Metastatic Potential Of Triple-negative Breast Cancer Research Articles

PD-L1 tumor-intrinsic signaling and its therapeutic implication in triple-negative breast cancer.

Although the immune checkpoint role of programmed death ligand 1 (PD-L1) has been established and targeted in cancer immunotherapy, the tumor-intrinsic role of PD-L1 is less appreciated in tumor biology and therapeutics development, partly because of the incomplete mechanistic understanding. Here we demonstrate a potentially novel mechanism by which PD-L1 promotes the epithelial-mesenchymal transition (EMT) in triple-negative breast cancer (TNBC) cells by suppressing the destruction of the EMT transcription factor Snail. PD-L1 directly binds to and inhibits the tyrosine phosphatase PTP1B, thus preserving p38-MAPK activity that phosphorylates and inhibits glycogen synthase kinase 3β (GSK3β). Via this mechanism, PD-L1 prevents the GSK3β-mediated phosphorylation, ubiquitination, and degradation of Snail and consequently promotes the EMT and metastatic potential of TNBC. Significantly, PD-L1 antibodies that confine the tumor-intrinsic PD-L1/Snail pathway restricted TNBC progression in immunodeficient mice. More importantly, targeting both tumor-intrinsic and tumor-extrinsic functions of PD-L1 showed strong synergistic tumor suppression effect in an immunocompetent TNBC mouse model. Our findings support that PD-L1 intrinsically facilitates TNBC progression by promoting the EMT, and this potentially novel PD-L1 signaling pathway could be targeted for better clinical management of PD-L1–overexpressing TNBCs.

Sulforaphane-cisplatin combination inhibits the stemness and metastatic potential of TNBCs via down regulation of sirtuins-mediated EMT signaling axis

BackgroundSulforaphane (SFN) is a naturally occurring organosulfur compound found in cruciferous vegetables such as broccoli, brussels sprouts and cabbage. SFN is known for its multiple therapeutic properties, such as HDAC inhibitory, chemo preventive and anti-cancer effects. Cisplatin (CIS) has limited effect against metastatic triple-negative breast cancer (TNBC). Additionally, CIS impose severe side effects to normal cells, and later TNBC cells develops resistance. Studies suggest that the overexpression of sirtuins (SIRTs) promotes CIS resistance and metastasis by activating epithelial-to-mesenchymal transition (EMT) pathway in TNBC. PurposeIn view of the above information, we investigated the therapeutic efficacy of SFN, in combination with CIS against TNBC metastasis and CIS resistance. MethodsThe anti-cancerous effect of SFN-CIS combination on human TNBC cell lines was demonstrated by utilizing MTT assay and, apoptosis and cell cycle assay followed by FACS analysis. The synergistic effect of SFN-CIS combination on the experimental metastasis was demonstrated by utilizing migration, invasion, chemotaxis, mammosphere and colony formation assay on human TNBC MDA-MB-231 and MDA-MB-468 cells. The role of SIRTs-mediated EMT signaling axis in the metastasis and chemoresistance was investigated by western blotting technique as well as sirtuin activity tests. This was further validated by using Chromatin immunoprecipitation (ChIP) analysis. ResultsWe found that SFN-CIS combination synergistically inhibits cellular growth of MDA-MB-231 and MDA-MB-468 cells. More importantly, SFN was found to protect normal kidney cells from CIS-induced toxicity. Further, SFN-CIS combination was found to synergistically inhibit metastatic-events via significantly altering EMT markers which was further associated with the suppression of SIRTs functions in TNBC cells. ChIP analysis validated that SFN-CIS combination suppresses EMT mechanism through altered chromatin modifications at E-cadherin promoter resulting in its re-expression. ConclusionThe results of the current study suggests that CIS when supplemented with SFN, inhibits metastasis and stemness potential of TNBC cells by down regulating SIRTs-mediated EMT cascade. Overall this study affirms that, this novel combination could be a promising strategy against SIRT-mediated TNBC metastasis and CIS-resistance.

