Population Of Breast Cancer Cells Research Articles

Altered TGF-α/β signaling drives cooperation between breast cancer cell populations.

The role of tumor heterogeneity in regulating disease progression is poorly understood. We hypothesized that interactions between subpopulations of cancer cells can affect the progression of tumors selecting for a more aggressive phenotype. We developed an in vivo assay based on the immortalized nontumorigenic breast cell line MCF10A and its Ras-transformed derivatives AT1 (mildly tumorigenic) and CA1d (highly tumorigenic). CA1d cells outcompeted MCF10A, forming invasive tumors. AT1 grafts were approximately 1% the size of CA1d tumors when initiated using identical cell numbers. In contrast, CA1d/AT1 mixed tumors were larger than tumors composed of AT1 alone (100-fold) or CA1d (3-fold), suggesting cooperation in tumor growth. One of the mechanisms whereby CA1d and AT1 were found to cooperate was by modulation of TGF-α and TGF-β signaling. Both of these molecules were sufficient to induce changes in AT1 proliferative potential in vitro. Reisolation of AT1 tumor-derived (AT1-TD) cells from these mixed tumors revealed that AT1-TD cells grew in vivo, forming tumors as large as tumorigenic CA1d cells. Cooperation between subpopulations of cancer epithelium is an understudied mechanism of tumor growth and invasion that may have implications on tumor resistance to current therapies.-Franco, O. E., Tyson, D. R., Konvinse, K. C., Udyavar, A. R., Estrada, L., Quaranta, V., Crawford, S. E., Hayward, S. W. Altered TGF-α/β signaling drives cooperation between breast cancer cell populations.

Phototheranostics of CD44-positive cell populations in triple negative breast cancer

Triple-negative breast cancer (TNBC) is one of the most lethal subtypes of breast cancer that has limited treatment options. Its high rates of recurrence and metastasis have been associated, in part, with a subpopulation of breast cancer stem-like cells that are resistant to conventional therapies. A compendium of markers such as CD44high/CD24low, and increased expression of the ABCG2 transporter and increased aldehyde dehydrogenase (ALDH1), have been associated with these cells. We developed a CD44-targeted monoclonal antibody photosensitizer conjugate for combined fluorescent detection and photoimmunotherapy (PIT) of CD44 expressing cells in TNBC. The CD44-targeted conjugate demonstrated acute cell killing of breast cancer cells with high CD44 expression. This cell death process was dependent upon CD44-specific cell membrane binding combined with near-infrared irradiation. The conjugate selectively accumulated in CD44-positive tumors and caused dramatic tumor shrinkage and efficient elimination of CD44-positive cell populations following irradiation. This novel phototheranostic strategy provides a promising opportunity for the destruction of CD44-positive populations that include cancer stem-like cells, in locally advanced primary and metastatic TNBC.

Bone marrow–derived fibroblasts are a functionally distinct stromal cell population in breast cancer and lung metastases

Bone marrow–derived fibroblasts are a functionally distinct stromal cell population in breast cancer and lung metastases

Increased autophagic response in a population of metastatic breast cancer cells.

Breast cancer cells are heterogeneous in their ability to invade and fully metastasize, and thus also in their capacity to survive the numerous stresses encountered throughout the multiple steps of the metastatic cascade. Considering the role of autophagy as a survival response to stress, the present study hypothesized that distinct populations of breast cancer cells may possess an altered autophagic capacity that influences their metastatic potential. It was observed that a metastatic breast cancer cell line, MDA-MB-231, that was sensitive to autophagic induction additionally possessed the ability to proliferate following nutrient deprivation. Furthermore, a selected subpopulation of these cells that survived multiple exposures to starvation conditions demonstrated a heightened response to autophagic induction compared to their parent cells. Although this subpopulation maintained a more grape-like pattern in three-dimensional culture compared to the extended spikes of the parent population, autophagic induction in this subpopulation elicited an invasive phenotype with extended spikes. Taken together, these results suggest that autophagic induction may contribute to the ability of distinct breast cancer cell populations to survive and invade.

