Claudin-low Breast Cancer Research Articles

Abstract 446: Differential effects of folate depletion on metabolic reprogramming and oxidative stress in nonmetastatic and metastatic claudin-low breast cancer cells

Abstract Folate coenzymes play an important role in biosynthesis and methylation reactions. Aberrant folate metabolism has been implicated in the development of several cancer types, though mechanisms underlying folate metabolism and cancer development remain unclear. We previously showed that a folate-restricted diet can exert differential effects on metastatic versus nonmetastatic murine claudin-low breast cancer cells in vivo. Specifically, folate restriction increased growth and invasion of orthotopically transplanted M-Wnt (nonmetastatic) tumor cells, but decreased growth and lung metastases of transplanted metM-Wnt cells, a metastatic subclone of M-Wnt cells. The current study set out to explore the underlying mechanism. To examine the effect of long-term folate depletion (LFD) on M-Wnt and metM-Wnt cell metabolism, oxidative stress and autophagic flux in vitro, the two cell lines were grown in standard and folate-depleted media for 14 days. metM-Wnt cells showed higher oxidative stress, as measured by ROS staining and Nrf2 expression, and phosphorylation of the key nutrient sensor, AMPK, compared to M-Wnt cells when grown in standard growth medium. LFD M-Wnt cells showed an increased dependence on glycolysis compared to those cultured in standard medium. Both M-Wnt and metM-Wnt cells displayed a high autophagy level in LFD, measured by LC3B cleavage, and AMPK phosphorylation. However, LFD metM-Wnt cells showed low viability, increased apoptosis and loss of redox defense, as measured by cleaved-caspase 3 and Nrf2 expressions. These results suggest that non-metastatic M-Wnt cells undergo metabolic reprogramming, including a shift from oxidative phosphorylation to glycolysis that may fuel cell growth and proliferation. Further, an elevated autophagic flux may mitigate nutrient stress induced by folate depletion, which allows them to withstand LFD and which may contribute to a more invasive primary tumor in response to folate restriction. In contrast, metM-Wnt cells are unable to undergo this metabolic adaptation, and display increased oxidative stress and cell death in response to LFD, preventing the development of metastatic lesions in vitro. This study highlights different responses of primary and metastatic breast cancer cells to folate depletion. The results provide additional rationale for targeting folate metabolism as a potential strategy for treating metastatic breast cancer. Citation Format: Xuewen Chen, Ciara H. O'Flanagan, Stephen D. Hursting. Differential effects of folate depletion on metabolic reprogramming and oxidative stress in nonmetastatic and metastatic claudin-low breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 446. doi:10.1158/1538-7445.AM2017-446

Glycoproteins in Claudin-Low Breast Cancer Cell Lines Have a Unique Expression Profile.

Claudin proteins are components of epithelial tight junctions; a subtype of breast cancer has been defined by the reduced expression of mRNA for claudins and other genes. Here, we characterize the expression of glycoproteins in breast cell lines for the claudin-low subtype using liquid chromatography/tandem mass spectrometry. Unsupervised clustering techniques reveal a group of claudin-low cell lines that is distinct from nonmalignant, basal, and luminal lines. The claudin-low cell lines express F11R, EPCAM, and other proteins at very low levels, whereas CD44 is expressed at a high level. Comparison of mRNA expression to glycoprotein expression shows modest correlation; the best agreement occurs when the mRNA expression level is lowest and little or no protein is detected. These findings from cell lines are compared to those for tumor samples by the Clinical Proteomic Tumor Analysis Consortium (CPTAC). The CPTAC samples contain a group low in CLDN3. The samples low in CLDN3 proteins share many differentially expressed glycoproteins with the claudin-low cell lines. In contrast to the situation for cell lines or patient samples classified as claudin-low by RNA expression, however, most of the tumor samples low in CLDN3 protein express the estrogen receptor or HER2. These tumor samples express CD44 protein at low rather than high levels. There is no correlation between CLDN3 gene expression and protein expression in these CPTAC samples; hence, the claudin-low subtype defined by gene expression is not the same group of tumors as that defined by low expression of CLDN3 protein.

Re-expression of miR-200c suppresses proliferation, colony formation and in vivo tumor growth of murine claudin-low mammary tumor cells.