PO-160 Downregulation of the oncofetal gene SALL4 in melanoma leads to invasion and metastasis by differential acetylation mediated through binding to HDACs

IntroductionIntegrin signalling regulates proliferation, migration, metastasis and cell death and has been suggested as a possible target in antitumor therapy. The aim of this study was to investigate the potential of different integrin α subunits knockdown in increasing sensitivity of triple-negative breast cancer (TNBC) and melanoma cell lines to cisplatin, microtubule poisons paclitaxel (PTX) and vincristine (VCR), and mitigating metastatic potential.Material and methodsThe knockdown of integrin subunits αv, β3, β5, α3 and α4 was achieved by transfection of siRNA. The sensitivity of cell lines to anticancer drugs was determined using MTT assay. For monitoring cell migration and invasion, migration or matrigel-coated invasion Transwell Cell Culture Inserts were used. Flow cytometry, western blot, and confocal microscopy were done using standard protocols.Results and discussionsWe analysed the proof of concept in melanoma cell line MDA-MB-435S that expresses integrins αvβ3 and αvβ5 but not αvβ1. Following transfection with integrin-specific siRNAs integrin subunit αv knockdown was selected since it increased sensitivity to PTX and VCR, and decreased metastatic potential. We observed an integrin switching effect upon β3, β5 and α4 knockdown that led to differing changes of sensitivity to antitumor drugs and cell migration. Using a panel of TNBC (MDA-MB-231,–468 and −436) and melanoma (RPMI-7951, MeWo and A375) cell lines we showed that knockdown of integrin αv increased sensitivity to PTX and VCR and inhibited metastatic potential in MDA-MB-231, MDA-MB-468, RPMI-7951 and MeWo, while no effect was observed in MDA-MB-436 and A375 cells. To assess the importance of integrins αvβ3/β5 in sensitisation to PTX we exposed all cell lines to combination of αvβ3/β5 inhibitor cilengitide and PTX and observed increased sensitivity to PTX in MDA-MB-231, MDA-MB-436, RPMI-7951 and A375 but surprisingly decreased sensitivity in MeWo and MDA-MB-468 cell lines, implying either involvement of other integrins αv or integrin switching effect. The correlation between loss of phosphorylated focal adhesion kinase pFAK(Y397) and increased sensitivity to PTX was observed in melanoma cell lines, however, sensitisation was not achieved using pFAK(Y397) inhibitor.ConclusionOur results highlight that knocking down integrin αv, rather than inhibiting αvβ3/β5 using cilengitide, could be more effective for increasing sensitivity to microtubule poisons and decreasing metastatic potential in TNBC and melanoma.

PO-156 Differential effect of integrin αV knockdown and cilengitide in sensitisation of triple-negative breast cancer and melanoma cell lines to microtubule poisons