Regulation of the T-box transcription factor Tbx3 by the tumour suppressor microRNA-206 in breast cancer

Background:The Tbx3 transcription factor is over-expressed in breast cancer, where it has been implicated in proliferation, migration and regulation of the cancer stem cell population. The mechanisms that regulate Tbx3 expression in cancer have not been fully explored. In this study, we demonstrate that Tbx3 is repressed by the tumour suppressor miR-206 in breast cancer cells.Methods:Bioinformatics prediction programmes and luciferase reporter assays were used to demonstrate that miR-206 negatively regulates Tbx3. We examined the impact of miR-206 on Tbx3 expression in breast cancer cells using miR-206 mimic and inhibitor. Gene/protein expression was examined by quantitative reverse-transcription–PCR and immunoblotting. The effects of miR-206 and Tbx3 on apoptosis, proliferation, invasion and cancer stem cell population was investigated by cell-death detection, colony formation, 3D-Matrigel and tumorsphere assays.Results:In this study, we examined the regulation of Tbx3 by miR-206. We demonstrate that Tbx3 is directly repressed by miR-206, and that this repression of Tbx3 is necessary for miR-206 to inhibit breast tumour cell proliferation and invasion, and decrease the cancer stem cell population. Moreover, Tbx3 and miR-206 expression are inversely correlated in human breast cancer. Kaplan–Meier analysis indicates that patients exhibiting a combination of high Tbx3 and low miR-206 expression have a lower probability of survival when compared with patients with low Tbx3 and high miR-206 expression. These studies uncover a novel mechanism of Tbx3 regulation and identify a new target of the tumour suppressor miR-206.Conclusions:The present study identified Tbx3 as a novel target of tumour suppressor miR-206 and characterised the miR-206/Tbx3 signalling pathway, which is involved in proliferation, invasion and maintenance of the cancer stem cell population in breast cancer cells. Our results suggest that restoration of miR-206 in Tbx3-positive breast cancer could be exploited for therapeutic benefit.

Antitumor activity of Cetuximab in combination with Ixabepilone on triple negative breast cancer stem cells.

BackgroundDeveloping novel strategies against treatment-resistant triple negative breast cancer (TNBC) cells remains a significant challenge. The ErbB family, including epidermal growth factor receptor (EGFR), plays key roles in metastasis, tumorigenesis, cell proliferation, and drug resistance. Recently, these characteristics have been linked to a small subpopulation of cells classified as cancer stem cells (CSC) which are believed to be responsible for tumor initiation and maintenance. Ixabepilone is a new generation microtubule-stabilizing agent, which has been expected to be more efficacious than conventional taxanes. Here we aim to investigate whether the EGFR monoclonal antibody Cetuximab, in combination with Ixabepilone, is more effective in eliminating CSC populations compared to chemotherapy alone in TNBC.MethodsRepresentative TNBC cell lines (MDA-MB-231 and SUM159) were used to evaluate breast CSC populations. We used fluorescence-activated cell sorter analysis (CD44+ and CD24-/low, or Aldefluor+) and a self-renewal assay called mammosphere formation efficiency (MSFE) to measure CSC population size after treatment with Cetuximab, or Cetuximab plus Ixabepilone in vitro.ResultsAlthough there was no significant decrease in cell viability, Cetuximab reduced MSFE and the CSC population in breast cancer cells in vitro and in vivo through inhibition of autophagy. Also, SUM159 and MDA-MB-231 orthotopic tumors demonstrated partial response to Centuximab or Ixabepilone monotherapy; however, the effect of the combination treatment was significant only in SUM159 tumors (p <0.0001), when compared to Ixabepilone alone.ConclusionsOverall, our findings demonstrate that EGFR-targeted therapy by Cetuximab effectively reduces the CSC population in TNBC tumors. However, combination therapy with Ixabepilone may be effective only in a small subset of TNBCs, warranting further investigation of alternative approaches to target multiple pathways for TNBC treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-015-0662-4) contains supplementary material, which is available to authorized users.

Evodiamine selectively targets cancer stem-like cells through the p53-p21-Rb pathway

In spite of the recent improvements, the resistance to chemotherapy/radiotherapy followed by relapse is the main hurdle for the successful treatment of breast cancer, a leading cause of death in women. A small population of breast cancer cells that have stem-like characteristics (cancer stem-like cells; CSLC) may contribute to this resistance and relapse. Here, we report on a component of a traditional Chinese medicine, evodiamine, which selectively targets CSLC of breast cancer cell lines MCF7 and MDAMB 231 at a concentration that does show a little or no cytotoxic effect on bulk cancer cells. While evodiamine caused the accumulation of bulk cancer cells at the G2/M phase, it did not hold CSLC in a specific cell cycle phase but instead, selectively killed CSLC. This was not due to the culture of CSLC in suspension or without FBS. A proteomic analysis and western blotting revealed that evodiamine changed the expression of cell cycle regulating molecules more efficiently in CSLC cells than in bulk cancer cells. Surprisingly, evodiamine selectively activated p53 and p21 and decreased inactive Rb, the master molecules in G1/S checkpoint. These data collectively suggest a novel mechanism involving CSLC-specific targeting by evodiamine and its possible use to the therapy of breast cancer.