Claudin-low breast cancer is a relatively rare breast cancer subtype. These cancers are typically ER−/PR−/HER2− and express high levels of mesenchymal genes as well as genes associated with inflammation, angiogenesis and stem cell function. In addition to alterations in gene expression, it was recently demonstrated that claudin-low breast cancers express very low levels of the miR-200 family of miRNAs. Given that each miRNA can regulate tens, hundreds or even thousands of genes, miRNAs are being evaluated as therapeutic targets. In this study we show that mammary tumors from MTB-IGFIR transgenic mice and cell lines derived from these tumors represent a model of human claudin-low breast cancer and murine claudin-low mammary tumors and cell lines express only very low levels of all five members of the miR-200 family. Reduced miR-200 family expression appears to be regulated via methylation as cells and tumors expressing low levels of miR-200 family members had higher levels of CpG methylation in a putative promoter region than tumors and cells expressing high levels of miR-200 family members. Re-expression of miR-200c in murine claudin-low mammary tumor cells inhibited tumor cell proliferation and colony formation in vitro and tumor growth in vivo. With respect to tumor growth in vivo, re-expression of miR-200c was associated with a reduction in tumor vasculature and expression of Flt1 and Vegfc. Therefore, miR-200c is an important regulator of mesenchymal tumor cell growth.

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.

Induced p53 loss in mouse luminal cells causes clonal expansion and development of mammary tumours

Most breast cancers may have a luminal origin. TP53 is one of the most frequently mutated genes in breast cancers. However, how p53 deficiency contributes to breast tumorigenesis from luminal cells remains elusive. Here we report that induced p53 loss in Krt8+ mammary luminal cells leads to their clonal expansion without directly affecting their luminal identity. All induced mice develop mammary tumours with 9qA1 (Yap1) and/or 6qA2 (Met) amplification(s). These tumours exhibit a mammary stem cell (MaSC)-like expression signature and most closely resemble claudin-low breast cancer. Thus, although p53 does not directly control the luminal fate, its loss facilitates acquisition of MaSC-like properties by luminal cells and predisposes them to development of mammary tumours with loss of luminal identity. Our data also suggest that claudin-low breast cancer can develop from luminal cells, possibly via a basal-like intermediate state, although further study using a different luminal promoter is needed to fully support this conclusion.

Metalloprotease-disintegrin ADAM12 actively promotes the stem cell-like phenotype in claudin-low breast cancer

BackgroundADAM12 is upregulated in human breast cancers and is a predictor of chemoresistance in estrogen receptor-negative tumors. ADAM12 is induced during epithelial-to-mesenchymal transition, a feature associated with claudin-low breast tumors, which are enriched in cancer stem cell (CSC) markers. It is currently unknown whether ADAM12 plays an active role in promoting the CSC phenotype in breast cancer cells.MethodsADAM12 expression was downregulated in representative claudin-low breast cancer cell lines, SUM159PT and Hs578T, using siRNA transfection or inducible shRNA expression. Cell characteristics commonly associated with the CSC phenotype in vitro (cell migration, invasion, anoikis resistance, mammosphere formation, ALDH activity, and expression of the CD44 and CD24 cell surface markers) and in vivo (tumor formation in mice using limiting dilution transplantation assays) were evaluated. RNA sequencing was performed to identify global gene expression changes after ADAM12 knockdown.ResultsWe found that sorted SUM159PT cell populations with high ADAM12 levels had elevated expression of CSC markers and an increased ability to form mammospheres. ADAM12 knockdown reduced cell migration and invasion, decreased anoikis resistance, and compromised mammosphere formation. ADAM12 knockdown also diminished ALDEFLUOR+ and CD44hi/CD24-/lo CSC-enriched populations in vitro and reduced tumorigenesis in mice in vivo. RNA sequencing identified a significant overlap between ADAM12- and Epidermal Growth Factor Receptor (EGFR)-regulated genes. Consequently, ADAM12 knockdown lowered the basal activation level of EGFR, and this effect was abolished by batimastat, a metalloproteinase inhibitor. Furthermore, incubation of cells with exogenously added EGF prevented the downregulation of CD44hi/CD24-/lo cell population by ADAM12 knockdown.ConclusionsThese results indicate that ADAM12 actively supports the CSC phenotype in claudin-low breast cancer cells via modulation of the EGFR pathway.

Abstract B47: Silver nanoparticles exhibit subtype specific cytotoxic and therapeutic effects in claudin low breast cancer in vitro and in vivo

