Claudin-low Breast Cancer Cells Research Articles

Systematic proteomics analysis revealed different expression of laminin interaction proteins in breast cancer: lower in luminal subtype and higher in claudin-low subtype.

Breast cancer is a major public health concern. Proteomics enables identification of proteins with aberrant properties. Here, we identified proteins with abnormal expression levels in breast cancer tissues and systematically analyzed and validated the data to locate potential diagnostic and therapeutic targets. Protein expression level in breast cancer tissues and para-carcinoma tissues were detected by Isobaric Tags for Relative and Absolute Quantification (iTRAQ) technology and further screened through Gene Expression Profiling Interactive Analysis (GEPIA) database. Cellular components, protein domain and Reactome pathway analysis were performed to screen functional targets. Abnormal expression levels of functional targets were validated by Oncomine database, quantitative real time polymerase chain reaction (qRT-PCR) and proteomics detection. Protein correlation analysis was performed to explain the abnormal expression levels of potential targets in breast cancer. Overall, 207 and 207 proteins were up- and down-regulated, respectively, in breast cancer tissues, and approximately 50% were also detected in the GEPIA database. The overlapping proteins were mainly extracellular proteins containing epidermal growth factor-like domain in leukocyte adhesion molecule (EGF-Lam) domain and enriched in laminin interaction pathway. Moreover, the downregulated laminin interaction proteins could be functional targets, which were also validated through Oncomine-Richardson and Oncomine-Curtis database. However, the lower expression level of laminin interaction proteins only fit for luminal breast cancer cells with no or low metastasis ability because the proteins achieved higher expression level in more invasive claudin-low breast cancer cells. In addition, when compared with corresponding in situ carcinoma tissues, above-mentioned proteins also showed higher expression levels in invasive carcinoma tissues. Finally, we have revealed the negative correlation between the laminin interaction proteins and the claudins. The laminin interaction protein, especially for laminins with β1 and γ1 subunits and their integrin receptors with α1 and α6 subunits, showed lower expression levels in luminal breast cancer with no or lower metastatic ability, but showed higher expression levels in claudin-low breast cancer with higher metastatic ability; and their higher expression could be related to the low claudin expression.

Abstract PO1-24-02: Enhancement of TGF-β Receptor Inhibitor Efficacy through CD44 Suppression in Claudin-low Breast Cancer

Abstract Introduction: Claudin-low breast cancer is mostly classified as triple-negative breast cancer and is known to possess cancer stem cell properties with high expression of epithelial-to-mesenchymal transition (EMT) markers. However, specific therapeutic targets for this subtype have not yet been identified. While reports suggest that inhibition of EMT using EMT inhibitors can suppress cell and tumor proliferation, monotherapy may not be sufficient. The expression of CD44, a surface marker of cancer stem cells, has been reported to be involved in multiple signaling pathways, promoting cancer growth and invasion. In this study, we focus on CD44 and the TGF-β receptor, which is involved in EMT, to investigate the effect of inhibiting TGF-β receptor through CD44 inhibition in Claudin-low breast cancer and validate its potential as an effective treatment. Methods: SUM159 and MDA-MB-231 cell lines were used as Claudin-low breast cancer cell models. CD44 knockdown cells were established to examine the effects of TGF-β receptor inhibition and downstream signaling. Results: CD44 knockdown did not affect the expression of TGF-β receptor. Both SUM159 and MDA-MB-231 cells showed comparable cell growth inhibition through CD44 knockdown and TGF-β receptor inhibition, with enhanced effects observed in CD44 knockdown cells in response to TGF-β receptor inhibition. Furthermore, CD44 knockdown combined with TGF-β receptor inhibition significantly reduced the expression of Smad2/3. Conclusion: In Claudin-low breast cancer cells, CD44 knockdown enhances the effectiveness of TGF-β receptor inhibition and significantly influences the expression level of Smad2/3. Citation Format: Ryoichi Matsunuma, Sae Imada, Shoko Sato, Ryosuke Hayami, Michiko Tsuneizumi. Enhancement of TGF-β Receptor Inhibitor Efficacy through CD44 Suppression in Claudin-low Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-02.