Tripartite motif 16 suppresses breast cancer stem cell properties through regulation of Gli-1 degradation via the ubiquitin-proteasome pathway.

Cancer stem cells (CSCs) are responsible for cancer progression and patient prognosis. Tripartite motif 16 (TRIM16) is a proteasome coactivator that regulates proteolytic activity in eukaryotic cells. Abundant evidence has shown that TRIM16 is lowly expressed in a number of human carcinomas. In a previous study, we demonstrated that TRIM16 suppressed cancer malignancy and that TRIM16 expression levels were associated with favorable prognostic parameters of patients with cancer. However, the precise role of this motif in the pathogenesis of breast cancer remains unknown. In the present study, we examined 29 human breast cancer specimens, and found that TRIM16 was lowly expressed in breast cancers; thus, TRIM16 expression is negatively correlated with metastasis in breast cancer patients. Moreover, we showed that TRIM16 suppressed CSC properties in a population of breast cancer cells. TRIM16 depletion resulted in an increased proportion of CSCs relative to breast cancer cells when several assays were used to assess CSC properties. Finally, we demonstrated that TRIM16 directly regulated the degradation of Gli‑1 protein via the ubiquitin‑proteasome pathway. In conclusion, we propose that inhibition of CSC properties may be one of the functions of TRIM16 as a suppressor of breast cancer progression.

DDX17 (P72), a Sox2 binding partner, promotes stem-like features conferred by Sox2 in a small cell population in estrogen receptor-positive breast cancer

We have previously demonstrated the existence of two phenotypically distinct cell subsets in estrogen receptor (ER)-positive breast cancer (BC) based on their differential response to a Sox2 reporter (SRR2), with reporter responsive (RR) cells being more tumorigenic and stem-like than reporter unresponsive (RU) cells. To delineate the molecular mechanisms underlying this phenotypic dichotomy, we tested our hypothesis that Sox2, which is a key regulator of the RR phenotype, is under the control of its binding partners. In this study, we focused on DDX17, known to be a transcription co-activator and found to be a Sox2 binding partner by liquid chromatography-mass spectrometry. Using immunoprecipitation, we confirmed the binding between DDX17 and Sox2, although this interaction was largely restricted to RR cells. While DDX17 was found in both the cytoplasm and nuclei in RU cells, it is confined to the nuclei in RR cells. siRNA knockdown of DDX17 in RR cells substantially decreased the Sox2-SRR2 binding and significantly decreased the SRR2 reporter activity without affecting the protein level of Sox2. Using ChIP-PCR, DDX17 knockdown also significantly decreased the binding of Sox2 to genomic SRR2, as well as 3 of its specific gene targets including MUC15, CCND1 and CD133. Correlating with these findings, siRNA knockdown of DDX17 significantly reduced soft agar colony formation and mammosphere formation in RR cells but not RU cells. To conclude, DDX17 is a Sox2-binding protein in ER-positive BC. In RR but not RU cells, DDX17 enhances the tumorigenic and stem-like features of Sox2 by promoting its binding to its target genes.

Abstract 2617: SUMOylation inhibition attenuates the cancer stem cell population in glioblastoma and basal breast cancer

Abstract INTRO: TFAP2A and mouse homolog Tcfap2a are transcription factors that play a critical role in development and cancer. We have recently shown that SUMOylation of TFAP2A is a key posttranslational modification that maintains the basal breast cancer phenotype. Treatment with anacardic acid (AA), a SUMOylation inhibitor, led to depletion of the CD44+/hi/CD24-/lo cancer stem cell population. Interestingly, glioblastoma has also been shown to express TFAP2A and demonstrates an expanded cancer stem cell population. METHODS: Mouse 4T1 basal mammary cancer, IOWA-1T human basal breast cancer and UG118 glioblastoma cell lines were analyzed. MTT was used to measure cell viability. Coulter Counter and hemocytometer were used for cell quantification. Matrigel invasion and western blot were used to further characterize the cell lines. RESULTS: Treatment of 4T1 and UG118 cells with AA in vitro resulted in a 67% and 65% decrease in cell viability by MTT assay, respectively (p &amp;lt; 0.05 for both). Quantification of cell number confirmed that AA treatment inhibited cell proliferation. At 48 hours, 4T1 cells demonstrated a decrease of 73-77% (p &amp;lt; 0.01), and UG 118 cells decreased by 97 to 98% (p &amp;lt; 0.001). Cell invasiveness assessed by Matrigel invasion assay was attenuated in 4T1 and UG118 cells after treatment (84%, p &amp;lt; 0.01 and 28%, p &amp;lt; 0.05, respectively). Western blot demonstrated a dose-dependent decrease in expression of the cancer stem cell marker CD44 or the mouse homolog CD49f following AA treatment of UG118 and 4T1 cells, respectively. Western blot confirmed that while total TFAP2A levels were unchanged, SUMO conjugated TFAP2A was decreased after AA treatment of 4T1 cells. To further establish relevance to human cancer, an aggressive chemotherapeutic resistant patient-derived xenograft, IOWA-1T, similarly demonstrated a 41% drop (p &amp;lt; 0.007) in cell invasiveness and a concomitant decrease in SUMOylated TFAP2A with AA treatment. CONCLUSION: Inhibition of the SUMO pathway attenuates the cancer stem cell population in basal breast cancer and glioblastoma. While further investigation is needed, SUMOylation may be a common biological posttranslational modification that defines cancer stem cells. Furthermore, basal breast cancer, brain cancer, and other cancers that express TFAP2A may be amenable to SUMOylation inhibitors such as AA. Citation Format: Jung M. Park, Tong Wu, Sarah Van Dorin, Ronald Weigel. SUMOylation inhibition attenuates the cancer stem cell population in glioblastoma and basal breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2617. doi:10.1158/1538-7445.AM2015-2617