IntroductionIntegrin signalling regulates proliferation, migration, metastasis and cell death and has been suggested as a possible target in antitumor therapy. The aim of this study was to investigate the potential of different integrin α subunits knockdown in increasing sensitivity of triple-negative breast cancer (TNBC) and melanoma cell lines to cisplatin, microtubule poisons paclitaxel (PTX) and vincristine (VCR), and mitigating metastatic potential.Material and methodsThe knockdown of integrin subunits αv, β3, β5, α3 and α4 was achieved by transfection of siRNA. The sensitivity of cell lines to anticancer drugs was determined using MTT assay. For monitoring cell migration and invasion, migration or matrigel-coated invasion Transwell Cell Culture Inserts were used. Flow cytometry, western blot, and confocal microscopy were done using standard protocols.Results and discussionsWe analysed the proof of concept in melanoma cell line MDA-MB-435S that expresses integrins αvβ3 and αvβ5 but not αvβ1. Following transfection with integrin-specific siRNAs integrin subunit αv knockdown was selected since it increased sensitivity to PTX and VCR, and decreased metastatic potential. We observed an integrin switching effect upon β3, β5 and α4 knockdown that led to differing changes of sensitivity to antitumor drugs and cell migration. Using a panel of TNBC (MDA-MB-231,–468 and −436) and melanoma (RPMI-7951, MeWo and A375) cell lines we showed that knockdown of integrin αv increased sensitivity to PTX and VCR and inhibited metastatic potential in MDA-MB-231, MDA-MB-468, RPMI-7951 and MeWo, while no effect was observed in MDA-MB-436 and A375 cells. To assess the importance of integrins αvβ3/β5 in sensitisation to PTX we exposed all cell lines to combination of αvβ3/β5 inhibitor cilengitide and PTX and observed increased sensitivity to PTX in MDA-MB-231, MDA-MB-436, RPMI-7951 and A375 but surprisingly decreased sensitivity in MeWo and MDA-MB-468 cell lines, implying either involvement of other integrins αv or integrin switching effect. The correlation between loss of phosphorylated focal adhesion kinase pFAK(Y397) and increased sensitivity to PTX was observed in melanoma cell lines, however, sensitisation was not achieved using pFAK(Y397) inhibitor.ConclusionOur results highlight that knocking down integrin αv, rather than inhibiting αvβ3/β5 using cilengitide, could be more effective for increasing sensitivity to microtubule poisons and decreasing metastatic potential in TNBC and melanoma.

The G\u03b1h-PLC\u03b41 signaling axis drives metastatic progression in triple-negative breast cancer

BackgroundDistant metastasis of triple-negative breast cancer (TNBC) to other organs, e.g., the lungs, has been correlated with poor survival rates among breast cancer patients. Therefore, the identification of useful therapeutic targets to prevent metastasis or even inhibit tumor growth of TNBC is urgently needed. Gαh is a novel GTP-binding protein and known as an inactive form of calcium-dependent tissue transglutaminase. However, the functional consequences of transamidating and G-protein activities of tissue transglutaminase in promoting cancer metastasis are still controversial.MethodsKaplan-Meier analyses were performed to estimate the prognostic values of Gαh and PLCδ1 by utilizing public databases and performing immunohistochemical staining experiments. Cell-based invasion assays and in vivo lung colony-forming and orthotropic lung metastasis models were established to evaluate the effectiveness of interrupting the protein-protein interaction (PPI) between Gαh and PLCδ1 in inhibiting the invasive ability and metastatic potential of TNBC cells.ResultsHere, we showed that the increased level of cytosolic, not extracellular, Gαh is a poor prognostic marker in breast cancer patients and correlates with the metastatic evolution of TNBC cells. Moreover, clinicopathological analyses revealed that the combined signature of high Gαh/PLCδ1 levels indicates worse prognosis in patients with breast cancer and correlates with lymph node metastasis of ER-negative breast cancer. Blocking the PPI of the Gαh/PLCδ1 complex by synthetically myristoylated PLCδ1 peptide corresponding to the Gαh-binding interface appeared to significantly suppress cellular invasiveness in vitro and inhibit lung metastatic colonies of TNBC cells in vivo.ConclusionsThis study establishes Gαh/PLCδ1 as a poor prognostic factor for patients with estrogen receptor-negative breast cancers, including TNBCs, and provides therapeutic value by targeting the PPI of the Gαh/PLCδ1 complex to combat the metastatic progression of TNBCs.

A Novel Isoform of CPEB2 Regulates the Metastasis of Triple Negative Breast Cancer