SP5.1 Paracrine Interferon signalling between cancer associated Fibroblasts and Cancer Cells correlates with poor survival after chemotherapy in Triple Negative Breast Cancer patients

Abstract Aim To determine if expression levels of Interferon-β1 (IFNβ1) in cancer associated fibroblasts, and of Myxovirus Resistance 1 (MX1) – a downstream marker of interferon activity, in cancer cells would be predictive markers of chemotherapy response and subsequent overall survival. Methods Breast cancer tissue was obtained from tissue microarrays of a triple negative cohort. Expression of IFNβ1 and MX1 was determined by immunohistochemistry in 109 patients treated with adjuvant chemotherapy. The cohort was further subdivided into claudin low and high subgroups, based on immunohistochemical assessment of relative expression of claudin-3. Results IFNβ1 in fibroblasts was significantly, positively associated with MX1 expression in tumour cells (Spearman’s correlation r=0.210; p=0.028). High expression of IFNβ1 in fibroblasts, and of MX1 in tumour cells were each significantly associated with poorer disease-free survival (p=0.01). Dividing the cohort into claudin-low (claudin-3 negative; n=49) and claudin-high (claudin-3 positive; n=60), resulted in a stronger correlation between fibroblast IFNβ1 and tumour cell MX1 in the claudin-low group (r=0.375; p=0.008), whereas it was lost in the claudin-high group (r=0.113; p=0.389). Likewise, correlations between survival and expression of IFNβ1 in fibroblasts and MX1 in tumour cells were maintained in claudin-low cases (p<0.05) but lost in claudin-highs. Conclusion IFNβ1 and MX1 may be of value as predictive biomarkers of chemo-response in breast cancer patients. These correlations imitate associations with chemoresistance identified in vitro using cancer associated fibroblasts and claudin-low breast cancer cell lines. Therefore, inhibiting the paracrine cross talk-between the cell types might be a viable strategy for chemo-sensitisation of breast cancers.

HER3 targeting augments the efficacy of panobinostat in claudin-low triple-negative breast cancer cells

Patients with triple-negative breast cancer (TNBC) have a poor prognosis and high relapse rate due to limited therapeutic options. This study was conducted to determine the mechanisms of action of panobinostat, a pan-inhibitor of histone deacetylase (HDAC) and FDA-approved medication for multiple myeloma, in TNBC and to provide a rationale for effective drug combinations against this aggressive disease. RNA sequencing analyses of the claudin-low (CL) TNBC (MDA-MB-231) cells untreated or treated with panobinostat were performed to identify the differentially expressed genes. Adaptive alterations in gene expression were analyzed and validated in additional CL TNBC cells. Tumor xenograft models were used to test the in vivo antitumor activity of panobinostat alone or its combinations with gefitinib, an EGFR-tyrosine kinase inhibitor (TKI). Panobinostat potently inhibited proliferation and induced apoptosis in all TNBC cells tested. However, in CL TNBC cells, this HDAC inhibitor markedly enhanced expression of HER3, which interacted with EGFR to activate both receptors and Akt signaling pathways. Combinations of panobinostat and gefitinib synergistically suppressed CL TNBC cell proliferation and promoted apoptosis in vitro and in vivo. Upregulation of HER3 compromises the efficacy of panobinostat in CL TNBC. Inactivation of HER3 combined with panobinostat represents a practical approach to combat CL TNBC.