Abstract 2379: The lung microenvironment influences the metastatic behavior of breast cancer cells in an innovative 3D ex vivo pulmonary metastasis model

Abstract Breast cancer remains a leading cause of mortality among women, with the majority of deaths attributed to metastatic disease. In particular, the lung is one of the most common and deadly sites of breast cancer metastasis, particular in women with more aggressive molecular subtypes such as Her2+ and triple-negative breast cancer. However, it still remains unclear whether the propensity of breast cancer cells to metastasize to the lung reflects properties of cancer cells themselves, properties of the lung microenvironment, or a combination of both. Both cancer initiation and progression can in part be attributed to small subsets of “stem cell-like” tumor cells, which in breast cancer are characterized by high aldehyde dehydrogenase (ALDH) activity and/or expression of the CD44+CD24- phenotype (ALDHhiCD44+). We have previously shown that ALDHhiCD44+ breast cancer cells demonstrate preferential migration and growth in response to lung-derived soluble factors, and an increased propensity to metastasize to the lung in vivo. The objective of this study was to use an innovative 3D ex vivo pulmonary metastasis model (PuMA) that incorporates the native 3D architecture of the lung in order to test the hypothesis that whole population and stem-like ALDHhiCD44+ breast cancer cell metastatic behavior is influenced by the lung microenvironment. Red fluorescent protein (RFP)-expressing MDA-MB-231 and MDA-MD-468 breast cancer cells were seeded (5×105 cells) to the lungs of female nude mice by tail vein injection. Following euthanasia, lungs were excised, sliced into 1 mm transverse sections, and grown in culture under serum-free conditions over 21 days. Sections were imaged at 0, 7, 14 and 21 days to observe growth and progression. Using H&amp;E and Masson's Trichrome staining, we confirmed that lung sections remained healthy with intact pulmonary architecture over 21 days in culture. Over a 14-day period, we observed significant growth of MDA-MB-231 whole breast cancer cell populations in this assay at day 14 relative to day 7 and day 0 (p≤0.05). Sorted populations of MDA-MB-231 ALDHhiCD44+ cells showed significant growth at days 14 (p≤0.001) and 21 (p≤0.0001) compared to their ALDHlowCD44- counterparts. ALDHhiCD44+ cells also progressed from the single cell state (&amp;lt;50 μm) to micrometastatic (200-400 μm) to macrometastatic (&amp;gt;400 μm) colonies during 21 days. Conversely, ALDHlowCD44- cells showed no such progression and remained predominantly as single cells throughout the assay. Together these results demonstrate that the PuMA assay sustains healthy lung architecture over 21 days, and that aggressive breast cancer cells interact with the lung to grow and progress as metastatic colonies. Ultimately, the results of this study will provide a greater understanding of the contribution of the lung microenvironment in mediating breast cancer metastasis. Citation Format: Matthew Piaseczny, David Goodale, Alison Allan. The lung microenvironment influences the metastatic behavior of breast cancer cells in an innovative 3D ex vivo pulmonary metastasis model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2379. doi:10.1158/1538-7445.AM2015-2379

Abstract 4112: Mechanisms of transendothelial migration by invasive breast carcinoma cells from patients