Abstract Triple negative breast cancers (TNBC) are characterized by loss of expression of hormone receptors and decreased expression of the human epidermal growth factor receptor 2 (HER2). TNBC patients do not benefit from current targeted breast cancer (BC) treatments. Molecular profiling of breast cancer has found that TNBC is largely comprised of basal-like and claudin-low intrinsic molecular subtypes. Claudin-low breast cancer (CLBC) accounts for approximately one third of TNBCs, and early evidence suggests CLBC tumors may be more resistant to neoadjuvant anthracycline/taxane-based chemotherapy compared to basal-like tumors. For the development of novel breast cancer therapeutics, attention must be paid to therapeutic efficacy in specific sub-types of the disease. We discovered a type of silver nanoparticle (AgNP) that is selectively cytotoxic for treatment of CLBC. We find that the increased sensitivity of CLBC cells as compared to non-cancerous cells was independent of nanoparticle size, and CLBC cell lines (MDA-MB-231, BT-549, SUM-159) are more sensitive to AgNP exposure than luminal A BC (MCF-7), HER2 positive BC (SKBR3), and basal-like BC (MDA-MB-468, BT-20), or non-cancerous breast cells (MCF-10A, 184B5, HMT-3522 S1) via MTT assay. By treating CLBC cells and non-cancer breast cells with AgNPs or silver ions, in the form of silver nitrate, we demonstrated that intact AgNPs are necessary for selective cytotoxicity in CLBC. To determine the mechanism of cell death caused by AgNPs, annexin V (AnnV) and propidium iodide (PI) co-staining was performed on non-cancerous MCF-10A breast cells and MDA-MB-231 CLBC cells treated with AgNPs for 48 hours. AgNPs induced a dose-dependent increase in late-stage apoptosis and an increase in necrosis in MDA-MB-231 compared to vehicle control. Conversely, AgNPs had a minimal effect on late-stage apoptosis and necrosis in non-cancerous MCF-10A. Utilizing western blots, we showed that AgNPs induce oxidative damage to protein thiols and activate the unfolded protein response (UPR) in CLBC, but not in non-cancerous breast cells. To better recapitulate the tumor volume in an in vitro setting, multicellular tumor nodules from MDA-MB-231 TNBC cells or HMT-3522 S1 non-transformed mammary epithelial cells were formed by culturing the cells on basement membrane. When grown using 3D culture techniques, HMT-3522 S1 cells can develop growth arrested, polarized spherical structures containing lumens resembling a normal breast acinar structure characterized by a central lumen, cell-cell junctional complexes (including apical tight junctions, TJ), and basal expression of hemidesmosomal α6/β4 integrins ligating an endogenous basement membrane. 48h treatment with AgNPs did not prevent apical localization of the TJ marker ZO-1, alter the basal deposition of collagen-IV, induce proliferation of the growth arrested acini, or induce DNA damage. Conversely, significant amounts of DNA damage were observed in the MDA-MB-231 tumor nodules following 48h AgNP treatment. We conducted a 3 month in vivo safety/efficacy study in CLBC tumor-bearing mice where we showed that AgNPs can be delivered repeatedly at substantial doses intravenously and are effective for treatment of tumors without systemic toxicity. This is the first time any nanomaterial has been demonstrated subtype-specific therapeutic efficacy. This work demonstrates that AgNPs may provide a significant benefit for the CLBC patient population. Citation Format: Jessica L. Swanner, Iliana Tenvooren, Brian W. Bernish, Cale D. Fahrenholtz, Pierre A. Vidi, Katherine L. Cook, Ravi N. Singh. Silver nanoparticles exhibit subtype specific cytotoxic and therapeutic effects in claudin low breast cancer in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr B47.

Claudin-Low Breast Cancer; Clinical & Pathological Characteristics.

Claudin-low breast cancer is a molecular type of breast cancer originally identified by gene expression profiling and reportedly associated with poor survival. Claudin-low tumors have been recognised to preferentially display a triple-negative phenotype, however only a minority of triple-negative breast cancers are claudin-low. We sought to identify an immunohistochemical profile for claudin-low tumors that could facilitate their identification in formalin fixed paraffin embedded tumor material. First, an in silico collection of ~1600 human breast cancer expression profiles was assembled and all claudin-low tumors identified. Second, genes differentially expressed between claudin-low tumors and all other molecular subtypes of breast cancer were identified. Third, a number of these top differentially expressed genes were tested using immunohistochemistry for expression in a diverse panel of breast cancer cell lines to determine their specificity for claudin-low tumors. Finally, the immunohistochemical panel found to be most characteristic of claudin-low tumors was examined in a cohort of 942 formalin fixed paraffin embedded human breast cancers with >10 years clinical follow-up to evaluate the clinico-pathologic and survival characteristics of this tumor subtype. Using this approach we determined that claudin-low breast cancer is typically negative for ER, PR, HER2, claudin 3, claudin 4, claudin 7 and E-cadherin. Claudin-low tumors identified with this immunohistochemical panel, were associated with young age of onset, higher tumor grade, larger tumor size, extensive lymphocytic infiltrate and a circumscribed tumor margin. Patients with claudin-low tumors had a worse overall survival when compared to patients with luminal A type breast cancer. Interestingly, claudin-low tumors were associated with a low local recurrence rate following breast conserving therapy. In conclusion, a limited panel of antibodies can facilitate the identification of claudin-low tumors. Furthermore, claudin-low tumors identified in this manner display similar clinical, pathologic and survival characteristics to claudin-low tumors identified from fresh frozen tumor material using gene expression profiling.