OR30-3 Direct and VGLL1-Mediated Gene Regulation by GRHL2 During Mesenchymal to Epithelial Transition in Breast Cancer

Abstract The transcription factor (TF) GRHL2 controls normal epithelial differentiation in different tissues. It is expressed in all breast cancer subtypes except those with a mesenchymal phenotype (claudin-low subtype), where its ectopic expression leads to induction of a mesenchymal-epithelial transition (MET) (1). Here we explored the mechanisms by which GRHL2 induces MET in claudin-low breast cancer cell lines using a combination of ChIP-seq and RNAseq analyses upon modulation of GRHL2 expression in mesenchymal and epithelial cell lines. Direct DNA binding by GRHL2 was enriched in proximity of genes positively regulated in mesenchymal cell lines, which include several epithelial TFs as well as an array of epithelial genes. Biotinylation and mass spectrometry analysis of membrane proteins confirmed surfaceome remodeling via induction of membrane proteins with roles in cell-cell contacts and adhesion. Repression of mesenchymal genes was mediated in part by direct induction of miRNAs targeting mesenchymal transcription factors. In addition, GRHL2 strongly induced expression of the cofactor VGLL1, whose expression is high in the triple-negative (TNBC) basal-like subtype. VGLL1 overexpression in mesenchymal cell lines and suppression in basal-like TNBC cells confirmed its role in mediating part of GRHL2 signaling, in particular via interaction with and modulation of TEAD transcription factors. In addition, GRHL2 also interacted with VGLL1 upon its induction in mesenchymal cells, recruiting it to some of its binding motifs, suggesting a multilayered cross-talk between GRHL2 and TEAD factors. Together, our results indicate that GRHL2 acts as a master regulator in the induction of TNBC epithelial differentiation via direct upregulation of epithelial transcription factors, miRNAs and TEAD cofactor VGLL1, resulting in both upregulation of epithelial genes and suppression of mesenchymal genes.(1) Cieply B. et al., Cancer Res. 2013 Oct. 1;73 (20): 6299-309 Presentation: Tuesday, June 14, 2022 10:15 a.m. - 10:30 a.m.

Elevated Expression of miR-200c/141 in MDA-MB-231 Cells Suppresses MXRA8 Levels and Impairs Breast Cancer Growth and Metastasis In Vivo.

Breast cancer cells with mesenchymal characteristics, particularly the claudin-low subtype, express extremely low levels of miR-200s. Therefore, this study examined the functional impact of restoring miR-200 expression in a human claudin-low breast cancer cell line MDA-MB-231. MDA-MB-231 cells were stably transfected with a control vector (MDA-231EV) or the miR-200c/141 cluster (MDA-231c141). Injection of MDA-231c141 cells into the 4th mammary gland of NCG mice produced tumors that developed significantly slower than tumors produced by MDA-231EV cells. Spontaneous metastasis to the lungs was also significantly reduced in MDA-231c141 cells compared to MDA-231EV cells. RNA sequencing of MDA-231EV and MDA-231c141 tumors identified genes including MXRA8 as being downregulated in the MDA-231c141 tumors. MXRA8 was further investigated as elevated levels of MXRA8 were associated with reduced distant metastasis free survival in breast cancer patients. Quantitative RT-PCR and Western blotting confirmed that MXRA8 expression was significantly higher in mammary tumors induced by MDA-231EV cells compared to those induced by MDA-231c141 cells. In addition, MXRA8 protein was present at high levels in metastatic tumor cells found in the lungs. This is the first study to implicate MXRA8 in human breast cancer, and our data suggests that miR-200s inhibit growth and metastasis of claudin-low mammary tumor cells in vivo through downregulating MXRA8 expression.

Quantitative Proteomics Reveals Knockdown of CD44 Promotes Proliferation and Migration in Claudin-Low MDA-MB-231 and Hs 578T Breast Cancer Cell Lines.

