Development Of Resistance In Breast Cancer Research Articles

Sphingomyelin synthase 2 promotes the stemness of breast cancer cells via modulating NF-κB signaling pathway

ObjectivesMulti-drug resistance (MDR) to chemotherapy is the main obstacle influencing the anti-tumor effect in breast cancer, which might lead to the metastasis and recurrence of cancer. Until now, there are still no effective methods that can overcome MDR. In this study, we aimed to investigate the role of sphingomyelin synthase 2 (SMS2) in breast cancer resistance.MethodsQuantitative RT-PCR analysis was performed to assess changes in mRNA expression. Western blot analysis was performed to detect protein expression. Inhibitory concentration value of adriamycin (ADR) was evaluated using CCK 8 assay. The stemness ability of breast cancer cells was assessed by spheroid-formation assay. Immunofluorescence staining was conducted to show the cellular distribution of proteins. Breast tumor masses were harvested from the xenograft tumor mouse model.ResultsSMS2 overexpression increased the IC50 values of breast cancer cells. SMS2 decreased the CD24 transcription level but increased the transcription levels of stemness-related genes including CD44, ALDH, OCT 4 and SOX2 in breast cancer cells. SMS2 overexpression promoted the nuclear translocation of phosphorylated NF-κB, while suppression of SMS2 could inhibit the NF-κB pathway.ConclusionsSMS2 increased the stemness of breast cancer cells via NF-κB signaling pathway, leading to resistance to the chemotherapeutic drug ADR. Thus, SMS2 might play a critical role in the development of breast cancer resistance, which is a previously unrecognized mechanism in breast cancer MDR development.

Tanshinone II A improves the chemosensitivity of breast cancer cells to doxorubicin by inhibiting β-catenin nuclear translocation.

Numerous evidence linkaberrant nuclear β-catenin accumulation to the development of breast cancer resistance, therefore, targeted inhibition of β-catenin nuclear translocation may effectively improve the chemosensitivity of breast cancer. Doxorubicin (Dox) is the most commonly used chemotherapeutic drug for breast cancer. Here, we determined that tanshinone II A (Tan II A) could improve the sensitivity of Dox-resistant breast cancer MCF-7/dox cells to Dox, and evaluated whether the sensitization effect of Tan II A on Dox was targeted to inhibit β-catenin nuclear translocation. The results showed that Tan II A not only significantly inhibited the nuclear translocation of β-catenin in MCF-7/dox cells treated by Dox but also inhibited the nuclear translocation of β-catenin in MCF-7 cells treated by Dox to a certain degree. Furthermore, when the above two cells treated by Dox combined with Tan II A were intervened with β-catenin agonist WAY-262611, with the re-nuclear translocation of β-catenin in the cells, the sensitization effect of Tan II A on Dox was greatly reduced. These results indicated that Tan II A could improve the chemosensitivity of breast cancer cells to Dox by inhibiting β-catenin nuclear translocation. Therefore, Tan II A could be used as a potential chemosensitizer in combination with Dox for breast cancer chemotherapy.

Abstract 4024: TRAF2 Ser-11 phosphorylation confers resistance to AKT inhibitors in breast cancer cells

Abstract The PI3K-AKT pathway is the most frequently aberrantly activated pathway in breast cancer (BC), with alterations in genes encoding the pathway components (e.g. RTKs, HER2, PTEN, PIK3C and AKT) occurring in ~80% of cases. However, inhibition of this pathway is either not effective or often results in development of resistance in BC. TRAF2 is a key effector protein downstream of the TNF receptor superfamily, and activates NF-κB to promote BC cell survival. We mapped two phosphorylation sites (Ser-11 and Ser-274) in TRAF2, and identified TBK1/IKKϵ and AKT as the kinases that directly phosphorylate TRAF2 at Ser-11 and Ser-274, respectively. TBK1 and IKKϵ are serine/threonine kinases activated by inflammatory stimuli and the oncogenic RTK-Ras-RALB pathway, and play a critical role in BC transformation and survival. IKKϵ and TRAF2 are overexpressed in a significant portion of BC, and their overexpression is associated with poor prognosis in patients with invasive breast carcinoma. We found that inhibition of AKT leads to increased TBK1 activation and TRAF2 Ser-11 phosphorylation in triple negative BC (TNBC) cells. Importantly, combination of AKT and TBK1/IKKϵ inhibitors synergistically induced apoptosis in all TNBC cell lines tested in vitro, suggesting that inhibition of PI3K-AKT derepresses the pro-survival TBK1-TRAF2 pathway, which in turn confers resistance to AKT inhibition in TNBC. We believe that any positive outcomes from our ongoing xenograft experiments will have an immediate translational impact in TNBC treatment. Citation Format: Hasem Habelhah, Laiqun Zhang. TRAF2 Ser-11 phosphorylation confers resistance to AKT inhibitors in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4024.

