TNBC tissue specimens compare to the normal adjacent tissues. Further analyses showed that ATL inhibits the activity of TrxR1 both in vitro and in vivo in TNBC and knockdown of TrxR1 in TNBC cells sensitized ATL‐induced cell apoptosis and ROS increase. These results will provide pre‐clinical evidences that ATL could be a potential therapeutic agent against TNBC by promoting ROS‐ER stress‐mediated apoptosis through partly targeting TrxR1.     

Circular RNA; a new biomarker for breast cancer: A systematic review

Circular RNAs (circRNAs) were recently discovered as a looped subset of competing endogenous RNAs, with an ability to regulate gene expression by microRNA sponging. There are several studies on their potential roles in cancer development, such as colorectal cancer and basal cell carcinoma. However, there is still a significant gap in the knowledge about circRNA functions in breast cancer (BC) progression. The current study systematically reviewed circRNA biogenesis and their potential roles as a novel biomarker in BC on published studies of the MEDLINE®/PubMed, Cochrane®, and Scopus® databases. The obtained results showed a general dysregulation of circRNAs expression in BC cells with a cell-type and stage-specific manner. The potential connection between circRNAs and BC cell proliferation, apoptosis, metastasis, and chemotherapy sensitivity and resistance were discussed.     

Sacituzumab govitecan in metastatic triple negative breast cancer (TNBC): Four design features in the ASCENT trial potentially favored the experimental arm

The ASCENT trial reports impressive results with a median overall survival (OS) increased from 6.7 months to 12.1 months with sacituzumab govitecan over single-agent chemotherapy, in metastatic triple negative breast cancer (TNBC) patients in second and subsequent line of therapy.We described design features in the ASCENT trial casting doubt on the extrapolation of the reported results to real world patients. First, the open-label design may exaggerate the effect of the experimental arm. Second, the choice of progression-free-survival (PFS) as a primary endpoint, debatable in metastatic TNBC, can lead to biases: early stopping rules may exaggerate efficacy results and informative censoring can bias PFS results interpretation. Third, the control arm was not a complete “physician''s choice”: it was restricted, preventing from using effective agents in this setting, and leading to a substandard control arm. Fourth and lastly, dose reduction and supportive care recommendations for the experimental drug were different between the trial protocol and the FDA labels, and favored the experimental arm as compared with the control arm.In conclusion, we described four design features in the ASCENT trial having the potential to favor the experimental arm or to penalize the control arm. It thus remains uncertain in which extent the reported outcomes will translate in the real world. Efforts should be made to avoid trial biases that will eventually prevent to conclude about their true impact in patients when applied broadly.     

Lamin B2 promotes the progression of triple negative breast cancer via mediating cell proliferation and apoptosis

Triple negative breast cancer (TNBC) is a more common type of breast cancer with high distant metastasis and poor prognosis. The potential role of lamins in cancer progression has been widely revealed. However, the function of lamin B2 (LMNB2) in TNBC progression is still unclear. The present study aimed to investigate the role of LMNB2 in TNBC. The cancer genome atlas (TCGA) database analysis and immunohistochemistry (IHC) were performed to examine LMNB2 expression levels. LMNB2 short hairpin RNA plasmid or lentivirus was used to deplete the expression of LMNB2 in human TNBC cell lines including MDA-MB-468 and MDA-MB-231. Alterations in cell proliferation and apoptosis in vitro and the nude mouse tumorigenicity assay in vivo were subsequently analyzed. The human TNBC tissues shown high expression of LMNB2 according to the bioinformation analysis and IHC assays. LMNB2 expression was correlated with the clinical pathological features of TNBC patients, including pTNM stage and lymph node metastasis. Through in vitro and in vivo assays, we confirmed LMNB2 depletion suppressed the proliferation and induced the apoptosis of TNBC cells, and inhibited tumor growth of TNBC cells in mice, with the decrease in Ki67 expression or the increase in caspase-3 expression. In conclusion, LMNB2 may promote TNBC progression and could serve as a potential therapeutic target for TNBC treatment.     

Cardamonin induces G2/M arrest and apoptosis via activation of the JNK–FOXO3a pathway in breast cancer cells