CD44 is a transmembrane glycoprotein that can regulate the oncogenic process. This is known to be a marker of the claudin-low subtype of breast cancer, as well as a cancer stem cell marker. However, its functional regulatory roles are poorly understood in claudin-low breast cancer. To gain comprehensive insight into the function of CD44, we performed an in-depth tandem mass tag-based proteomic analysis of two claudin-low breast cancer cell lines (MDA-MB-231 and Hs 578T) transfected with CD44 siRNA. As a result, we observed that 2736 proteins were upregulated and 2172 proteins were downregulated in CD44-knockdown MDA-MB-231 cells. For Hs 578T CD44-knockdown cells, 412 proteins were upregulated and 443 were downregulated. Gene ontology and network analyses demonstrated that the suppression of this marker mediates significant functional alterations related to oncogenic cellular processes, including proliferation, metabolism, adhesion, and gene expression regulation. A functional study confirmed that CD44 knockdown inhibited proliferation by regulating the expression of genes related to cell cycle, translation, and transcription. Moreover, this promoted the expression of multiple cell adhesion-associated proteins and attenuated cancer cell migration. Finally, our proteomic study defines the landscape of the CD44-regulated proteome of claudin-low breast cancer cells, revealing changes that mediate cell proliferation and migration. Our proteomics data set has been deposited to the ProteomeXchange Consortium via the PRIDE repository with the data set identifier PXD015171.

A tetraprenylated benzophenone 7-epiclusianone induces cell cycle arrest at G1/S transition by modulating critical regulators of cell cycle in breast cancer cell lines

Breast cancer is a complex disease and encompassing different types of tumor. Although advances in understanding of the molecular bases of breast cancer biology, the therapeutic proposals available still are not effective. In this scenario, the present study aimed to evaluate the mechanisms associated to antitumor activity of 7-Epiclusianone (7-Epi), a tetraprenylated benzophenone, on luminal A (MCF-7) and claudin-low (Hs 578T) breast cancer cell lines. We found that 7-Epi efficiently inhibited cell proliferation and migration of these cells; however MCF-7 was slightly more responsive than Hs 578T. Cell cycle analysis showed accumulation of cells at G0/G1 phase with drastic reduction of S population in treated cultures. This effect was associated to downregulation of CDKN1A (p21) and cyclin E in both cell lines. In addition, 7-Epi reduced cyclin D1 and p-ERK expression levels in MCF-7 cell line. Cytotoxic effect of 7-Epi on breast cancer cell lines was associated to its ability to increase BAX/BCL-2 ratio. In conclusion, our findings showed that 7-Epi is a promising antitumor agent against breast cancer by modulating critical regulators of the cell cycle and apoptosis.

Targeting PIK3CG in Combination with Paclitaxel as a Potential Therapeutic Regimen in Claudin-Low Breast Cancer.

PurposeMolecular targeting is a powerful approach for aggressive claudin-low breast cancer (CLBC). Overexpression of PI3K catalytic subunit gamma (PIK3CG) in human CLBC is offering a promising opportunity for targeted therapies. We utilized a specific inhibitor of PIK3CG combined with paclitaxel (PTX) to treat CLBC cells in vitro and in vivo.Patients and MethodsThe tumor cells growth and apoptosis in vitro were analyzed by CCK8, plate clone formation assay, tumorsphere assay, Hoechst staining and flow cytometry. The invasion and metastasis ability of tumor cells in vitro were investigated by wound healing and transwell experiments. Critical gene expression levels were checked by qRT-PCR and Western blot. Xenograft models with CLBC cell lines in SCID mice were established to investigate the effect of combined drugs in vivo.ResultsWe identified that PIK3CG was a potential therapeutic target for CLBC patients. Targeting PIK3CG potentiated CLBC cells growth inhibition in 2D and 3D cultures by PTX. Inhibition of PIK3CG activation could enhance CLBC cells apoptosis and migration suppression induced by PTX. Manipulating autophagy was a validated approach for the use of PIK3CG inhibitor. Using CLBC xenograft mice model, we found that CLBC tumors in vivo could be well treated by combined drugs of PIK3CG inhibitor and PTX.ConclusionWe demonstrated that PIK3CG was a potential target for the therapy of CLBC and inhibition of PIK3CG activation could reinforce the therapeutic effect of this aggressive disease by PTX. The combined use of PIK3CG inhibitor and PTX might be a potential regimen for treating this subtype of breast cancer.

Metastasis inhibition by BRMS1 and miR-31 replacement therapy in claudin-low cell lines.

