Basal Breast Cancer Cell Research Articles

Constitutive NOTCH3 Signaling Promotes the Growth of Basal Breast Cancers.

Notch ligands signal through one of four receptors on neighboring cells to mediate cell-cell communication and control cell fate, proliferation, and survival. Although aberrant Notch activation has been implicated in numerous malignancies, including breast cancer, the importance of individual receptors in distinct breast cancer subtypes and the mechanisms of receptor activation remain unclear. Using a novel antibody to detect active NOTCH3, we report here that NOTCH3 signals constitutively in a panel of basal breast cancer cell lines and in more than one third of basal tumors. Selective inhibition of individual ligands revealed that this signal does not require canonical ligand induction. A NOTCH3 antagonist antibody inhibited growth of basal lines, whereas a NOTCH3 agonist antibody enhanced the transformed phenotype in vitro and in tumor xenografts. Transcriptomic analyses generated a Notch gene signature that included Notch pathway components, the oncogene c-Myc, and the mammary stem cell regulator Id4 This signature drove clustering of breast cancer cell lines and tumors into the common subtypes and correlated with the basal classification. Our results highlight an unexpected ligand-independent induction mechanism and suggest that constitutive NOTCH3 signaling can drive an oncogenic program in a subset of basal breast cancers. Cancer Res; 77(6); 1439-52. ©2017 AACR.

Abstract P6-08-01: A directed siRNA screen identifies INHBA as a major regulator of tumor aggressiveness in basal HER2 breast cancer

Abstract HER2+ breast cancers can be treated with several HER2-targeted inhibitors, but tumors often acquire or have de novo resistance to the therapies, limiting the long term efficacy of these treatments. To identify genes associated with resistance, we performed drug screens on a panel of HER2 positive breast cancer cell lines using lapatinib, then performed statistical analyses to identify differentially expressed genes. We procured siRNA against the top 20 targets expressed in the resistant subset for subsequent viability screening, both in the presence and absence of lapatinib. This screen identified INHBA, a member of the TGF-B superfamily, as a strong mediator of cell growth and lapatinib response in two resistant HER2+ cell lines. Both these lines were of the basal subtype, which tend to be more resistant in our cell line panel. We tested 8 different HER2+ breast cancer cell lines (4 basal and 4 luminal subtype) with siRNA against INHBA. We observed significant inhibition of growth in 3 out of 4 basal lines, with growth rates of 40-55% of scramble control lines. In contrast, none of the luminal lines showed significant growth inhibition when treated with the INHBA siRNA. Furthermore, treatment with INHBA siRNA sensitized cells to lapatinib, with GI50 (dose required to inhibit growth by 50%) dropping from 10uM to less than 2 uM in JIMT1 and 21-MT1 basal lines. We also tested the effects of knockdown in 3-d matrigel cultures, which recapitulated the 2-d findings with decreased growth and increased sensitivity. For the cell line 21-MT1, growth following INHBA siRNA treatment was 12.6% of scramble control, and lapatinib treatment further reduced this to 7.8%, in contrast to scramble control/lapatinib treated cells, which grew at 97.7% of control rate. We performed RNAseq on 21-MT1 and JIMT1 cells treated with siRNA and looked at both relative changes in expression as well as absolute magnitude changes in expression. There were a large number of mitochondrial genes induced in the INHBA siRNA knockdown cells and loss of glycolytic genes such as LDHA compared to scramble control cells, suggesting a change in metabolic activity. We tested this by treating cells with the oxidative phosphorylation inhibitor oligomycin or the glycolysis inhibitor 2-deoxyglucose (2-DG). We found that JIMT1 cells treated with INHBA siRNA were more sensitive to oligomycin but less sensitive to 2-DG compared to scramble treated controls, suggesting a shift from glycolytic to oxidative phosphorylation metabolism. Since this type of metabolic shift has also been associated with reduced invasive capacity, we performed invasion assays on cells treated with INHBA or scramble-control siRNA. We found that knockdown of INHBA reduced invasion of both 21-MT1 and JIMT1 cells, with invasion rates less than 10% of control cells. Finally, we mined expression data sets to determine associations between INHBA levels and outcome, and found that high levels of expression of INHBA were associated with poor outcome in both HER2+ and the basal subtype of breast tumors, but not in luminal tumors. In conclusion, we identified INHBA as a major regulator of metabolism and aggressiveness in HER2+ basal breast cancer cells that is associated with poor outcome in patients. Citation Format: Korkola JE, Liu M, Smith R, Liby T, Heiser L, Gray JW. A directed siRNA screen identifies INHBA as a major regulator of tumor aggressiveness in basal HER2 breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-08-01.

Abstract P1-07-01: A pan-cancer perspective of functional proteomics provides novel information content for uncommon breast cancer subtypes