Abstract The majority of breast cancer related deaths are not due to the primary tumor, but rather to the dissemination of metastatic tumor cells from it to distant sites. Our lab has previously identified the tumor microenvironment of metastasis (TMEM) in mouse and human mammary tumors, sites where transendothelial migration, intravasation and dissemination occur. The constituent cells of TMEM are an endothelial cell, a perivascular TIE2-expressing macrophage (TEM) and an invasive Mena-over expressing tumor cell in direct contact. TMEM are present in human invasive breast tumors and the density of TMEM is positively associated with the risk of developing metastases. Using invasive ductal carcinoma cells of the breast obtained from patients by fine needle aspiration (FNA), we demonstrated that intravasation-directed transendothelial migration (iTEM) of these cancer cells requires macrophages and, depending on clinical subtype, involves either paracrine (macrophage CSF1-R; tumor cell EGFR), or both paracrine and autocrine (tumor cell EGFR + CSF1-R) signaling. Compared to the total population of primary breast cancer cells assayed, cells capable of transendothelial migration expressed relatively high MenaINV and low Mena11a levels, independently of clinical subtype. MenaINV and Mena11a are functionally distinct isoforms of Mena, a key regulator of motility and invasion. Depletion of MenaINV using siRNA showed that MenaINV is required for efficient macrophage-dependent iTEM of tumor cells. Furthermore, relative MenaINV expression correlated with the density of TMEM, which were previously shown to correlate with risk of metastasis in patients. From these observations we have identified the signaling pathways involved in macrophage-dependent tumor cell iTEM. Two novel small molecule inhibitors, Rebastinib (Tie2 inhibitor) and Altiratinib (c-MET inhibitor), have been identified that show great promise in preventing key steps in iTEM. Our data reveal targetable signaling events required for transendothelial migration of human breast cancer cells, and indicate that relative MenaINV levels and TMEM frequency are correlated prognostic markers of metastasis and therapeutic targets for most human breast cancers. Citation Format: Jeanine Pignatelli, Joan Jones, Xiaoming Chen, Bryan Smith, Daniel Flynn, John Condeelis, Maja Oktay. Mechanisms of transendothelial migration by invasive breast carcinoma cells from patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4112. doi:10.1158/1538-7445.AM2015-4112

Histone deacetylase inhibitor entinostat in combination with a retinoid downregulates HER2 and reduces the tumor initiating cell population in aromatase inhibitor-resistant breast cancer.

Resistance to aromatase inhibitors (AIs) involves increased HER2. One mechanism by which HER2 may mediate resistance is through expansion of the tumor initiating cell (TIC) population. This study investigates whether combining all-trans retinoic acid (ATRA) and histone deacetylase inhibitor entinostat (ENT) can inhibit TICs and HER2 in AI-resistant cells and tumors. Modulation of cell viability and HER2 expression were assessed in AI-resistant cells treated with ATRA + ENT. Letrozole-resistant LTLT-Ca cells treated with ATRA + ENT were assayed for changes in TIC characteristics, such as TIC markers (BCRP, ALDH, and BMI-1), side population (SP), and mammosphere formation. Xenograft tumors of MCF-7Ca cells made resistant to letrozole were treated with ATRA, ATRA + letrozole, ATRA + ENT, or ATRA + ENT + letrozole. Resulting tumors were assayed for changes in TIC characteristics. Patient samples taken pre- and post-AI treatment were analyzed for changes in ERα and HER2 protein expression. Treatment with ATRA + ENT reduced HER2 expression and viability (P < 0.001) in AI-resistant cells, as well as decreased SP (P < 0.0001), mammosphere formation (P < 0.01), and expression of TIC molecular markers (P < 0.01) in LTLT-Ca. A reduction in tumor growth rate was observed in mice treated with ENT + ATRA + letrozole when compared to mice treated with single agents (P < 0.0001) or ENT + ATRA (P = 0.02). Decreased TIC characteristics, including mammosphere formation (P < 0.05), were observed in tumors from the triple combination. An increase in HER2 and downregulation in ERα protein expression was observed in patients upon resistance to AI (P < 0.005). These studies indicate that the combination of ATRA and ENT inhibits the TIC population of AI-resistant cells and may be effective in reducing tumor recurrence.