Objective(s):The growing trend of research demonstrates that dynamic expression of two metastasis repressor classes (metastasis suppressor genes and anti-metastatic miRNA) has a close relationship with tumor invasion and metastasis. Using different strategies, it was revealed that cellular levels of miR-31 and Breast cancer Metastasis Suppressor1 (BRMS1) protein, which are among the most significant modulators of metastasis, have a correlation with the cell’s capability for invading and metastasizing; cells containing higher levels of miR-31 or BRMS1 were less metastatic. This project was carried out to determine whether the combinations of miR-31 and BRMS1 genes are able to enhance the capability of repressing the claudin-low breast cancer cell (MDA-MB-231) invasion. Materials and Methods:This study used a restoration-based approach by miR-31 mimic and optimized BRMS1 gene sequences, which were cloned into a chimeric construct and transfected to the MDA-M231cells. Results:Our data revealed that the simultaneous expression of anti-metastasis miR and metastasis suppressor might inhibit migration and invasion in MDA-MB-231 cells efficiently.Conclusion:This combinatorial use of anti-metastatic miR and gene suggests a new therapeutic intervention for metastasis inhibition in MDA-MB-231.

Nup93 regulates breast tumor growth by modulating cell proliferation and actin cytoskeleton remodeling.

Nucleoporin 93 (Nup93) expression inversely correlates with the survival of triple-negative breast cancer patients. However, our knowledge of Nup93 function in breast cancer besides its role as structural component of the nuclear pore complex is not understood. Combination of functional assays and genetic analyses suggested that chromatin interaction of Nup93 partially modulates the expression of genes associated with actin cytoskeleton remodeling and epithelial to mesenchymal transition, resulting in impaired invasion of triple-negative, claudin-low breast cancer cells. Nup93 depletion induced stress fiber formation associated with reduced cell migration/proliferation and impaired expression of mesenchymal-like genes. Silencing LIMCH1, a gene responsible for actin cytoskeleton remodeling and up-regulated upon Nup93 depletion, partially restored the invasive phenotype of cancer cells. Loss of Nup93 led to significant defects in tumor establishment/propagation in vivo, whereas patient samples revealed that high Nup93 and low LIMCH1 expression correlate with late tumor stage. Our approach identified Nup93 as contributor of triple-negative, claudin-low breast cancer cell invasion and paves the way to study the role of nuclear envelope proteins during breast cancer tumorigenesis.

DOCK1 Regulates Growth and Motility through the RRP1B-Claudin-1 Pathway in Claudin-Low Breast Cancer Cells.

Dedicator of cytokinesis 1 (DOCK1) is a critical regulator of cancer metastasis. Claudins are transmembrane proteins that play a role in epithelial barrier integrity. Due to a loss or low expression of claudins (CLDN), the claudin-low type of triple-negative breast cancer (TNBC) is characterized by a mesenchymal-like phenotype with strong metastatic potential. In order to elucidate the mechanism of DOCK1 in cancer metastasis, we first analyzed the transcriptomic changes using a clinical database of human TNBC and found that the increase in DOCK1 expression was highly correlated with the poor survival rate of TNBC patients. Interference with DOCK1 expression by shRNA resulted in re-expression of claudin-1 in conjunction with significant inhibition of cell viability and motility of claudin-low breast cancer cells. Accordingly, overexpression of claudin-1 suppressed cell viability and migration. Genetic knockdown and pharmacological blockade of Rac1/Rac2 up-regulated claudin-1. DOCK1 knockdown also caused a decrease in DNA methyltransferase (DNMT) expression and an increase in claudin-1 transcript and promoter activity. Furthermore, RRP1B mediated DOCK1 depletion, which up-regulated claudin-1 expression, cell viability, and motility in claudin-low breast cancer cells. This study demonstrated that DOCK1 mediates growth and motility through down-regulated claudin-1 expression via the RRP1B–DNMT–claudin-1 pathway and that claudin-1 serves as an important effector in DOCK1-mediated cancer progression and metastasis in claudin-low breast cancer cells.