Abstract Cancer cell lines (CCLs) serve as models to study the functional consequences of the genomic lesions in patients and as screening platforms for prediction of drug response. While genomic and transcriptomic data have proven to be useful predictors, the ability of these omics platforms to predict protein level and function is limited. Furthermore, since proteins are the targets of the majority of the targeted therapies, protein levels and importantly protein function would be expected to provide more powerful predictions than DNA or RNA data. While large scale genomic and transcriptomic data linked to drug sensitivity are available for over a thousand CCLs, proteomic data is available for only a small subset of lines. Here we performed proteomic profiling of 736 cell lines using reverse-phase protein arrays (RPPAs) with approximately 300 antibodies providing an unbiased sparse representation of the majority of signaling pathways. The functional proteomic analysis revealed 10 protein-based clusters across all cell lines. Similar to human tumors, the breast cell lines fell into three major clusters representing basal-like, luminal/Her2-amplified and claudin-low breast cancer subtypes. The basal-like and claudin-low clusters contained all of the representative breast cancer cell lines as well as a much larger number of other CCLs. For example, the 6 claudin-low breast cancers analyzed reside in an EMT cluster, in which only 8/126 are breast cell lines. However, the complete cluster including multiple non-breast cancer cell lines recapitulated mRNA and protein features of claudin-low breast tumors, including a high EMT signature and low level of hormone receptor pathway activity. We thus explored whether we could gain power for linking the limited number of basal and claudin-low breast cancer cell lines to therapeutic sensitivity by assessing patterns of drug sensitivity in each cluster for both the breast and non-breast cancer cell lines in the cluster. We explored drug sensitivity of 481 therapeutic compounds from the Cancer Therapeutic Response Portal (CTRP v2) and demonstrated that the non- breast cancer and breast cancer cell lines in each cluster provided similar patterns of drug sensitivity. For example, Claudin-low/EMT cell lines of both breast cancer and non-breast cancer origin showed decreased sensitivity to PI3K/mTOR inhibitors compared to luminal breast cancers (p<0.05 for 4 mTOR inhibitors) and drugs targeting EGFR family compared to basal cell lines (p<0.05 for 7 EGFR/ERBB2 inhibitors). Thus it is possible to gain information by characterizing cell lines with similar patterns of protein expression and provide important information related to drug sensitivity of uncommon breast cancer lineages. The functional proteomic analysis provides a wealth of information that complements the genomic and transciptomic studies of cancer cell lines, and demonstrates the opportunity to leverage cell line 'pan-cancer' proteomic patterns to improve characterization of specific breast cancer subtypes. To facilitate broad access to these data, we developed a user-friendly data portal, the MD Anderson Cell Lines Project (MCLP), that provides both data analysis and download (http://ibl.mdanderson.org/mclp/). Citation Format: Zhao W, Li J, Lu Y, Akbani R, Liang H, Mills GB. A pan-cancer perspective of functional proteomics provides novel information content for uncommon breast cancer subtypes [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-07-01.

Multiple drug resistance-associated protein (MRP4) exports prostaglandin E2 (PGE2) and contributes to metastasis in basal/triple negative breast cancer

Cyclooxygenase-2 (COX-2) and its primary enzymatic product, prostaglandin E2 (PGE2), are associated with a poor prognosis in breast cancer. In order to elucidate the factors contributing to intratumoral PGE2 levels, we evaluated the expression of COX-2/PGE2 pathway members MRP4, the prostaglandin transporter PGT, 15-PGDH (PGE2 metabolism), the prostaglandin E receptor EP4, COX-1, and COX-2 in normal, luminal, and basal breast cancer cell lines. The pattern of protein expression varied by cell line reflecting breast cancer heterogeneity. Overall, basal cell lines expressed higher COX-2, higher MRP4, lower PGT, and lower 15-PGDH than luminal cell lines resulting in higher PGE2 in the extracellular environment. Genetic or pharmacologic suppression of MRP4 expression or activity in basal cell lines led to less extracellular PGE2. The key finding is that xenografts derived from a basal breast cancer cell line with stably suppressed MRP4 expression showed a marked decrease in spontaneous metastasis compared to cells with unaltered MRP4 expression. Growth properties of primary tumors were not altered by MRP4 manipulation. In addition to the well-established role of high COX-2 in promoting metastasis, these data identify an additional mechanism to achieve high PGE2 in the tumor microenvironment; high MRP4, low PGT, and low 15-PGDH. MRP4 should be examined further as a potential therapeutic target in basal breast cancer.

Cystine addiction of triple-negative breast cancer associated with EMT augmented death signaling.

Despite the advances in the diagnosis and treatment of breast cancer, breast cancers still cause significant mortality. For some patients, especially those with triple negative breast cancer (TNBC), current treatments continue to be limited and ineffective. Therefore, there remains an unmet need for a novel therapeutic approach. One potential strategy is to target the altered metabolic state that is rewired by oncogenic transformation. Specifically, this rewiring may render certain outside nutrients indispensable. To identify such a nutrient, we performed a nutrigenetic screen by removing individual amino acids to identify possible addictions across a panel of breast cancer cells. This screen revealed that cystine deprivation triggered rapid programmed necrosis, but not apoptosis, in the basal-type breast cancer cells mostly seen in TNBC tumors. In contrast, luminal-type breast cancer cells are cystine-independent and exhibit little death during cystine deprivation. The cystine addiction phenotype is associated with a higher level of cystine-deprivation signatures noted in the basal type breast cancer cells and tumors. We found that the cystine-addicted breast cancer cells and tumors have strong activation of TNFα and MEKK4-p38-Noxa pathways that render them susceptible to cystine deprivation-induced necrosis. Consistent with this model, silencing of TNFα and MEKK4 dramatically reduces cystine-deprived death. In addition, the cystine addiction phenotype can be abrogated in the cystine-addictive cells by miR-200c, which converts the mesenchymal-like cells to adopt epithelial features. Conversely, the introduction of inducers of epithelial-mesenchymal transition (EMT) in cystine-independent breast cancer cells conferred the cystine-addiction phenotype by modulating the signaling components of cystine addiction. Together, our data reveal that cystine-addiction is associated with EMT in breast cancer during tumor progression. These findings provide the genetic and mechanistic basis to explain how cystine deprivation triggers necrosis by activating pre-existing oncogenic pathways in cystine-addicted TNBC with prominent mesenchymal features.