Heat Shock Factor 1 (HSF1) Controls Chemoresistance and Autophagy through Transcriptional Regulation of Autophagy-related Protein 7 (ATG7)

Heat shock factor 1 (HSF1), a master regulator of heat shock responses, plays an important role in tumorigenesis. In this study we demonstrated that HSF1 is required for chemotherapeutic agent-induced cytoprotective autophagy through transcriptional up-regulation of autophagy-related gene ATG7. Interestingly, this is independent of the HSF1 heat shock response function. Treatment of cancer cells with the FDA-approved chemotherapeutic agent carboplatin induced autophagy and growth inhibition, which were significantly increased upon knockdown of HSF1. Mechanistic studies revealed that HSF1 regulates autophagy by directly binding to ATG7 promoter and transcriptionally up-regulating its expression. Significantly, breast cancer patient sample study revealed that a higher ATG7 expression level is associated with poor patient survival. This novel finding was further confirmed by analysis of two independent patient databases, demonstrating a prognostic value of ATG7. Furthermore, a strong positive correlation was observed between levels of HSF1 and ATG7 in triple-negative breast cancer patient samples, thus validating our in vitro findings. This is the first study identifying a critical role for HSF1 in controlling cytoprotective autophagy through regulation of ATG7, which is distinct from the HSF1 function in the heat shock response. This is also the first study demonstrating a prognostic value of ATG7 in breast cancer patients. These findings strongly argue that combining chemotherapeutic agents with autophagy inhibition by repressing HSF1/ATG7 axis represents a promising strategy for future cancer treatment.

Abstract P6-04-01: Global analysis of breast cancer metastasis suggests cellular reprogramming is central to the endocrine resistant phenotype.

Abstract The development of breast cancer resistance to endocrine therapy results from altered cellular plasticity leading to the emergence of a hormone independent tumour. We have previously shown that the endocrine resistant phenotype is poorly differentiated and has greater metastatic potential when compared with an endocrine sensitive phenotype. The underlying mechanism of how an ER positive, endocrine sensitive tumour can turn on these adaptive mechanisms remains unresolved. We hypothesise that cellular reprogramming can occur as a result of long-term exposure to endocrine treatment which manifests clinically as tumour recurrence. Our aim was to identify the mechanism of breast cancer cellular reprogramming in a clinical context. In this study we adopted a global approach to define transcriptional networks significantly elevated in the breast cancer metastatic setting. Using RNAseq we determined the gene expression profile of three ER positive patients who developed tumour recurrence on tamoxifen treatment. RNAseq was performed on primary tumours, axillary node metastases and subsequent distant metastases. Following bioinformatic analysis, we defined the genes that were significantly elevated in the metastatic tumours. In the metastases, genes clustered away from those in the primary and node, with 209 genes uniquely elevated in the metastatic context, suggesting altered gene expression in response to endocrine therapy. Pathway analysis of genes significantly elevated in the metastatic setting included developmental pathways (WNT signalling and TGFbeta), growth factor signalling pathways (MAPkinase), cell adhesion molecules, junction adherens and pathways in cancer. Further analysis provided a list of clinically relevant transcriptional networks which may facilitate tumour cell de-differentiation. Transcription factors including Myc, SMAD2, ESR, TCF3, CUX1 and CLOCK were identified which potentially drive reprogramming in response to endocrine treatment. We interrogated this data to identify downstream targets of this ‘de-differentiation’ network for new predictive markers of response to endocrine treatment. Bioinformatic analysis identified PAWR, an inhibitor of progenitor cell expansion as a potential SMAD2 target. In a xenograft model of endocrine resistance, PAWR expression was found to be reduced in metastatic tissue from lung, liver and bone compared with the primary tumour. In human breast cancer patients PAWR significantly associated with a good response to endocrine treatment and luminal A status (p = 0.0008), establishing PAWR as a predictor of good response to endocrine therapies. We have identified a transcriptional network which may drive a de-differentiated phenotype following endocrine treatment. Data presented here proposes a mechanism by which apparently less aggressive Luminal A type tumours may fail endocrine treatment and develop subsequent recurrence. This represents a fundamental shift in how we approach the development of resistance to endocrine therapy, establishing a number of biomarkers which will allow tracking of response to endocrine therapy or allow accurate early prediction of those who will respond to treatment. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-04-01.