Abstract P3-05-10: Glucocorticoid and aldosterone mimic progestin-induction of a therapy-resistant cytokeratin-5 positive cell population in estrogen receptor-positive breast cancer through a Bcl6-dependent mechanism

Abstract Resistance to anti-estrogen therapy remains a significant problem in patients diagnosed with estrogen receptor-α (ERα) positive breast cancer. Recent progress has defined a "lumino-basal" subclass of ERα-positive breast cancer characterized by mosaic presence of a minor population of ERα-negative cells expressing the basal cytokeratin-5 (CK5). The CK5-positive cells are therapy-resistant and have increased tumorigenic potential. Initial studies have suggested that progestins but not other steroids expand this CK5+ cell population. Unexpectedly, we discovered that at least two 3-ketosteroids other than progestins, glucocorticoids and mineralocorticoids, are capable of inducing the CK5+/ERα- cell population. CK5+ cells induced by glucocorticoid or aldosterone showed increased clonogenicity in soft agar, expressed the stem cell marker CD44, showed loss of ERα and PR expression, and demonstrated therapy-resistance with reduced apoptosis in response to chemotherapy, and were further enriched following adjuvant antiestrogen or chemotherapies. Induction of CK5+ cells by 3-ketosteroids was consistently preceded by induction of Bcl6, a transcriptional repressor implicated in breast cancer progression. Suppression of Bcl6 by shRNA or the Bcl6 suppressor, prolactin, abolished 3-ketosteroid-induction of CK5+ cells. Prolactin also blocked 3-ketosteroid-induced colony formation in vitro and suppressed progestin-induction of the CK5+ cell population in T47D xenograft tumors in vivo. Survival analyses with recursive partitioning revealed that CK5 and Bcl6 transcripts or protein levels in ERα+ breast cancer identify patients at high or low risk for tumor in two independent cohorts. The observations provide a mechanism by which stress-related or pharmacologic elevation of glucocorticoids may adversely affect patients with ERα+/CK5+ breast cancer, and may justify further exploring of inhibitors to 3-ketosteroid receptors or Bcl6 for therapeutic benefit. Citation Format: Hallgeir Rui, Takahiro Sato, Amy Peck, Melanie A Girondo, Chengbao Liu, Albert J Kovatich, Jeffery A Hooke, Craig D Shriver, Edith Mitchell, Terry Hyslop, Chelain Goodman. Glucocorticoid and aldosterone mimic progestin-induction of a therapy-resistant cytokeratin-5 positive cell population in estrogen receptor-positive breast cancer through a Bcl6-dependent mechanism [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-05-10.

Abstract PD3-3: Impact of neoadjuvant chemotherapy on the clonal composition of breast cancer

Abstract Background Cancer genomic investigations have identified recurrent genomic aberrations critical for cancer initiation, progression, and metastases. However, these investigations are typically performed in isolation, and the effects of treatment on the clonal selection of tumor cells are mostly unknown. We hypothesized that molecular profiling of residual tumors after neoadjuvant chemotherapy (NAC) would identify new drug targets/pathways in patients at high risk for disease recurrence. To better identify clonal populations of resistant breast cancer cells, we utilized DNA content-based flow sorting of nuclei to identify and isolate clonal populations for aCGH and next generation sequencing (NGS) both before and after NAC. Methods The Breast Cancer Genome Guided Therapy Study (BEAUTY) (NCT 02022202) is a prospective study of patients with high-risk breast cancer treated with neoadjuvant 12 weekly paclitaxel (T) +/- trastuzumab followed by 4 cycles of anthracycline based chemotherapy. Tumor tissue from baseline, residual disease from surgery, distant metastases, and patient derived xenografts (PDX) are obtained for cell sorting by DNA ploidy, aCGH, RNA and exome sequencing. Results: 140 patients have been enrolled, 104 have completed surgery and 30 unique PDX have been established corresponding to 26 patients prior to chemotherapy and 4 from residual disease at surgery. Baseline exome and RNA sequencing is complete in 140. Currently, genomic analyses of flow sorted matched baseline, surgical, PDX, and distant disease samples are available in 6 patients. Substantial genomic variation was observed in the surgical sample compared to the primary tumor including gain of oncogenic drivers (EGFR) and loss of negative regulators (ATG5) (Table). The PDX recapitulated these events with excellent fidelity compared to the corresponding human tumor. In patients with TNBC, RNA seq obained from matched samples demonstrated changes in immune related pathways. Evaluation of drug targets/pathways identified in the resistant tumors are ongoing using the PDX and sequencing of the remaining matched baseline/surgical disease will be reported. Clonal changes occurring over time in patients with residual disease or disease recurrence after NACTumor SubtypeResidual Cancer BurdenDisease StatusClonal Aberration Changes (baseline and post NAC)TNBC (AR subtype)3Contralateral lymph node recurrence at 150 days2p25.2 - p25.1 amplicon lost at recurrence; 6p21.32 -p21.31 amplicon lost at recurrenceTNBC (BL 1)3Bone/liver/lymph node recurrence at 135 days5q11.2 amplicon gained at surgery; 12p13.33 - p13.2 amplicon gained at surgeryTNBC (BL 1)3Disease-free at day 1506q21 amplicon lost at sugeryTNBC (BL 2)0Brain recurrence at 390 daysChr 2 chromothripsis at surgeryLuminal B3Progression during chemotherapy7p12.1 - p11.2 amplicon gained at surgeryLuminal HER23Disease-free at day 3609q33.2 amplicon lost at surgery; 15q13.1 - q13.3 amplicon lost at surgery; 15q22.2 - q24.1 amplicon lost at surgery Conclusions: We observed substantial evolutionary changes in residual breast tumors remaining after NAC. Our findings suggest that a comprehensive assessment of the mutational landscape that has evolved during NAC can inform drug development in high risk breast cancer patients. Citation Format: Matthew P Goetz, Michael T Barrett, Krishna R Kalari, Vera J Suman, Sarah A McLaughlin, Alvaro Moreno-Aspitia, Ann M Moyer, Donald W Northfelt, Richard J Gray, Jason Sinnwell, Douglas Mahoney, Poulami Barman, Peter Vedell, Xiaojia Tang, Kevin Thompson, Travis Dockter, Katie Jones, Sara J Felten, Amy Conners, Jeanette Eckel-Passow, Hughes Sicotte, Steven N Hart, Jia Yu, Daniel W Visscher, Eric D Wieben, Cloann Schultz, Minetta C Liu, James N Ingle, Liewei Wang, Richard W Weinshilboum, Judy C Boughey. Impact of neoadjuvant chemotherapy on the clonal composition of breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD3-3.