Treg depletion potentiates checkpoint inhibition in claudin-low breast cancer

Treg depletion potentiates checkpoint inhibition in claudin-low breast cancer

Differential response to exercise in claudin-low breast cancer

Differential response to exercise in claudin-low breast cancer

Abstract 3689: Inactivation of p53 in mammary luminal cells leads to their clonal expansion and facilitates development of mammary tumors with loss of luminal identity

Abstract Mammary epithelium is hierarchically organized, with multipotent basal mammary stem cells (MaSCs) sit at the top of the hierarchy, giving rise to both luminal and basal mammary epithelial cells (MECs) during fetal development or upon transplantation to cleared mammary fat pad. Recent studies suggested that most breast cancers, including basal-like breast cancer, might originate from luminal cells, rather than from basal MaSCs, and that oncogenic events, such as ectopic expression of PIK3CA(H1047R) mutation, could induce multipotency in committed luminal cells, leading to acquisition of a MaSC-like phenotype by luminal MECs. TP53 is the most commonly mutated gene in human breast cancer; in particular, its inactivating mutations have been found in most basal-like and claudin-low breast cancer cases, raising a question as to whether TP53 loss-of-function mutations play a key role in dedifferentiation of committed luminal cells to multipotent MaSC-like cells. To study this, we induced disruption of Trp53 conditional knockout alleles in a small number of luminal MECs, either by transient Cre expression from intraductally injected Cre adenovirus under the control of the Keratin 8 (K8) promoter (Ad-K8-Cre virus) or by tamoxifen-induced transient activation of the CreER fusion driven by the same K8 promoter (K8-CreER mice). By in situ lineage-tracing, we found that induced loss of p53 in K8+ luminal cells led to their clonal expansion (by outcompeting their wild-type neighbors), due to increased cell cycle progression and impaired apoptosis control. However, p53-loss in luminal MECs did not directly affect their luminal identity, as determined by flow cytometry, immunostaining, and microarray expression profiling. Intriguingly, all induced female mice eventually developed mammary tumors. The majority of these tumors were large, fast growing claudin-low mammary tumors, based on histological and microarray analyses; a small number of these tumors exhibited features of basal-like mammary tumors. These data demonstrate that although p53 does not dictate the luminal MEC fate directly (i.e., loss of p53 does not directly lead to luminal-to-basal or mesenchymal transition), its inactivation facilitates loss of the luminal identity and predisposes luminal cells to development of claudin-low or basal-like breast tumors, thus in a way similar to the role of p53 in restricting reprogramming of somatic cells to induced pluripotent cells. Lastly, although it has been suggested that claudin-low breast cancer may originate from transformation of basal cells, our data support that upon p53-loss, claudin-low breast cancer can also have a luminal origin. Citation Format: Luwei Tao, Ying Xie, Zhe Li. Inactivation of p53 in mammary luminal cells leads to their clonal expansion and facilitates development of mammary tumors with loss of luminal identity. [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 3689.

Abstract 3661: ALDH1A3-inducible RARRES1 is a tumor suppressor in triple-negative breast cancer and is methylated in claudin-low breast cancers

Abstract The retinoic acid (RA) signalling pathway plays an important role in breast cancer progression and has either a pro-tumorigenic or tumor-suppressive role depending upon the effector function of RA-inducible genes that are expressed or epigenetically silenced. To study this paradigm in breast cancer, we focused on a controversial RA-inducible gene, the retinoic acid receptor responder 1 (RARRES1) protein that is often hypermethylated in cancer and has been reported to have tumor-suppressive function in prostate and nasopharyngeal carcinomas. However, in a study focused on a rare subtype of breast cancer, inflammatory breast cancer, RARRES1 is pro-tumorigenic. This functional discrepancy requires further investigation to determine its role in breast cancer in general. First, analysis of patient data sets revealed that RARRES1 is predominantly expressed in triple-negative breast cancers (TNBCs). Knockdown of RARRES1 in claudin-low MDA-MB-231 and basal-like MDA-MB-468 and HCC1937 significantly increased tumor growth and cell proliferation, suggesting RARRES1 has a tumor suppressive function in (TNBC), regardless of position on the differentiation hierarchy. Expression analyses of 24 breast cancer cell lines (including 18 TNBC and 2 normal-like cell lines) revealed that RARRES1 is predominantly expressed in basal-like TNBC cells We found that RARRES1 expression is dependent on, and strongly correlates with, the cancer stem cell marker, and RA-producing, ALDH1A3 in fixed breast cancer patient samples. Immunohistochemistry of the same patient tumor samples revealed RARRES1 expression is localized to the endoplasmic reticulum. Importantly, however, the presence of ALDH1A3 or RA is not sufficient to induce RARRES1 expression. RARRES1 is hypermethylated in claudin-low breast cancer cell lines, and release of this silencing is required for full induction of RARRES1 expression. We have identified sites of regulation by methylation in RARRES1 using Illumina 450K methylation arrays and 5-methylcytosine ChIP. We conclude that RARRES1 is an ALDH1A3/RA-inducible tumor suppressor in TNBC with methylation and expression profiles distinct to the differentiation hierarchy observed in breast cancer. Citation Format: Krysta M. Coyle, Patrick Murphy, Dejan Vidovic, Cheryl A. Dean, Margaret L. Thomas, Derek Clements, Mohammad Sultan, Ahmad Vaghar-Kashani, Carman Giacomantonio, Lucy Helyer, Ian Weaver, Shashi Gujar, Patrick WK Lee, Paola Marcato. ALDH1A3-inducible RARRES1 is a tumor suppressor in triple-negative breast cancer and is methylated in claudin-low breast cancers. [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 3661.