Global Characterization of the SRC-1 Transcriptome Identifies ADAM22 as an ER-Independent Mediator of Endocrine-Resistant Breast Cancer

The development of breast cancer resistance to endocrine therapy results from an increase in cellular plasticity that permits the emergence of a hormone-independent tumor. The steroid coactivator protein SRC-1, through interactions with developmental proteins and other nonsteroidal transcription factors, drives this tumor adaptability. In this discovery study, we identified ADAM22, a non-protease member of the ADAM family of disintegrins, as a direct estrogen receptor (ER)-independent target of SRC-1. We confirmed SRC-1 as a regulator of ADAM22 by molecular, cellular, and in vivo studies. ADAM22 functioned in cellular migration and differentiation, and its levels were increased in endocrine resistant-tumors compared with endocrine-sensitive tumors in mouse xenograft models of human breast cancer. Clinically, ADAM22 was found to serve as an independent predictor of poor disease-free survival. Taken together, our findings suggest that SRC-1 switches steroid-responsive tumors to a steroid-resistant state in which the SRC-1 target gene ADAM22 has a critical role, suggesting this molecule as a prognostic and therapeutic drug target that could help improve the treatment of endocrine-resistant breast cancer.

P4-01-06: Global Characterization of the SRC-1 Transcriptome Identifies Disintegrin C as an ER-Independent Mediator of Endocrine Resistant Breast Cancer.

Abstract Background: The development of breast cancer resistance to endocrine therapy results from an increase in cellular plasticity leading to the development of a steroid independent tumour. The p160 steroid coactivataor protein SRC-1 drives this tumour adaptability. SRC-1 is central to the development of the endocrine resistant phenotype and is an independent predictor of disease free survival in tamoxifen-treated patients. Here, using discovery studies we identify Disintegrin C (DC), a multidomain transmembrane glycoprotein as a direct, ER-independent target of SRC-1. Methods: SRC-1-ChIP sequencing was performed in endocrine resistant (LY2) cells. Microarray experiments were performed on LY2 cells transfected with scrambled siRNA or SRC-1 siRNA, +/−4-OHT to assess SRC-1-dependent gene expression. Combining the ChIP-seq and expression array datasets, a total of 2,065 genes were significantly downregulated (p<0.05) following SRC-1 knockdown. DC was selected for validation as a potential novel metastatic oncogene. DC transcipt levels were measured following knockdown and over-expression of SRC-1 and ERα. Mouse xenograft and SRC-1 knockout models were used to examine DC expression in vivo. A TMA comprising 560 patients was stained for DC. Functional validation studies were performed using 3D culture and migration assays, with transient knockdown of DC and treatment with JC1, an inhibitory peptide against DC. Results: Treatment with 4-OHT significantly increased the number of ChIP-enriched intervals identified. The majority of peaks in the 4-OHT treated sample were close to the transcriptional start site, in the promoter, first exon or upstream of the promoter. As a defined ER coactivator, an overlap between ER binding sites and those identified for SRC-1 is expected. 43% of high confidence SRC-1 peaks in 4-OHT-treated LY2 cells contained an ERE binding motif. This raises the possibility that, in endocrine resistance, SRC-1 can drive transcription independent of ER. Treatment with 4-OHT resulted in substantial recruitment of SRC-1 to the DC promoter. In primary tumours there was a significant association between transcript levels of SRC-1 and DC. Cells derived from mammary tumours of the SRC-1-/−/PyMT mouse lacked DC, but there was high expression in the wild type PyMT mouse. Knockdown of ERα in LY2 cells did not alter DC expression. Knockdown of DC reduced cell migration and restored cell differentiation in resistant cells. In xenograft studies treatment with 4-OHT increased tumour volume in the resistant tumours and induced DC expression. Treatment with recombinant JC1 reduced cellular migration in endocrine resistant cells. Kaplan Meier estimates of disease free survival show that DC is a strong predictor of disease free survival in breast cancer (p<0.0001). SRC-1 and DC are significant independent predictors of disease recurrence (odds ratios 2.18 and 2.4). Conclusion: Our discovery studies have uncovered a steroid independent SRC-1 mediated network in endocrine resistant breast cancer. We identified a new SRC-1 target, DC. DC represents a rational new therapeutic target with a robust companion biomarker for treatment of endocrine resistant tumours. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-01-06.