G Protein-coupled Receptor Kinase 2 (GRK2) Promotes Breast Tumorigenesis Through a HDAC6-Pin1 Axis

In addition to oncogenic drivers, signaling nodes can critically modulate cancer-related cellular networks to strength tumor hallmarks. We identify G-protein-coupled receptor kinase 2 (GRK2) as a relevant player in breast cancer. GRK2 is up-regulated in breast cancer cell lines, in spontaneous tumors in mice, and in a proportion of invasive ductal carcinoma patients. Increased GRK2 functionality promotes the phosphorylation and activation of the Histone Deacetylase 6 (HDAC6) leading to de-acetylation of the Prolyl Isomerase Pin1, a central modulator of tumor progression, thereby enhancing its stability and functional interaction with key mitotic regulators. Interestingly, a correlation between GRK2 expression and Pin1 levels and de-acetylation status is detected in breast cancer patients. Activation of the HDAC6-Pin1 axis underlies the positive effects of GRK2 on promoting growth factor signaling, cellular proliferation and anchorage-independent growth in both luminal and basal breast cancer cells. Enhanced GRK2 levels promote tumor growth in mice, whereas GRK2 down-modulation sensitizes cells to therapeutic drugs and abrogates tumor formation. Our data suggest that GRK2 acts as an important onco-modulator by strengthening the functionality of key players in breast tumorigenesis such as HDAC6 and Pin1.

Loss of Dickkopf 3 Promotes the Tumorigenesis of Basal Breast Cancer.

Dickkopf 3 (DKK3) has been associated with tumor suppression of various tumor entities including breast cancer. However, the functional impact of DKK3 on the tumorigenesis of distinct molecular breast cancer subtypes has not been considered so far. Therefore, we initiated a study analyzing the subtype-specific DKK3 expression pattern as well as its prognostic and functional impact with respect to breast cancer subtypes. Based on three independent tissue cohorts including one in silico dataset (n = 30, n = 463 and n = 791) we observed a clear down-regulation of DKK3 expression in breast cancer samples compared to healthy breast tissue controls on mRNA and protein level. Interestingly, most abundant reduction of DKK3 expression was detected in the highly aggressive basal breast cancer subtype. Analyzing a large in silico dataset comprising 3,554 cases showed that low DKK3 mRNA expression was significantly associated with reduced recurrence free survival (RFS) of luminal and basal-like breast cancer cases. Functionally, DKK3 re-expression in human breast cancer cell lines led to suppression of cell growth possibly mediated by up-regulation of apoptosis in basal-like but not in luminal-like breast cancer cell lines. Moreover, ectopic DKK3 expression in mesenchymal basal breast cancer cells resulted in partial restoration of epithelial cell morphology which was molecularly supported by higher expression of epithelial markers like E-Cadherin and down-regulation of mesenchymal markers such as Snail 1. Hence, we provide evidence that down-regulation of DKK3 especially promotes tumorigenesis of the aggressive basal breast cancer subtype. Further studies decoding the underlying molecular mechanisms of DKK3-mediated effects may help to identify novel targeted therapies for this clinically highly relevant breast cancer subtype.

Abstract 1585: Molecular subtypes of breast cancers and normal mammary epithelial cells show hierarchical heterogeneity and complexity at single-cell resolution

Abstract The analysis of average gene expression in cell populations may obscure the heterogeneity of rare cells such as cancer stem cells (CSCs). The use of CSC markers to enrich for these cell populations has proven valuable in their molecular characterization. However, conventional gene expression analysis of these CSC enriched populations is still not conducive to elucidating their heterogeneity. We exploited single-cell resolution methods using C1 and BioMark HD platform and TaqMan assays in the analysis of CSCs and non-CSCs/differentiated tumor cells. The expression of 96 target genes was studied at single cell level in a multiplex RT-qPCR setting. Breast cancer cell lines including MCF7 (luminal), BT474 (HER2+), SUM149 (Basal), and MDA-MB-231 (Basal-Claudin low) that represent major different subtypes of breast cancers and normal mammary epithelial cells (NM) were studied at single cell resolution. Despite similarities, expression profiles of 96 target genes were distinctly different between subtypes of breast cancers and also with that of NM cells. Interestingly, results of single cell studies revealed significant levels of hierarchical heterogeneities within and between each analyzed population of cells. The observed heterogeneous signatures were due to both different expression levels as well as presence/absence of certain genes. We could identify CSCs with EMT (i.e. CD44+/CD24-/ALDH-/CDH2+/Vimentin+), MET (i.e. ALDH+/CD44-/CD24+/CDH1+/Vimentin-), and dual EMT-MET (CD44+/CD24-/ALDH+/CDH1+ or CDH2+/Vimentin+) characteristics. This methodology enabled us to also identify a range of several different non-CSCs/differentiated single cells with heterogeneous expression patterns for CD44, CD24, ALDH, ESR, PGR, HER2, Vimentin, CDH1, CDH2, EpCAM, and Cytokeratins that were similar to luminal, HER2 overexpressed, basal, and basal-Claudin low breast cancer cells. Additionally, NM single cells also showed significant heterogeneity that was similar to the wide spectrum patterns of some single breast cancer cells described above. This study highlights the use of single cell technologies to redefine tumor cell characterization, revealing heterogeneity of single tumor cells with respect to stem cell properties, metastatic and drug resistance characteristics. This will further improve the identification of new markers/pathways that can be used to develop new target therapies for better therapeutic responses in cancer patients. Citation Format: Ebrahim Azizi, Justin Colacino, Shamileh Fouladdel, Max S. Wicha. Molecular subtypes of breast cancers and normal mammary epithelial cells show hierarchical heterogeneity and complexity at single-cell resolution. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1585.