Cardamonin (CD), a naturally occurring chalcone isolated from large black cardamom, was previously reported to suppress the proliferation of breast cancer cells. However, its precise molecular anti‐tumor mechanisms have not been well elucidated. In this study, we found that CD markedly inhibited the proliferation of MDA‐MB 231 and MCF‐7 breast cancer cells through the induction of G2/M arrest and apoptosis. Reactive oxygen species (ROS) plays a pivotal role in the inhibition of CD‐induced cell proliferation. Treatment with N‐acetyl‐cysteine (NAC), an ROS scavenger, blocked CD‐induced G2/M arrest and apoptosis in this study. Quenching of ROS by overexpression of catalase also blocked CD‐induced cell cycle arrest and apoptosis. We showed that CD enhanced the expression and nuclear translocation of Forkhead box O3 (FOXO3a) via upstream c‐Jun N‐terminal kinase, inducing the expression of FOXO3a and its target genes, including p21, p27, and Bim. This process led to the reduction of cyclin D1 and enhancement of activated caspase‐3 expression. The addition of NAC markedly reversed these effects, knockdown of FOXO3a using small interfering RNA also decreased CD‐induced G2/M arrest and apoptosis. In vivo, CD efficiently suppressed the growth of MDA‐MB 231 breast cancer xenograft tumors. Taken together, our data provide a molecular mechanistic rationale for CD‐induced cell cycle arrest and apoptosis in breast cancer cells.     

Triple negative breast cancer: Key role of Tumor-Associated Macrophages in regulating the activity of anti-PD-1/PD-L1 agents

Triple-negative breast cancer (TNBC) is associated with a poor prognosis, due to its aggressive behaviour and lack of effective targeted therapies. Immunocheckpoint inhibitors, such as anti-programmed cell death 1 (PD-1) and anti-PD-ligand(L)1 agents, are in course of investigation in TNBC, used alone or in combination with other systemic or local approaches. However, the high cost of these drugs and the lack of validated predictive biomarkers support the development of strategies aimed to overcome resistance and optimize the efficacy of these approaches.Tumor-Associated Macrophages (TAMs) derive from peripheral blood monocytes recruited into the TNBC microenvironment and, in response to several stimuli, undergo M1 (classical) or M2 (alternative) activation. In TNBC, TAMs promote tumor growth and progression by several mechanisms that include the secretion of inhibitory cytokines, the reduction of effector functions of Tumor Infiltrating Lymphocytes (TILs) and the promotion of Regulatory T cell (Treg). Interestingly, TAMs have been shown to directly and indirectly modulate PD-1/PD-L1 expression in tumor environment. On this scenario, several TAM-centered strategies have been proposed, such as the suppression of TAM recruitment, the depletion of their number, the switch of M2 TAMs into antitumor M1 phenotype and the inhibition of TAM-associated molecules. In this review, we will illustrate the activity of TAMs and associated molecules in TNBC, focusing on their role in modulating the expression of PD-1/PD-L1 and on the emerging TAM-tailored strategies for TNBC patients.     

Down-regulating GRP78 reverses pirarubicin resistance of triple negative breast cancer by miR-495-3p mimics and involves the p-AKT/mTOR pathway

Due to the lack of known therapeutic targets for triple-negative breast cancer (TNBC), chemotherapy is the only available pharmacological treatment. Pirarubicin (tetrahydropyranyl Adriamycin, THP) is the most commonly used anthracycline chemotherapy agent. However, TNBC has a high recurrence rate after chemotherapy, and the mechanisms of chemoresistance and recurrence are not entirely understood. To study the chemoresistance mechanisms, we first screened compounds on a pirarubicin-resistant cell line (MDA-MB-231R) derived from MDA-MB-231. The drug resistance index of MDA-MB-231R cells was approximately five times higher than that of MDA-MB-231 cells. MDA-MB-231R cells have higher GRP78 and lower miR-495-3p expression levels than MDA-MB-231 cells. Transfecting MDA-MB-231R cells with a siGRP78 plasmid reduced GRP78 expression, which restored pirarubicin sensitivity. Besides, transfecting MDA-MB-231R cells with miR-495-3p mimics increased miR-495-3p expression, which also reversed pirarubicin chemoresistance. Cell counting kit-8 (CCK-8), EdU, wound healing, and Transwell assays showed that the miR-495-3p mimics also inhibited cell proliferation and migration. Based on our results, miR-495-3p mimics could down-regulate GRP78 expression via the p-AKT/mTOR signaling pathway in TNBC cells. Remarkably, chemo-resistant and chemo-sensitive TNBC tissues had opposite trends in GRP78 and miR-495-3p expressions. The lower the GRP78 and the higher the miR-495-3p expression, the better prognosis in TNBC patients. Therefore, the mechanism of pirarubicin resistance might involve the miR-495-3p/GRP78/Akt axis, which would provide a possible strategy for treating TNBC.     