Abstract 1020: Autophagy forms part of a metabolic switch during epithelial-to-mesenchymal transition and metastasis in a murine claudin-low breast cancer model

Abstract Epithelial-to-mesenchymal transition (EMT), the process through which epithelial cells gain mesenchymal characteristics including reduced adhesion and enhanced invasion, is a critical step in progression of cancer cells toward metastasis. However, the molecular mechanisms governing this process remain poorly understood. Furthermore, though metastasis is the cause of 90% of cancer deaths, no targeted therapy is currently available for metastatic disease. Metabolic reprogramming is a key feature of most cancer cells, with oxidative phosphorylation often being switched off in favor of glycolytic and synthetic pathways. Autophagy is a catabolic process in which protein complexes, damaged organelles and other macromolecules are lysosomally degraded. Autophagy can promote cancer cell survival, providing protein, lipid, nucleic acid and membrane precursors as well as auxiliary energy during periods of energy stress. The role of metabolism in EMT and metastasis has not been well studied. We hypothesized that the transition to metastasis involves metabolic alterations, including activation of autophagy. Previously, we derived (from a spontaneous mammary tumor in an MMTV-Wnt transgenic mouse) epithelial-like (E-Wnt) and mesenchymal-like (M-Wnt) cell lines, which recapitulate basal-like and claudin-low and breast tumors, respectively. Here, a metastatic derivative of M-Wnt cells was generated from lung metastases following serial passages in a severe combined immunodeficient (SCID) mouse. metM-Wnt cells were more proliferative, invasive and formed more colonies in soft agar than their parental M-Wnt cell line. Tail vein injection or mammary fat pad orthotopic implantation of metM-Wnt cells resulted in metastatic tumor formation in either the lungs or liver as detected by IVIS imaging and histological analysis. metM-Wnt cells displayed higher rates of glycolysis and oxidative phosphorylation, indicating that these cells are highly energetic. Furthermore, M-Wnt and metM-Wnt cells and tumors were found to have increased autophagic flux, as measured by LC3B expression and cleavage compared to E-Wnt cells and tumors. metM-Wnt cells were more sensitive to autophagy inhibition (either via chloroquine treatment or knockout of Atg5, a key component of the autophagic machinery) than M-Wnt or E-Wnt cells. Conversely, metM-Wnt cells were resistant to treatment with rapamycin, concomitant with sustained activation of mTOR, which controls many metabolic pathways including autophagy and protein synthesis. These results indicate that metabolic alteration is a feature of EMT in claudin-low breast cancer, including increased glycolysis, oxidative phosphorylation and autophagy. Furthermore, metastatic breast cancer cells may be more reliant on autophagy than non-metastatic cells, and autophagy may therefore be a therapeutic target in which to treat metastatic breast cancers. Citation Format: Ciara H. O’Flanagan, Emily L. Rossi, Stephen D. Hursting. Autophagy forms part of a metabolic switch during epithelial-to-mesenchymal transition and metastasis in a murine claudin-low breast cancer model. [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 1020.