Abstract 5059: Cancer cell invasion dynamics in microchannels during stromal cell coculture

Abstract Metastasizing cancer cells must escape their tumor microenvironment and invade into surrounding healthy stromal tissue. During this process, they must balance intercellular crosstalk with ECM degradation, physical force generation, and cytoskeletal rearrangement. To mimic this process in vitro, PDMS microchannels fabricated with photolithography and replica molding were used to examine the interaction of cancer cells with healthy stromal cells during physical confinement in three dimensional environments. A focus was placed on understanding the variations in invasion behavior between healthy cells and cancer cells, then attempting to explain these differences by cell mechanical properties and gene expression. Distinct differences in cancer cell invasion characteristics were observed between wide 10 μm channels and narrow 3 μm channels, with the latter exhibiting smooth velocity profiles and increased channel permeation. Live cell actin staining, ECM protein alteration, and chemical inhibition of both Rac1 and RhoA mechanical pathways were all used to better understand these differences, leading to the conclusion that the actin cytoskeleton is greatly altered during invasion into very confined channels. To better understand the role of stromal cells in this process, both indirect and direct coculture of MDA MB-231 basal breast cancer cells and stromal cells were performed in microchannel chips with the aid of live cell permeable tracking dyes. To reflect a diverse range of stromal cell types, cocultures were performed with MCF 10A non-tumorigenic breast epithelial cells, macrophage-differentiated THP-1 monocytes, and HN-CAFs (head and neck carcinoma-associated fibroblasts). Both the cancer cells and the stromal cells exhibited increases in invasion speed upon direct co-culture of the two cancer cell lines, both in wide and narrow channels. The use of stromal cell-conditioned media as well as observed differences in migration speed and persistence when unconfined provided further understanding of the complex processes underlying cancer cell and stromal cell invasion. Citation Format: Andrew W. Holle, Verena Kast, Ralf Kemkemer, Joachim Spatz. Cancer cell invasion dynamics in microchannels during stromal cell coculture. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5059.

Abstract 1574: Neuropilin 2 regulates lung disseminated tumor cell escape from dormancy and progression into metastasis

Abstract Despite considerable advances in the treatment of cancer, about 50% of patients will still eventually develop metastatic disease which remains the main cause of cancer-related death. Disseminated tumor cells (DTCs) are the precursors of metastasis and understanding their biology is one of the most important challenges for the future of cancer research. Our previous studies have shown that TGFβ2 through binding to TGFβR3 regulates DTCs quiescence through the activation of p38αMAPK and induction of the dormant genes DEC2 and p27. However, these DTCs dormancy may be reversed when micro-environmental conditions shift to support DTCs expansion. Nerve fibers and neural mediators are present in organs that serve as key targets for breast and head and neck cancer metastasis, including lungs and bone. In addition, neuropeptides and neurotransmitters receptors are expressed by both, nerve and cancer cells, which suggests that neurotrophic factors can act as messengers between the nervous system and DTCs and influence metastasis progression. Therefore, we hypothesized that in situations of chronic stress or inflammation, neural mediators might influence DTCs fate at secondary organs. Using bioinformatics tools we have found that the semaphorins receptor Neuropilin 2 (NRP2), a co-receptor for TGFβ ligands, is overexpressed in basal and HER2+ breast cancer patient samples and its expression in primary tumors correlate with worst prognosis. In basal breast cancer cell lines, NRP2 expression correlates with lower levels of DEC2 and p27 dormancy genes. Furthermore, we have found that NRP2 is downregulated in dormant cells and overexpressed in proliferative lung DTCs derived cell lines that express low levels of P-p38, DEC2 and p27 dormancy markers. Our results show that in lung DTCs derived cell lines, NRP2 inhibits TGFβR3 expression favoring TGFβ1 signaling and promoting DTCs growth. In agreement with this, breast cancer lung metastasis express higher levels of NRP2 than quiescence lung DTCs in vivo, suggesting that NRP2 might play a role in lung DTCs proliferation. Therefore, we conclude that NRP2 can regulate breast cancer lung DTCs progression from a dormant state to a proliferative state and promote metastasis formation. Citation Format: Paloma Bragado Domingo, Raul Alonso, Gemma Fuster, Mario Mancino, Patricia Fernandez-Nogueira, Julio Aguirre-Ghiso, Pere Gascon. Neuropilin 2 regulates lung disseminated tumor cell escape from dormancy and progression into metastasis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1574.