GALNT3 Maintains the Epithelial State in Trophoblast Stem Cells

SUMMARYO-GalNAc glycosylation is initiated in the Golgi by glycosyltransferases called GALNTs. Proteomic screens identified >600 O-GalNAc-modified proteins, but the biological relevance of these modifications has been difficult to determine. We have discovered a conserved function for GALNT3 in trophoblast stem (TS) cells, blastocyst trophectoderm, and human mammary epithelial cells (HMECs). The loss of GALNT3 expression in these systems reduces O-GalNAc glycosylation and induces epithelial-mesenchymal transition. Furthermore, Galnt3 expression is reduced in aggressive, mesenchymal claudin-low breast cancer cells. We show that GALNT3 expression controls the O-GalNAc glycosylation of multiple proteins, including E-cadherin in both TS cells and HMECs. The loss of GALNT3 results in the intracellular retention of E-cadherin in the Golgi. Significantly, re-expression of GALNT3 in TS cells increases O-GalNAc glycosylation and restores the epithelial state. Together, these data demonstrate the critical biological role of GALNT3 O-GalNAc glycosylation to promote the epithelial phenotype in TS cells, blastocyst trophectoderm, and HMECs.

Proteomic analysis defines kinase taxonomies specific for subtypes of breast cancer.

Multiplexed small molecule inhibitors covalently bound to Sepharose beads (MIBs) were used to capture functional kinases in luminal, HER2-enriched and triple negative (basal-like and claudin-low) breast cancer cell lines and tumors. Kinase MIB-binding profiles at baseline without perturbation proteomically distinguished the four breast cancer subtypes. Understudied kinases, whose disease associations and pharmacology are generally unexplored, were highly represented in MIB-binding taxonomies and are integrated into signaling subnetworks with kinases that have been previously well characterized in breast cancer. Computationally it was possible to define subtypes using profiles of less than 50 of the more than 300 kinases bound to MIBs that included understudied as well as metabolic and lipid kinases. Furthermore, analysis of MIB-binding profiles established potential functional annotations for these understudied kinases. Thus, comprehensive MIBs-based capture of kinases provides a unique proteomics-based method for integration of poorly characterized kinases of the understudied kinome into functional subnetworks in breast cancer cells and tumors that is not possible using genomic strategies. The MIB-binding profiles readily defined subtype-selective differential adaptive kinome reprogramming in response to targeted kinase inhibition, demonstrating how MIB profiles can be used in determining dynamic kinome changes that result in subtype selective phenotypic state changes.

Neutralization of IL-8 decreases tumor PMN-MDSCs and reduces mesenchymalization of claudin-low triple-negative breast cancer.

The complex signaling networks of the tumor microenvironment that facilitate tumor growth and progression toward metastatic disease are becoming a focus of potential therapeutic options. The chemokine IL-8 is overexpressed in multiple cancer types, including triple-negative breast cancer (TNBC), where it promotes the acquisition of mesenchymal features, stemness, resistance to therapies, and the recruitment of immune-suppressive cells to the tumor site. The present study explores the utility of a clinical-stage monoclonal antibody that neutralizes IL-8 (HuMax-IL8) as a potential therapeutic option for TNBC. HuMax-IL8 was shown to revert mesenchymalization in claudin-low TNBC models both in vitro and in vivo as well as to significantly decrease the recruitment of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) at the tumor site, an effect substantiated when used in combination with docetaxel. In addition, HuMax-IL8 enhanced the susceptibility of claudin-low breast cancer cells to immune-mediated lysis with NK and antigen-specific T cells in vitro. These results demonstrate the multifaceted way in which neutralizing this single chemokine reverts mesenchymalization, decreases recruitment of MDSCs at the tumor site, assists in immune-mediated killing, and forms the rationale for using HuMax-IL8 in combination with chemotherapy or immune-based therapies for the treatment of TNBC.

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.

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.