Abstract 678: Lunatic Fringe and p53 cooperatively prevent development of claudin-low breast cancer

Abstract Claudin-low breast cancer (CLBC) is a poor prognosis disease that disproportionately affects younger patients and women with African ancestry, causing significant morbidity and mortality due to chemoresistance and lack of targeted therapy. CLBC shows stemness and mesenchymal features, and is thought to have originated from mammary stem cells. However, little is known on how these tumors initiate and progress. We previously reported that deletion of Lunatic Fringe (Lfng), a modulator of Notch signaling, in the moue mammary epithelial cells induced development of mammary tumors, among them two-thirds are basal-like breast cancer and one-third resemble human CLBC. Deletion of p53 in combination with other mutations (inactivation of Rb or overexpression of oncogenic c-Met) also resulted in mammary tumors with mesenchymal features. We hypothesize that Lfng and p53 may cooperate to suppress CLBC initiation and progression. To this end, we have generated mammary-specific deletion of Lfng and p53 using MMTV-Cre. The vast majority of Lfngflox/flox;p53flox/flox;MMTV-Cre mice succumbed to lymphoma by 3-5 months of age before mammary tumor onset, likely due to the leaky deletion in B- and T cells. Interestingly, deletion of both copies of Lfng and one copy of p53 (Lfngflox/flox;p53flox/+;MMTV-Cre) dramatically accelerated mammary tumor development compared to the Lfngflox/flox;MMTV-Cre model, with the median tumor-free survival shortened from 600 days to 191 days. Of note, all mammary tumors from the Lfngflox/flox;p53flox/+;MMTV-Cre mice were consisted of mesenchymal/spindle-shaped cells and expressed markers of epithelial-mesenchymal transition (EMT), suggesting that deletion of one copy of p53 caused a shift of tumor subtype from basal-like to claudin-low. We found that deletion of Lfng caused up-regulation of E-cadherin in the mammary gland, whereas additional deletion of one copy of p53 decreased E-cadherin but dramatically increased vimentin levels, suggesting that p53 haploinsufficiency promotes EMT in this context. Compared with the wild type gland at 3 months of age, Lfngflox/flox;p53flox/+;MMTV-Cre mutant gland showed down-regulation of Notch downstream targets including Hes5, Hey1, and Hey2, suggesting reduced Notch signaling. Given that Notch signaling is known to restrict mammary stem cell expansion, down-regulation of Notch signaling may lead to inappropriate accumulation of mammary stem cells during CLBC initiation. Taken together, our results reveal genetic interaction between Notch and p53 in the pathogenesis of CLBC. Moreover, we have established a new mouse model for CLBC with complete penetrance and accelerated tumor onset, providing an ideal tool in defining molecular events leading to the initiation and progression of CLBC as well as identifying potential therapeutic targets for this poor prognosis disease. Citation Format: Wen-Cheng Chung, Shubing Zhang, Keli Xu. Lunatic Fringe and p53 cooperatively prevent development of claudin-low breast cancer. [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 678.

Induction of HOXA9 expression in three-dimensional organotypic culture of the Claudin-low breast cancer cells.

The gene expression signatures of the molecular intrinsic subtypes of breast cancer are regulated by epigenetic mechanisms such as methylation of CpG islands in gene promoters. Epigenetic codes can be regulated by the tumor microenvironment. The Claudin-low subtype is associated with triple-negative invasive ductal carcinomas in patients. Herein we explored epigenetic regulation of gene expression in the Claudin-low breast cancer cells by extracellular matrix (ECM), a key component of the tumor microenvironment. We modeled attachment to ECM using laminin rich ECM three-dimensional organotypic culture (lrECM 3D). In 2D and lrECM 3D cultures we examined expression of the homeobox (HOX) genes that epigenetically regulated in development and cancer. We demonstrated induction of the selected HOX genes in lrECM 3D culture of the Claudin-low breast cancer cells MDA-MB-231 and Hs578T. In particular activation of HOXA9 expression in lrECM 3D culture required binding of bromodomain containing 4 to the HOXA9 promoter and involved CpG hypomethylation. Our findings warrant further investigation of the ECM-regulated epigenetic coding of gene expression in the Claudin-low breast cancer.

Osteopontin regulates proliferation, apoptosis, and migration of murine claudin-low mammary tumor cells.