Abstract 1656: Subtype-specific radiation response in human breast cancer and potential therapeutic effect of FAS death receptor modulation

Abstract Breast cancer is the most common malignancy diagnosed among women worldwide and represents a heterogeneous group of subtypes. Radiation therapy is an important component of multimodal treatment for women with breast cancer. However, little is known about radiation response among these subtypes. Recent clinical data suggests that distinct patterns of response to systemic therapy may exist in association with each phenotype. Therefore, understanding the intrinsic variation of radiation response in breast cancer subtypes is important in prescribing individualized radiotherapy that maximizes the therapeutic ratio. Our previous studies have identified marked changes in gene expression in breast patient tumors and breast tumor cell lines in response to radiation. Among candidate genes, FAS was consistently and significantly induced after radiation in luminal breast tumors and cell lines. Modulation of FAS in basal breast cancer cell lines favorably impacted radiation response. In this study, we further investigated the role of FAS modulation and radiation response in a mouse model of breast cancer. MCF7 (luminal), HCC1954 (HER2+) or SUM159 (basal) cells were implanted orthotopically into the dorsal mammary fat pad of nude mice. These mice were then treated with different doses of radiation. Our results showed that radiation treatment inhibited MCF7, and to a less extent HCC1954 tumor growth in a dose-dependent manner, while SUM159 tumors were resistant to radiation. The estimated TCD50 value was 19.30 Gy for MCF7 and 44.88 Gy for SUM159. FAS was induced in MCF7 tumors after radiation but showed no change in SUM159 tumors. To investigate whether FAS modulation can affect radiation response, we silenced FAS in MCF7 tumors and activated FAS in SUM159 tumors. We found that silencing of FAS did not negatively impact radiation response in MCF7 tumors, possibly due to compensation by other apoptotic pathways. On the other hand, FAS activating antibody in combination with radiation delayed SUM159 and HCC1954 tumor growth (p = NS). In conclusion, these results suggest that there is intrinsic variation in radiation response among breast cancer subtypes. FAS concurrent with radiation slows tumor growth in the radiation resistant subtypes, but the effect was not significant. Alternative subtype-specific modulators of radiation response are under investigation. Citation Format: Chen-Ting Lee, Yingchun Zhou, Kingshuk R. Choudhury, Sharareh Siamakpour Reihani, Mark W. Dewhirst, Janet K. Horton. Subtype-specific radiation response in human breast cancer and potential therapeutic effect of FAS death receptor modulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1656.

Abstract B47: Methylation differences in breast tumor DNA from African American and European women are predominant in estrogen receptor (ER) negative breast cancer and are associated with childbearing

Abstract Background: Although European American (EA) women have higher incidence of breast cancer than African American (AA) women, AA women are more often diagnosed with aggressive estrogen receptor negative (ER-) tumors, the risk of which increases with parity, lack of breastfeeding, and an early age at menarche. DNA methylation could be affected by reproductive factors and may be a potential molecular mechanism driving differences in tumor etiology. In a small study using fresh frozen breast tissue (n=58 AA, 80 EA), we previously found more differentially methylated loci (DMLs) in ER- tumors from AA and EA women than in ER+ tumors. Methods: To follow up on these preliminary findings, we used the Illumina 450K platform to determine genome-wide DNA methylation profiles in formalin fixed paraffin embedded (FFPE) breast tumors from 383 AA and 350 EA women who participated in the Women's Circle of Health Study, a case control study conducted in NY and NJ. We identified DMLs by race and ER status, as well as differentially methylated regions (DMRs). DMLs in eight genes were validated using the Sequenom EpiTYPER platform. Recursively partitioned mixture modeling (RPMM) package in R was used to examine relationships between methylation clusters and reproductive factors, information available from in-person interviews. In addition, using fresh frozen tumor tissue from 50 patients treated at Roswell Park Cancer Institute, we performed RNA sequencing on samples with methylation data available from a prior study, and used Spearmen's correlation to compare methylation and gene expression of DMLs and DMRs. Results: In assessing average methylation by location relative to CpG-islands (CGIs), we found that CGIs and shores in ER- tumors were significantly hypomethylated compared to CGI and shores in ER+ tumors from AA women, but not in tumors from EA women. We also identified 410 DMLs (Δβ>0.10 & FDR<0.05) between AA and EA breast tumors (race-associated DMLs, raDMLs), the majority of which were unique to ER- tumors (n = 260) and hypomethylated in AAs. These loci were enriched for immune response genes and several cell adhesion and inflammatory pathways. RPMM showed that parity, but not breastfeeding or age at menarche, was significantly associated with methylation class of ER- breast tumors. Of the genes that had ER- specific raDMLs, FOXA1 and THSD4 DNA methylation was highly correlated with gene expression (rho=-0.80, rho=+0.87, respectively; pvalue< 2.2x10-16). Summary: ER- tumors from AA women, but not from EA women, exhibited global hypomethylation at CGI and shores compared to ER+ tumors and contained the majority of raDMLs, many of which were associated with genes involved in immune and inflammatory response. The FOXA1 gene, which encodes a pioneer factor previously implicated in suppressing the molecular phenotype of basal breast cancer cells, was found to be methylated and silenced in the majority of ER- tumors analyzed for expression, consistent with aggressiveness of these tumors. These results, and the novel finding of associations between parity and DNA methylation signature, bring us a step forward in understanding the heterogeneous population of ER- tumors and provide insight into mechanisms of racial disparities in breast cancer. Citation Format: Allyson C. Espinal, Dan Wang, Lara Sucheston-Campbell, Song Liu, Qiang Hu, Li Tang, Gary Zirpoli, Thaer Khoury, Song Yao, Kitaw Demissie, Elisa V. Bandera, Christine B. Ambrosone, Michael J. Higgins. Methylation differences in breast tumor DNA from African American and European women are predominant in estrogen receptor (ER) negative breast cancer and are associated with childbearing. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr B47.