While breast cancer has an overall 5‐year survival rate of 90%, triple negative breast cancer (TNBC) represents an anomalous subset of the disease with a greatly reduced (30%) 5‐year survival rate. The enhanced mortality and morbidity associated with TNBC arises from the high metastatic rate, which requires the acquisition of anoikis resistance (AnR). As the dysregulation of mRNA splicing is becoming a recognized phenomenon in cancer and aberrant cell signaling, the alternative mRNA splicing events were interrogated in AnR TNBC cells using next generation sequencing. The mRNA splicing of cytoplasmic polyadenylation element binding 2 (CPEB2), a translational regulator, was identified as dysregulated in AnR TNBC. Specifically, the inclusion of exon 4 into the mature mRNA to produce high levels of the CEPB2B isoform correlated with AnR, which translated to human breast cancer tumors. Furthermore, the molecular manipulation of CPEB2 isoforms had a robust effect on AnR and the metastatic potential of TNBC. Finally, next generation sequencing indicated a subset of mRNA species related to both tumor suppression and oncogenesis were differentially regulated by specific CPEB2 splice variants. Overall, our findings demonstrate that the regulation of CPEB2 mRNA splicing is a key step in the acquisition of AnR for TNBC as well as a major driving force in metastasis.

AP-1-mediated chromatin looping regulates ZEB2 transcription: new insights into TNFα-induced epithelial-mesenchymal transition in triple-negative breast cancer.

The molecular determinants of malignant cell behaviour in triple-negative breast cancer (TNBC) are poorly understood. Recent studies have shown that regulators of epithelial-mesenchymal transition (EMT) are potential therapeutic targets for TNBC. In this study, we demonstrate that the inflammatory cytokine TNFα induces EMT in TNBC cells via activation of AP-1 signaling and subsequently induces expression of the EMT regulator ZEB2. We also show that TNFα activates both the PI3K/Akt and MAPK/ERK pathways, which act upstream of AP-1. We further investigated in detail AP-1 regulation of ZEB2 expression. We show that two ZEB2 transcripts derived from distinct promoters are both expressed in breast cancer cell lines and breast tumor samples. Using the chromosome conformation capture assay, we demonstrate that AP-1, when activated by TNFα, binds to a site in promoter 1b of the ZEB2 gene where it regulates the expression of both promoter 1b and 1a, the latter via mediating long range chromatin interactions. Overall, this work provides a plausible mechanism for inflammation-induced metastatic potential in TNBC, involving a novel regulatory mechanism governing ZEB2 isoform expression.

Abstract P6-01-03: Decreasing the metastatic potential in Triple Negative Breast Cancer through the miR-17 cluster

Abstract Background: In animal models of breast cancer, caloric restriction (CR), or a 25–30% reduction of calories, has decreased tumor initiation and progression. We sought to determine if CR could be used as a novel therapeutic intervention to enhance cytotoxic therapies such as radiation (RT) and alter the molecular profile of triple negative breast cancer (TNBC), which displays a poor prognosis. Methods/Results: In two murine models of TNBC (4T1 which is highly metastatic and 67NR which is locally aggressive), mice with palpable tumors were treated with radiation, caloric restriction (30% intake reduction) or both. 3 times per week measurements revealed significant tumor regression with RT or diet modification, and an even greater regression is observed combining diet modification with RT. Concurrently, many reports have shown that TNBC is most distinct from other breast tumor subtypes due to up-regulation of the miR-17-92 cluster which we have shown by RT-PCR is downregulated with both CR and radiation (RT) and even further decreased with the combination. cDNA arrays done on all conditions revealed that the top 5 statistically significant GO terms with high fold changes were all associated with extracellular matrix and metastases. In silico analysis demonstrated 5 ECM related genes that were targets of the miR-17 cluster: collagen 4 alpha 1 (COL4A1), collagen 4 alpha 3 (COL4A3), laminin alpha 3 (LAMA3), metallopeptidase inhibitor 2 and 3 (TIMP2 and TIMP3) and these were inversely correlated with the miRs. Conclusions: CR, or altering the metabolism of breast cancer, can be combined with RT to decrease progression of TNBC tumors and that the miR-17-92 cluster is also decreased with these conditions. cDNA arrays suggest that extracellular matrix (ECM) proteins play a large role in the mechanism of action of CR and RT combined. Combination therapy with CR and RT can decrease the metastatic potential in TNBC and in part does this through the miR-17-92 cluster by way of controlling ECM. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-01-03.