Autophagy Facilitates the Development of Breast Cancer Resistance to the Anti-HER2 Monoclonal Antibody Trastuzumab

Autophagy has been emerging as a novel cytoprotective mechanism to increase tumor cell survival under conditions of metabolic stress and hypoxia as well as to escape chemotherapy-induced cell death. To elucidate whether autophagy might also protect cancer cells from the growth inhibitory effects of targeted therapies, we evaluated the autophagic status of preclinical breast cancer models exhibiting auto-acquired resistance to the anti-HER2 monoclonal antibody trastuzumab (Tzb). We first examined the basal autophagic levels in Tzb-naive SKBR3 cells and in two pools of Tzb-conditioned SKBR3 cells (TzbR), which optimally grow in the presence of Tzb doses as high as 200 µg/ml Tzb. Fluorescence microscopic analyses revealed that the number of punctate LC3 structures -a hallmark of autophagy- was drastically higher in Tzb-refractory cells than in Tzb-sensitive SKBR3 parental cells. Immunoblotting analyses confirmed that the lipidation product of the autophagic conversion of LC3 was accumulated to high levels in TzbR cells. High levels of the LC3 lipidated form in Tzb-refractory cells were accompanied by decreased p62/sequestosome-1 protein expression, a phenomenon characterizing the occurrence of increased autophagic flux. Moreover, increased autophagy was actively used to survive Tzb therapy as TzbR pools were exquisitely sensitive to chemical inhibitors of autophagosomal formation/function. Knockdown of LC3 expression via siRNA similarly resulted in reduced TzbR cell proliferation and supra-additively interacted with Tzb to re-sensitize TzbR cells. Sub-groups of Tzb-naive SKBR3 parental cells accumulated LC3 punctate structures and decreased p62 expression after treatment with high-dose Tzb, likely promoting their own resistance. This is the first report showing that HER2-overexpressing breast cancer cells chronically exposed to Tzb exhibit a bona fide up-regulation of the autophagic activity that efficiently works to protect breast cancer cells from the growth-inhibitory effects of Tzb. Therapeutic targeting autophagosome formation/function might represent a novel molecular avenue to reduce the emergence of Tzb resistance in HER2-dependent breast carcinomas.

17-β-Estradiol Inhibits Transforming Growth Factor-β Signaling and Function in Breast Cancer Cells via Activation of Extracellular Signal-Regulated Kinase through the G Protein-Coupled Receptor 30

Breast cancer development and breast cancer progression involves the deregulation of growth factors leading to uncontrolled cellular proliferation, invasion and metastasis. Transforming growth factor (TGF)-beta plays a crucial role in breast cancer because it has the potential to act as either a tumor suppressor or a pro-oncogenic chemokine. A cross-communication between the TGF-beta signaling network and estrogens has been postulated, which is important for breast tumorigenesis. Here, we provide evidence that inhibition of TGF-beta signaling is associated with a rapid estrogen-dependent nongenomic action. Moreover, we were able to demonstrate that estrogens disrupt the TGF-beta signaling network as well as TGF-beta functions in breast cancer cells via the G protein-coupled receptor 30 (GPR30). Silencing of GPR30 in MCF-7 cells completely reduced the ability of 17-beta-estradiol (E2) to inhibit the TGF-beta pathway. Likewise, in GPR30-deficient MDA-MB-231 breast cancer cells, E2 achieved the ability to suppress TGF-beta signaling only after transfection with GPR30-encoding plasmids. It is most interesting that the antiestrogen fulvestrant (ICI 182,780), which possesses agonistic activity at the GPR30, also diminished TGF-beta signaling. Further experiments attempted to characterize the molecular mechanism by which activated GPR30 inhibits the TGF-beta pathway. Our results indicate that GPR30 induces the stimulation of the mitogen-activated protein kinases (MAPKs), which interferes with the activation of Smad proteins. Inhibition of MAPK activity prevented the ability of E2 from suppressing TGF-beta signaling. These findings are of great clinical relevance, because down-regulation of TGF-beta signaling is associated with the development of breast cancer resistance in response to antiestrogens.