BackgroundOsteopontin is a secreted phosphoglycoprotein that is expressed by a number of normal cells as well as a variety of tumor cells. With respect to breast cancer, osteopontin has been implicated in regulating tumor cell proliferation and migration/metastasis and may serve as a prognostic indicator. However it remains unclear whether osteopontin has the same impact in all breast cancer subtypes and in particular, osteopontin’s effects in claudin-low breast cancer are poorly understood.MethodscDNA microarrays and qRT-PCR were used to evaluate osteopontin expression in mammary tumors from MTB-IGFIR transgenic mice and cell lines derived from these tumors. siRNA was then used to determine the impact of osteopontin knockdown on proliferation, apoptosis and migration in vitro in two murine claudin-low cell lines as well as identify the receptor mediating osteopontin’s physiologic effects.ResultsOsteopontin was expressed at high levels in mammary tumors derived from MTB-IGFIR transgenic mice compared to normal mammary tissue. Evaluation of cell lines derived from different mammary tumors revealed that mammary tumor cells with claudin-low characteristic expressed high levels of osteopontin whereas mammary tumor cells with mixed luminal and basal-like features expressed lower levels of osteopontin. Reduction of osteopontin levels using siRNA significantly reduced proliferation and migration while increasing apoptosis in the claudin-low cell lines. Osteopontin’s effect appear to be mediated through a receptor containing ITGAV and not through CD44.ConclusionsOur data suggests that mammary tumors with a mixed luminal/basal-like phenotype express high levels of osteopontin however this osteopontin appears to be largely produced by non-tumor cells in the tumor microenvironment. In contrast tumor cells with claudin-low characteristics express high levels of osteopontin and a reduction of osteopontin in these cells impaired proliferation, survival and migration.

A high capacity polymeric micelle of paclitaxel: Implication of high dose drug therapy to safety and invivo anti-cancer activity.

The poor solubility of paclitaxel (PTX), the commercially most successful anticancer drug, has long been hampering the development of suitable formulations. Here, we present translational evaluation of a nanoformulation of PTX, which is characterized by a facile preparation, extraordinary high drug loading of 50% wt. and PTX solubility of up to 45g/L, excellent shelf stability and controllable, sub-100nm size. We observe favorable invitro and invivo safety profiles and a higher maximum tolerated dose compared to clinically approved formulations. Pharmacokinetic analysis reveals that the higher dose administered leads to a higher exposure of the tumor to PTX. As a result, we observed improved therapeutic outcome in orthotopic tumor models including particularly faithful and aggressive "T11" mouse claudin-low breast cancer orthotopic, syngeneic transplants. The promising preclinical data on the presented PTX nanoformulation showcase the need to investigate new excipients and is a robust basis to translate into clinical trials.

Snail-induced epithelial-to-mesenchymal transition of MCF-7 breast cancer cells: systems analysis of molecular changes and their effect on radiation and drug sensitivity.

BackgroundEpithelial-to-mesenchymal transition (EMT) has been associated with the acquisition of metastatic potential and the resistance of cancer cells to therapeutic treatments. MCF-7 breast cancer cells engineered to constitutively express the zinc-finger transcriptional repressor gene Snail (MCF-7-Snail cells) have been previously shown to display morphological and molecular changes characteristic of EMT. We report here the results of a comprehensive systems level molecular analysis of changes in global patterns of gene expression and levels of glutathione and reactive oxygen species (ROS) in MCF-7-Snail cells and the consequence of these changes on the sensitivity of cells to radiation treatment and therapeutic drugs.MethodsSnail-induced changes in global patterns of gene expression were identified by microarray profiling using the Affymetrix platform (U133 Plus 2.0). The resulting data were processed and analyzed by a variety of system level analytical methods. Levels of ROS and glutathione (GSH) were determined by fluorescent and luminescence assays, and nuclear levels of NF-κB protein were determined by an ELISA based method. The sensitivity of cells to ionizing radiation and anticancer drugs was determined using a resazurin-based cell cytotoxicity assay.ResultsConstitutive ectopic expression of Snail in epithelial-like, luminal A-type MCF-7 cells induced significant changes in the expression of >7600 genes including gene and miRNA regulators of EMT. Mesenchymal-like MCF-7-Snail cells acquired molecular profiles characteristic of triple-negative, claudin-low breast cancer cells, and displayed increased sensitivity to radiation treatment, and increased, decreased or no change in sensitivity to a variety of anticancer drugs. Elevated ROS levels in MCF-7-Snail cells were unexpectedly not positively correlated with NF-κB activity.ConclusionsEctopic expression of Snail in MCF-7 cells resulted in morphological and molecular changes previously associated with EMT. The results underscore the complexity and cell-type dependent nature of the EMT process and indicate that EMT is not necessarily predictive of decreased resistance to radiation and drug-based therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2274-5) contains supplementary material, which is available to authorized users.