PKC activation sensitizes basal-like breast cancer cell lines to Smac mimetics

There is a need for novel strategies to initiate cancer cell death. One approach is the use of Smac mimetics, which antagonize inhibitor of apoptosis proteins (IAPs). Recent studies have shown that combinations of Smac mimetics such as LBW242 or LCL161 in combination with chemotherapeutic agents increase cancer cell death. Here we show that the protein kinase C (PKC) activator TPA together with the Smac mimetic LBW242 induces cell death in two basal breast cancer cell lines (MDA-MB-468 and BT-549) that are resistant to Smac mimetic as single agent. Ten other LBW242-insensitive cancer cell lines were not influenced by the TPA+LBW242 combination. The TPA+LBW242 effect was suppressed by the PKC inhibitor GF109203X, indicating dependence on PKC enzymatic activity. The PKC effect was mediated via increased synthesis and release of TNFα, which can induce death in the presence of Smac mimetics. The cell death, coinciding with caspase-3 cleavage, was suppressed by caspase inhibition and preceded by the association of RIP1 with caspase-8, as seen in complex II formation. Smac mimetics, but not TPA, induced the non-canonical NF-κB pathway in both MDA-MB-231 and MDA-MB-468 cells. Blocking the canonical NF-κB pathway suppressed TPA induction of TNFα in MDA-MB-468 cells whereas isolated downregulation of either the canonical or non-canonical pathways did not abolish the Smac mimetic induction of the NF-κB driven genes TNFα and BIRC3 in MDA-MB-231 cells although the absolute levels were suppressed. A combined downregulation of the canonical and non-canonical pathways further suppressed TNFα levels and inhibited Smac mimetic-mediated cell death. Our data suggest that in certain basal breast cancer cell lines co-treatment of TPA with a Smac mimetic induces cell death highlighting the potential of using these pathways as molecular targets for basal-like breast cancers.

Histone deacetylase 9 regulates breast cancer cell proliferation and the response to histone deacetylase inhibitors.

Histone lysine acetylation is an epigenetic mark regulated by histone acetyltransferases and histone deacetylases (HDAC) which plays an important role in tumorigenesis. In this study, we observed a strong overexpression of class IIa HDAC9, at the mRNA and protein levels, in the most aggressive human breast cancer cell lines (i.e. in basal breast cancer cells vs luminal ones or in malignant vs begnin MCF10A breast epithelial cell lines). HDAC9 overexpression was associated with higher rates of gene transcription and increased epigenetic marks on the HDAC9 promoter. Ectopic expression of HDAC9 in MCF7 luminal breast cancer cells led to an increase in cell proliferation and to a decrease in apoptosis. These effects were associated with a deregulated expression of several genes controlled by HDAC inhibitors such as CDKN1A, BAX and TNFRSF10A. Inversely, knock-down of HDAC9 expression in MDA-MB436 basal breast cancer cells reduced cell proliferation. Moreover, high HDAC9 expression decreased the efficacy of HDAC inhibitors to reduce cell proliferation and to regulate CDKN1A gene expression. Interestingly, the gene encoding the transcription factor SOX9 was identified by a global transcriptomic approach as an HDAC9 target gene. In stably transfected MCF7 cells, SOX9 silencing significantly decreased HDAC9 mitogenic activity. Finally, in a large panel of breast cancer biopsies, HDAC9 expression was significantly increased in tumors of the basal subtype, correlated with SOX9 expression and associated with poor prognosis. Altogether, these results indicate that HDAC9 is a key factor involved in mammary carcinogenesis and in the response to HDAC inhibitors.

Abstract A19: CCR5 contributes to breast cancer stem cell expansion by enhancing DNA damage repair

Abstract Tumors are highly heterogeneous with a few cells capable of generating a new tumor referred to as cancer stem cells. Cancer stem cells contribute to metastasis and therapy resistance (chemotherapy, radiation). Therapy resistance in some circumstances is attributable to induction of the DNA repair machinery. Herein the role of endogenous CCR5 in cancer stem cells was assessed in the SUM159 basal breast cancer cell line. By FACS analysis, 1-10% of the SUM159 cells were CCR5+. CCR5+ cells formed more mammospheres, a surrogate measurement of progenitor cell expansion. CCR5+ SUM159 cells preferentially gave rise to tumors when injected subcutaneously. The tumors formed by CCR5+ cells were >40 times larger. When CCR5 was overexpressed in CCR5- cells and injected subcutaneously to the mice, the tumors were ten-thousand times larger than vector-control cells. In order to characterize the functional pathways regulated by CCR5, total RNA was prepared from both SUM159 CCR5+ and CCR5- cells isolated by FACS sorting. Microarray analysis showed that 321 genes were differently regulated in CCR5+ cells vs CCR5- cells. GOTERM_BP_FAT analysis revealed 18 Gene Ontology pathways were regulated by CCR5 including “DNA repair” and “response to DNA damage stimulus”. Genes elated to DNA repair (FANCB, LIG3, POLE and CRY1) were confirmed by qtPCR. Histone H2AX phosphorylation at Serine 139 (p-γH2AX) serves to recruit proteins that sense or signal the presence of DNA damage and can be used as surrogate marker of DNA damage/repair. SUM159 cells stably transfected with CCR5 or control vector were compared for the DNA damage/repair response. γ-radiation of SUM159 cells induced p-γH2AX, however CCR5-overexpressing cells showed reduced p-γH2AX at 24 hours. Treatment of SUM159 cells with the DNA intercalating anthracycline doxorubicin induced γH2AX phosphorylation, and CCR5-overexpressing cells showed less p-γH2AX than its control with 24 hours treatment. The DNA repair reporter, DR-GFP is used to measure homology-directed DNA repair (HDR). In order to measure HDR activity CCR5+ SUM159 cells, the cells were co-transfected with the plasmid encoding I-SceI and the I-SceI based DNA repair reporter DR-GFP and stained with APC labeled anti-CCR5 antibody. GFP+ cells, generated by HDR of I-SceI induced double-strand DNA, were sorted by FACS into CCR5- and CCR5+ populations. The percentage of DR-GFP+ cells was increased in CCR5+ or CCR5-overexpressing cells compared with CCR5- or vector control cells. Together these studies demonstrate that CCR5+ breast cancer cells possess cancer stem cell-like properties and have increased DNA damage repair activity. CCR5 promote stem cell expansion and breast cancer tumor formation through enhancing DNA damage repair. CCR5 antagonists were approved by the FDA for HIV treatment. These results suggest potential clinical value of these compounds for breast cancer metastasis management. Citation Format: Xuanmao Jiao, Marco Velasco, Zhiping Li, Shaohua Xu, Massimo Cristofanilli, Hallgeir Rui, Richard G. Pestell. CCR5 contributes to breast cancer stem cell expansion by enhancing DNA damage repair. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A19.