磷酸化蛋白质组学在三阴性乳腺癌诊治研究中的进展

三阴性乳腺癌是乳腺癌中恶性程度最高的亚型,其治疗仍以化疗为主,但容易出现耐药,且患者预后较差。随着蛋白质组学技术的发展,磷酸化蛋白质组学研究取得了长足的进步,并在肿瘤发生发展机制和诊治研究中得到了广泛的应用。同样,磷酸化蛋白质组学在三阴性乳腺癌的发生发展、靶向治疗和耐药机制研究等方面也发挥着重要作用。本文主要对目前磷酸化蛋白质组学在三阴性乳腺癌中的研究进展进行综述,旨在为基于磷酸化蛋白质组学的三阴性乳腺癌发生发展机制和诊治研究提供指导和帮助。     

GPC3对肝癌细胞糖酵解的调控作用研究

目的:探讨GPC3(glypican 3)在肝癌细胞糖酵解中的调控作用。方法:采用si RNA(small interfering RNA)干扰肝癌细胞中GPC3的表达后,采用q PCR(quantitative PCR)与Western blot实验检测肿瘤糖酵解关键调控分子Glut1(glucose transporter-1)、HK2(hexokinase 2)与LDH-A(Lactate Dehydrogenase A)的表达,通过检测培养液中葡萄糖的减少量分析GPC3对细胞葡萄糖摄取情况,通过检测培养液中乳酸含量与PH值分析GPC3对细胞乳酸产生的影响,通过检测细胞的氧耗速率,分析GPC3对线粒体氧化磷酸化功能的影响。结果:干扰肝癌细胞中GPC3的表达可抑制糖酵解关键调控分子Glut1、HK2与LDH-A表达,降低肝癌细胞葡萄糖摄取速率和细胞氧耗速率,且细胞培养液PH升高,乳酸产生减少。结论:肝癌细胞中GPC3高表达通过上调糖酵解关键调控分子Glut1、HK2与LDH-A表达而促进肝癌细胞糖酵解效应,同时抑制线粒体氧化磷酸化活性。这些结果进一步提示糖代谢重编程可能是GPC3促进肝癌增殖与转移的重要机制。     

Human breast cancer histoid: an in vitro 3-dimensional co-culture model that mimics breast cancer tissue

Progress in our understanding of heterotypic cellular interaction in the tumor microenvironment, which is recognized to play major roles in cancer progression, has been hampered due to unavailability of an appropriate in vitro co-culture model. The aim of this study was to generate an in vitro 3-dimensional human breast cancer model, which consists of cancer cells and fibroblasts. Breast cancer cells (UACC-893) and fibroblasts at various densities were co-cultured in a rotating suspension culture system to establish co-culture parameters. Subsequently, UACC-893, BT.20, or MDA.MB.453 were co-cultured with fibroblasts for 9 days. Co-cultures resulted in the generation of breast cancer histoid (BCH) with cancer cells showing the invasion of fibroblast spheroids, which were visualized by immunohistochemical (IHC) staining of sections (4 μm thick) of BCH. A reproducible quantitative expression of C-erbB.2 was detected in UACC-893 cancer cells in BCH sections by IHC staining and the Automated Cellular Imaging System. BCH sections also consistently exhibited qualitative expression of pancytokeratins, p53, Ki-67, or E-cadherin in cancer cells and that of vimentin or GSTPi in fibroblasts, fibronectin in the basement membrane and collagen IV in the extracellular matrix. The expression of the protein analytes and cellular architecture of BCH were markedly similar to those of breast cancer tissue.     

Featured Article: Teriflunomide,an immunomodulatory drug,exerts anticancer activity in triple negative breast cancer cells

Triple-negative breast cancer (TNBC) is defined as a group of primary breast cancers lacking expression of estrogen, progesterone, and human epidermal growth factor receptor-2 (HER-2) receptors, characterized by higher relapse rate and lower survival compared with other subtypes. Due to lack of identified targets and molecular heterogeneity, conventional chemotherapy is the only available option for treatment of TNBC, but non-discordant positive therapeutic efficacy could not be achieved. Here, we demonstrated that these TNBC cells were sensitive to teriflunomide, which was a well-known immunomodulatory drug for treatment of relapsing multiple sclerosis (MS). Potent anti-cancer effects in TNBC in vitro, including proliferation inhibition, cell cycle delay, cell apoptosis, and suppression of cell motility and invasiveness, could be achieved with this agent. Of note, we showed that multiple signals involved in TNBC proliferation, survival, migratory, and invasive potential were under regulation by teriflunomide. Among them, we identified down-regulation of growth factor receptors to abolish growth maintenance, suppression of c-Myc, and cyclin D1 to contribute to its anti-proliferative effect, modulation of components of cell cycle to induce S-phase arrest, degradation of Bcl-xL, and up-regulation of BAX via activation of MAPK pathway to induce apoptosis, and inhibition of epithelial-mesenchymal transition (EMT) process, matrix metalloproteinase-9 (MMP9) expression, and inactivation of Src/FAK to reduce TNBC migration and invasion. The results identified teriflunomide may be of therapeutic benefit for the more aggressive and difficult-to-treat breast cancer subtype, indicating the use of teriflunomide for clinical trials for treatment of TNBC patients.