Abstract A18: Expression of the tumor suppressor gene RARRES1 in the differentiation hierarchy of breast cancer is regulated by DNA methylation

Abstract The tumor-suppressive function of the retinoic acid (RA)-inducible gene, retinoic acid receptor responder 1 (RARRES1), has been reported in many cancer types; however, in inflammatory breast cancer, RARRES1 is pro-tumorigenic. Furthermore, high transcript levels of RARRES1 are associated with more aggressive triple-negative breast cancers (TNBCs) and poorer patient outcomes. This suggests that RARRES1 may also be oncogenic and requires investigation to determine its role in breast cancer. Expression analyses of 20 breast cancer cell lines (including 18 TNBC and two estrogen-receptor-positive cell lines) revealed that RARRES1 is predominantly expressed in basal-like breast cancer cells, but is often methylated and silenced in claudin-low breast cancer cells. We have identified possible sites of regulation by methylation in RARRES1 using Illumina 450K methylation arrays and 5-methylcytosine ChIP. Basal-like cells express higher levels of the cancer stem cell (CSC) marker ALDH1A3. Expression of RARRES1 is dependent on, and strongly correlates with ALDH1A3 expression in fixed breast cancer patient samples. Immunohistochemistry of the same patient tumor samples revealed RARRES1 expression is localized to the endoplasmic reticulum. Finally, knockdown of RARRES1 in claudin-low MDA-MB-231 and basal-like MDA-MB-468 and HCC1937 significantly increased cell proliferation and tumor growth, suggesting RARRES1 has a tumor suppressive function regardless of position on the differentiation hierarchy. We conclude that RARRES1 is a tumor suppressor in TNBC with methylation and expression profiles distinct to the differentiation hierarchy observed in breast cancer. Citation Format: Krysta Mila Coyle, Dejan Vidovic, Cheryl A. Dean, Margaret Lois Thomas, Mohammad Sultan, Derek Clements, Ahmad Vaghar-Kashani, Melissa Wallace, Ian Weaver, Carman A. Giacomantonio, Lucy Helyer, Paola Marcato. Expression of the tumor suppressor gene RARRES1 in the differentiation hierarchy of breast cancer is regulated by DNA methylation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A18.

Abstract B24: Triple-negative breast cancer adaptive response to MEK inhibition is regulated by the induction of super-enhancers

Abstract A predominant mechanism by which cancer cells circumvent the action of targeted kinase inhibitors is the activation of bypass signaling networks that reactivate cellular proliferation. In triple negative breast cancer (TNBC), a chief component of the adaptive response to MEK inhibition is the transcriptional upregulation and activation of receptor tyrosine kinases (RTKs), which contributes to drug resistance by reactivating ERK signaling as well as by initiating growth signaling through other kinase nodes. The cohort of RTKs activated differs between TNBC molecular subtypes and is heterogeneous even among cell lines of the same subtype; thus designing effective inhibitor combinations is challenging. Targeting the chromatin reader protein BRD4 via BET bromodomain inhibition attenuates the transcriptional adaptive response to MEK inhibition, yielding a more durable growth suppression than kinase inhibitor combinations. Yet, a detailed understanding of molecular mechanisms underlying BET bromodomain inhibitor synergy with MEK inhibitor remain unclear. We show by ChIP-seq the dynamic formation of BRD4-dense super-enhancers in response to 24 h MEK inhibition. In SUM159PT claudin-low breast cancer cells, de novo super-enhancers, whose identity were validated by histone H3 lysine 27 acetylation and MED1 co-occupancy with BRD4, were found proximal to genomic loci of DDR1, KDR, and MYLK kinases previously implicated in the adaptive MEK inhibitor response. In sum we identified 110 putative MEK inhibitor-induced SUM159PT super-enhancers, 40% of which reside within 100 kilobases 5' or 3' of a gene whose transcript is upregulated 2 fold or greater by MEK inhibition as assessed by RNA-seq, suggesting that de novo superenhancer formation is a general mode of transcriptional regulation of the adaptive response. Importantly, the MEK inhibitor-induced super-enhancers are disrupted by the BET bromodomain inhibitor JQ1, providing one potential mechanism for the growth suppression synergism obtained by combining MEK + BET bromodomain inhibition. To identify candidate direct DNA binding factors that may be seeding the observed super-enhancer formation, Multiple EM for Motif Elicitation (MEME) analysis identified an enrichment of consensus DNA binding motifs for the enhancer factors CEBPB/D in de novo super-enhancer regions, and the presence of CEBPB was confirmed to be a component of these regions by ChIP-seq. Additionally, we have achieved MEK inhibitor growth suppression synergy with inhibitors of the lysine acetyl transferases CBP/p300, known super-enhancer components. These data provide a new mechanism underlying the transcriptional control of the adaptive response to MEK inhibitor and suggest that targeting drug-induced super-enhancer elements may be a potential therapeutic avenue for TNBC. Citation Format: Jon S. Zawistowski, Samantha M. Miller, Daniel R. Goulet, Timothy J. Stuhlmiller, Darshan Singh, Noah Sciaky, Sara H. Velarde, Steven P. Angus, Gary L. Johnson. Triple-negative breast cancer adaptive response to MEK inhibition is regulated by the induction of super-enhancers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B24.