Investigation into local cell mechanics by atomic force microscopy mapping and optical tweezer vertical indentation

Investigating the mechanical properties of cells could reveal a potential source of label-free markers of cancer progression, based on measurable viscoelastic parameters. The Young’s modulus has proved to be the most thoroughly studied so far, however, even for the same cell type, the elastic modulus reported in different studies spans a wide range of values, mainly due to the application of different experimental conditions. This complicates the reliable use of elasticity for the mechanical phenotyping of cells. Here we combine two complementary techniques, atomic force microscopy (AFM) and optical tweezer microscopy (OTM), providing a comprehensive mechanical comparison of three human breast cell lines: normal myoepithelial (HBL-100), luminal breast cancer (MCF-7) and basal breast cancer (MDA-MB-231) cells. The elastic modulus was measured locally by AFM and OTM on single cells, using similar indentation approaches but different measurement parameters. Peak force tapping AFM was employed at nanonewton forces and high loading rates to draw a viscoelastic map of each cell and the results indicated that the region on top of the nucleus provided the most meaningful results. OTM was employed at those locations at piconewton forces and low loading rates, to measure the elastic modulus in a real elastic regime and rule out the contribution of viscous forces typical of AFM. When measured by either AFM or OTM, the cell lines’ elasticity trend was similar for the aggressive MDA-MB-231 cells, which were found to be significantly softer than the other two cell types in both measurements. However, when comparing HBL-100 and MCF-7 cells, we found significant differences only when using OTM.

ELK3 Expression Correlates With Cell Migration, Invasion, and Membrane Type 1-Matrix Metalloproteinase Expression in MDA-MB-231 Breast Cancer Cells.

ELK3 is a member of the Ets family of transcription factors. Its expression is associated with angiogenesis, vasculogenesis, and chondrogenesis. ELK3 inhibits endothelial migration and tube formation through the regulation of MT1-MMP transcription. This study assessed the function of ELK3 in breast cancer (BC) cells by comparing its expression between basal and luminal cells in silico and in vitro. In silico analysis showed that ELK3 expression was higher in the more aggressive basal BC cells than in luminal BC cells. Similarly, in vitro analysis showed that ELK3 mRNA and protein expression was higher in basal BC cells than in normal cells and luminal BC cells. To investigate whether ELK3 regulates basal cell migration or invasion, knockdown was achieved by siRNA in the basal BC cell line MDA-MB-231. Inhibition of ELK3 expression decreased cell migration and invasion and downregulated MT1-MMP, the expression of which is positively correlated with tumor cell invasion. In silico analysis revealed that ELK3 expression was associated with that of MT1-MMP in several BC cell lines (0.98 Pearson correlation coefficient). Though MT1-MMP expression was upregulated upon ELK3 nuclear translocation, ELK3 did not directly bind to the 1.3-kb promoter region of the MT1-MMP gene. These results suggest that ELK3 plays a positive role in the metastasis of BC cells by indirectly regulating MT1-MMP expression.

Bromoenol Lactone Attenuates Nicotine-Induced Breast Cancer Cell Proliferation and Migration.

ObjectivesCalcium independent group VIA phospholipase A2 (iPLA2β) and Matrix Metalloproteinase-9 (MMP-9) are upregulated in many disease states; their involvement with cancer cell migration has been a recent subject for study. Further, the molecular mechanisms mediating nicotine-induced breast cancer cell progression have not been fully investigated. This study aims to investigate whether iPLA2β mediates nicotine-induced breast cancer cell proliferation and migration through both in-vitro and in-vivo techniques. Subsequently, the ability of Bromoenol Lactone (BEL) to attenuate the severity of nicotine-induced breast cancer was examined.Method and ResultsWe found that BEL significantly attenuated both basal and nicotine-induced 4T1 breast cancer cell proliferation, via an MTT proliferation assay. Breast cancer cell migration was examined by both a scratch and transwell assay, in which, BEL was found to significantly decrease both basal and nicotine-induced migration. Additionally, nicotine-induced MMP-9 expression was found to be mediated in an iPLA2β dependent manner. These results suggest that iPLA2β plays a critical role in mediating both basal and nicotine-induced breast cancer cell proliferation and migration in-vitro. In an in-vivo mouse breast cancer model, BEL treatment was found to significantly reduce both basal (p<0.05) and nicotine-induced tumor growth (p<0.01). Immunohistochemical analysis showed BEL decreased nicotine-induced MMP-9, HIF-1alpha, and CD31 tumor tissue expression. Subsequently, BEL was observed to reduce nicotine-induced lung metastasis.ConclusionThe present study indicates that nicotine-induced migration is mediated by MMP-9 production in an iPLA2β dependent manner. Our data suggests that BEL is a possible chemotherapeutic agent as it was found to reduce both nicotine-induced breast cancer tumor growth and lung metastasis.