Accumulation of GD1α Ganglioside in MDA-MB-231 Breast Cancer Cells Expressing ST6GalNAc V.

α-Series gangliosides define a particular sub-class of glycosphingolipids containing sialic acid α2,6-linked to GalNAc residue that was isolated as a minor compound from the brain. The sialyltransferase ST6GalNAc V was cloned from mouse brain and showed α2,6-sialyltransferase activity almost exclusively for GM1b, to form GD1α and is considered as the main enzyme involved in the biosynthesis of α-series gangliosides. Recently, ST6GALNAC5 was identified as one of the genes over-expressed in breast cancer cell populations selected for their ability to produce brain metastasis. However, the capacity of human breast cancer cells to produce α-series gangliosides has never been clearly demonstrated. Here, we show by stable transfection and MS-MS analysis of total glycosphingolipids that ST6GALNAC5 expressing MDA-MB-231 breast cancer cells accumulate GD1α ganglioside (IV3Neu5Ac1, III6Neu5Ac1Gg4-Cer).

Autophagy Supports Breast Cancer Stem Cell Maintenance by Regulating IL6 Secretion.

Autophagy is a mechanism by which cells degrade cellular material to provide nutrients and energy for survival during stress. The autophagy is thought to be a critical process for cancer stem cell (CSC) or tumor-initiating cell maintenance but the mechanisms by which autophagy supports survival of CSCs remain poorly understood. In this study, inhibition of autophagy by knockdown of ATG7 or BECN1 modified the CD44(+)/CD24(low/-) population in breast cancer cells by regulating CD24 and IL6 secretion. In a breast cancer cell line that is independent of autophagy for survival, autophagy inhibition increased IL6 secretion to the media. On the other hand, in an autophagy-dependent cell line, autophagy inhibition decreased IL6 secretion, cell survival, and mammosphere formation. In these cells, IL6 treatment or conditioned media from autophagy-competent cells rescued the deficiency in mammosphere formation induced by autophagy inhibition. These results reveal that autophagy regulates breast CSC maintenance in autophagy-dependent breast cancer cells by modulating IL6 secretion implicating autophagy as a potential therapeutic target in breast cancer. Modulation of autophagy in breast cancer has different and even opposite effects, indicating the need for a selection strategy when trying to manipulate autophagy in the context of cancer therapy.

CD44(+)/CD24(-) breast cancer cells exhibit phenotypic reversion in three-dimensional self-assembling peptide RADA16 nanofiber scaffold.