Iroquois homeobox 2 suppresses cellular motility and chemokine expression in breast cancer cells.

BackgroundDisseminated tumor cells (DTCs) can be detected using ultrasensitive immunocytochemical assays and their presence in the bone marrow can predict the subsequent occurrence of overt metastasis formation and metastatic relapse. Using expression profiling on early stage primary breast tumors, low IRX2 expression was previously shown to be associated with the presence of DTCs in the bone marrow, suggesting a possible role of IRX2 in the early steps of metastasis formation. The purpose of this study is to gain insights into the significance of IRX2 protein function in the progression of breast cancer.MethodsTo assess the physiological relevance of IRX2 in breast cancer, we evaluated IRX2 expression in a large breast cancer cohort (n = 1992). Additionally, constitutive IRX2 over expression was established in BT-549 and Hs578T breast cancer cell lines. Subsequently we analyzed whether IRX2 overexpression effects chemokine secretion and cellular motility of these cells.ResultsLow IRX2 mRNA expression was found to correlate with high tumor grade, positive lymph node status, negative hormone receptor status, and basal type of primary breast tumors. Also in cell lines low IRX2 expression was associated with mainly basal breast cancer cell lines. The functional studies show that overexpression of the IRX2 transcription factor in basal cell lines suppressed secretion of the pro-metastatic chemokines and inhibited cellular motility but did not influence cell proliferation.ConclusionOur results imply that the IRX2 transcription factor might represent a novel metastasis associated protein that acts as a negative regulator of cellular motility and as a repressor of chemokine expression. Loss of IRX2 expression could therefore contribute to early hematogenous dissemination of breast cancer by sustaining chemokine secretion and enabling mobilization of tumor cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1907-4) contains supplementary material, which is available to authorized users.

Abstract LB-207: ΔNp63α regulates quiescence, stem or progenitor activity of normal and malignant breast cells in a cell type-specific manner

Abstract [Introduction] Despite apparent resection of tumors, breast cancer patients often suffer relapse years after due to remnant dormant tumor cells. Nevertheless, the molecular mechanism regulating dormancy of breast cancer cell is still unclear. On the other hand, relapsed tumor is generally believed to be derived from cancer stem cells retaining normal stem cell property, which can undergo quiescence. ΔNp63α, an N-terminally truncated isoform of p51/p63 protein was shown to play crucial roles in the maintenance of stem cells within mammary epithelium. Furthermore, both tumor suppressive and oncogenic roles of ΔNp63α have been reported in mammary carcinogenesis. Thus, we investigated the role of ΔNp63α in the regulation of normal and malignant breast cells. [Method] MCF10A, normal human mammary epithelial cells and breast cancer cell lines of different subtypes were introduced with doxycycline-inducible constructs of ΔNp63α. These cells were subjected to cell cycle analysis, western blot, BrdU-Ki-67 immunostaining. Stemness was determined by mammosphere formation assay combined with flow cytometry analysis of cell surface markers. Drug response was studied using MTT assay. Also luminal breast cancer cell line, MCF7, was subjected to combined mRNA-miRNA microarray analysis. [Result] The induction of ΔNp63α in MCF7 luminal breast cancer cell line led the cells to acquire phenotype typical to quiescent luminal progenitor-like cells. Furthermore, the BMP and Wnt signaling pathways emerged as regulators of ΔNp63α-induced quiescence by microarray analysis. Interestingly, these quiescent cells exhibited down-regulation of BRCA1-dependent DNA repair pathways making them more sensitive to DNA damaging drug, Doxorubicin, but more resistant to anti-mitotic drug, Paclitaxel. Conversely, induction of ΔNp63α in MCF10A normal basal cells resulted in increased cell proliferation and expansion of mammary stem-like cells. ΔNp63α expression also led to the increase of cells expressing breast cancer stem cell markers in both MCF7 and MCF10A cells. However, induction of ΔNp63α affected neither cell proliferation nor the increase of mammary stem- or luminal progenitor-like cells in more aggressive luminal (T47D) and basal (MDA-MB-231) breast cancer cells containing mutant p53. This result interestingly suggested a cell type-specific function of ΔNp63α. We also identified a microRNA network that might regulate quiescence in ΔNp63α-expressing breast cancer cells which will be presented at the meeting in detail. [Conclusion] Our results suggest that the role of ΔNp63α in regulating quiescence, stem or progenitor activity in normal and breast cancer cells is cell type-specific. Given the heterogeneity in the cellular origin of breast cancer subtypes, ΔNp63α-directed chemotherapeutic strategy might be instructive to target therapeutically resistant breast cancer cells. Citation Format: Md. Ruhul Amin, Yuiko Morita-Fujimura, Shuntaro Ikawa. ΔNp63α regulates quiescence, stem or progenitor activity of normal and malignant breast cells in a cell type-specific manner. [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 LB-207. doi:10.1158/1538-7445.AM2015-LB-207