BackgroundSelf-assembling peptide nanofiber scaffolds have been shown to be a permissive biological material for tissue repair, cell proliferation, differentiation, etc. Recently, a subpopulation (CD44+/CD24−) of breast cancer cells has been reported to have stem/progenitor cell properties. The aim of this study was to investigate whether this subpopulation of cancer cells have different phenotypes in self-assembling COCH3-RADARADARADARADA-CONH2 (RADA16) peptide nanofiber scaffold compared with Matrigel® (BD Biosciences, Two Oak Park, Bedford, MA, USA) and collagen I.MethodsCD44 and CD24 expression was determined by flow cytometry. Cell proliferation was measured by 5-bromo-2′-deoxyuridine assay and DNA content measurement. Immunostaining was used to indicate the morphologies of cells in three-dimensional (3D) cultures of different scaffolds and the localization of β-catenin in the colonies. Western blot was used to determine the expression of signaling proteins. In vitro migration assay and inoculation into nude mice were used to evaluate invasion and tumorigenesis in vivo.ResultsThe breast cancer cell line MDA-MB-435S contained a high percentage (>99%) of CD44+/CD24− cells, which exhibited phenotypic reversion in 3D RADA16 nanofiber scaffold compared with collagen I and Matrigel. The newly formed reverted acini-like colonies reassembled a basement membrane and reorganized their cytoskeletons. At the same time, cells cultured and embedded in RADA16 peptide scaffold exhibited growth arrest. Also, they exhibited different migration potential, which links their migration ability with their cellular morphology. Consistent with studies in vitro, the in vivo tumor formation assay further supported of the functional changes caused by the reversion in 3D RADA16 culture. Expression levels of intercellular surface adhesion molecule-1 were upregulated in cells cultured in RADA16 scaffolds, and the NF-kappa B inhibitor pyrrolidine dithiocarbamate could inhibit RADA16-induced upregulation of intercellular surface adhesion molecule-1 and the phenotype reversion of MDA-MB-453S cells.ConclusionCulturing a CD44+/CD24−-enriched breast cancer cell population in 3D RADA16 peptide nanofiber scaffold led to a significant phenotypic reversion compared with Matrigel and collagen I.

PNIPAAm-MAA nanoparticles as delivery vehicles for curcumin against MCF-7 breast cancer cells

Breast cancer is the most frequently occurring cancer among women throughout the world. Natural compounds such as curcumin hold promise to treat a variety of cancers including breast cancer. However, curcumin's therapeutic application is limited, due to its rapid degradation and poor aqueous solubility. On the other hand, previous studies have stated that drug delivery using nanoparticles might improve the therapeutic response to anticancer drugs. Poly(N-isopropylacrylamide-co-methacrylic acid) (PNIPAAm–MAA) is one of the hydrogel copolymers utilized in the drug delivery system for cancer therapy. The aim of this study was to examine the cytotoxic potential of curcumin encapsulated within the NIPAAm-MAA nanoparticle, on the MCF-7 breast cancer cell line. In this work, polymeric nanoparticles were synthesized through the free radical mechanism, and curcumin was encapsulated into NIPAAm-MAA nanoparticles. Then, the cytotoxic effect of curcumin-loaded NIPAAm-MAA on the MCF-7 breast cancer cell line was measured by MTT assays. The evaluation of the results showed that curcumin-loaded NIPAAm-MAA has more cytotoxic effect on the MCF-7 cell line and efficiently inhibited the growth of the breast cancer cell population, compared with free curcumin. In conclusion, this study indicates that curcumin-loaded NIPAAm-MAA suppresses the growth of the MCF-7 cell line. Overall, it is concluded that encapsulating curcumin into the NIPAAm-MAA copolymer could open up new avenues for breast cancer treatment.

Reprogramming of the ERRα and ERα target gene landscape triggers tamoxifen resistance in breast cancer.

Endocrine treatment regimens for breast cancer that target the estrogen receptor-α (ERα) are effective, but acquired resistance remains a limiting drawback. One mechanism of acquired resistance that has been hypothesized is functional substitution of the orphan receptor estrogen-related receptor-α (ERRα) for ERα. To examine this hypothesis, we analyzed ERRα and ERα in recurrent tamoxifen-resistant breast tumors and conducted a genome-wide target gene profiling analysis of MCF-7 breast cancer cell populations that were sensitive or resistant to tamoxifen treatment. This analysis uncovered a global redirection in the target genes controlled by ERα, ERRα, and their coactivator AIB1, defining a novel set of target genes in tamoxifen-resistant cells. Beyond differences in the ERα and ERRα target gene repertoires, both factors were engaged in similar pathobiologic processes relevant to acquired resistance. Functional analyses confirmed a requirement for ERRα in tamoxifen- and fulvestrant-resistant MCF-7 cells, with pharmacologic inhibition of ERRα sufficient to partly restore sensitivity to antiestrogens. In clinical specimens (n = 1041), increased expression of ERRα was associated with enhanced proliferation and aggressive disease parameters, including increased levels of p53 in ERα-positive cases. In addition, increased ERRα expression was linked to reduced overall survival in independent tamoxifen-treated patient cohorts. Taken together, our results suggest that ERα and ERRα cooperate to promote endocrine resistance, and they provide a rationale for the exploration of ERRα as a candidate drug target to treat endocrine-resistant breast cancer.