Subtype-specific divergent roles of calpain-1 and calpain-2 in basal A triple-negative breast cancer

<p>Breast cancer is currently the second most common form of cancer and the second leading cause of death due to cancer in the United States. Breast cancer itself is subdivided into at least four subtypes, luminal A, luminal B, HER2-enriched, and basal-like, based on genomewide molecular expression patterns. Luminal A is the most common form and typically characterized by high levels of estrogen receptor (ER). HER2-enriched cancers usually, but not always, harbor amplified copies of the HER2 oncogene. Luminal B cancers share characteristics with the luminal A and HER2-enriched subtypes. Finally, basal-like cancers are more oftentimes defined by their lack of any markers or molecular targets. Thus, they are often called triple-negative breast cancer. Recent evidence suggests that there are a number transcription factors that play critical roles in the cancer progression of these malignancies. Indeed, TFAP2C has been clearly shown to positively regulate ER in luminal A cancers. Alternatively, TFAP2A appears to play an interesting, but as of yet incompletely, understood role in basal-like cancer. There has been additional evidence that suggests TFAP2C regulates multiple members of the ErbB family of receptor tyrosine kinases. Thus, we hypothesize that the TFAP2 family of transcription factors play a critical role in breast cancer progression. More specifically, we will show that TFAP2A and TFAP2C not only regulate a few critical genes in luminal and basal-like cancer, but instead are responsible for the genomewide expression pattern of these two breast cancer subtypes. Moreover, we argue that TFAP2C's regulation of certain receptor tyrosine kinases in luminal A cancers indicates promising therapeutic targets, particularly with small molecule inhibitors that are already FDA-approved. In addition, we provide data suggesting that TFAP2C likely plays an oncogenic role in HER2-positive breast cancer, possibly through the regulation of certain members of the ErbB family of receptor tyrosine kinases, such as EGFR. To address these points, we use a combination of genetically engineered mouse models, xenografts, siRNA mediated knockdown technology, western blot, qPCR, and number of additional molecular biological techniques. These results will not only establish the family of TFAP2 family of proteins as critical regulators of cancer progression, but our findings will specify how and to what extent each subtype of breast cancer is affected by individual members of the TFAP2 family of transcription factors.</p>

Background: Although Del-1 was recently proposed as a new biomarker for early breast cancer in our previous studies, the mechanisms of Del-1 expression are barely understood. In the current study, we selected two microRNAs (miR-137 and - 496), potentially affecting Del-1 expression in breast cancer and examined their impact on Del-1 expression in a variety of breast cancer cell lines to identify their potential role in Del-1 expression and thereby breast cancer development or progression. Methods: Del-1 mRNA and miR-137/- 496 levels were measured by qRT-PCR among breast epithelial (MCF10A) and cancer cells (MDA-MB-231, MCF7, SK-BR3 and T-47D). The effects of miR-137/- 496 on cell proliferation and invasion were detected using MTT, wound healing and Transwell assays. Furthermore, luciferase reporter assay was used to identify the direct regulation of Del-1 by miR-137 or - 496 in MDA-MB-231 cells. Plus, we analyzed the expressions of miR-137 or - 496 and Del-1 mRNA from 20 patients with triple negative early breast cancer. Results: miR-137 and - 496 levels were low in all breast cancer cell lines. As Del-1 mRNA expression was remarkably higher in MDA-MB-231 compared to the other breast cancer cell lines, further functional analyses were done with MDA-MB-231 representing triple negative breast cancer subtype. Both miR-137 and miR-496 were revealed to directly bind at the 3’-UTR of Del-1. Del-1 by Luciferase reporter assay and Del-1 expression was upregulated by inhibitors and reversed by both mimics of both miR-137 and miR-496. Furthermore, both miR-137 and miR-496 were also demonstrated to inhibit cell proliferation, migration and invasion of MDA-MB-231, suggesting that these miRNAs affect cancer progression via Del-1. MiR-137 and miR-496 were remarkably down-regulated in 7 out of 12 triple negative breast cancer tissues, in particular with high Ki67 and high histologic grade. Conclusion: Although Del-1 was recently introduced as a new biomarker for triple negative breast cancer, the mechanisms of Del-1 expression were barely identified. The current study firstly demonstrated that microRNA 137 and 496 are involved in Del-1 regulation by binding at Del-1 gene, affecting cancer progression by altering Del-1 expression. Citation Format: Jeeyeon Lee, Yee Soo Chae, Soo Jung Lee, Jin Hyang Jung, Ho Yong Park, Moon-Chang Baek. MiR-137 and MiR-496 target Del-1 and affect triple negative breast cancer progression [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 2797. doi:10.1158/1538-7445.AM2017-2797

Inflammation and immune response play a key role in triple-negative breast cancer (TNBC) progression and metastasis. Previous studies have shown that CCL2 plays a significant role in breast cancer cell signaling and can increase cell proliferation and differentiation, immune suppression, epithelial-to-mesenchymal transition, and angiogenesis. CCL2 expression is modulated via mitogen-activated protein kinase (MAPK) and nuclear factor-kappa beta (NFĶB) signaling pathways. Compounds that can inhibit the release of CCL2 have been shown to prevent cancer-associated inflammatory and pro-oncogenic processes. Diallyl Trisulfide (DATS), an organosulfide found in garlic, has been previously reported to be effective in chemoprevention, apoptosis, and cell cycle arrest in DNA damaged normal breast epithelial cells, breast cancer cells, as well as other cancers. The current study explored the effect of DATS on the genetically different TNBC MDA-MB-231 and MDA-468 cells, stimulated by TNF-α. Cytokine arrays, ELISA, and RT-PCR using individual primers were used. DATS downregulated CCL2 protein expression in MDA-MB-231, but not in MDA-MB-468 cells. These results were confirmed with ELISA analysis that showed a reduction in the expression of CCL2 in the TNF-α stimulated MDA-MB-231 cells treated with DATS. The data also showed that DATS treatment reduced by 50% the mRNA expression of CCL2 in MDA-MB-231cells after a 24-h period. To study a possible mechanism for DATS inhibitory effect over CCL2 expression, this compound's effect on the mRNA expression of IKBKE and MAPK8, which belong to the NFĶB and MAPK signaling pathways, respectively, was also investigated. DATS downregulated both genes in the TNF-α stimulated MDA-MB-231 and MDA-MB-468 cells. In conclusion, this study showed that DATS could downregulate CCL2 expression in the protein and transcription level in MDA-MB-231 cells. A possible mechanism for DATS inhibitory effect may involve the downregulation of IKBKE and MAPK8 mRNA expression. IKBKE gene was reported to be overexpressed in approximately 30% of human breast tumors and promotes cytokine release and pro-survival signaling through the activation of NFĶB and JAK-STAT pathways. Our findings demonstrate that DATS may have potential in breast cancer treatment, slowing down cancer progression by the downregulation of CCL2 expression and that genetically different TNBC cells may respond differently to DATS treatment.

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.

e23058 Background: Although Del-1 was recently proposed as a new biomarker for early breast cancer in our previous studies, the mechanisms of Del-1 expression are barely understood. In the current study, we selected two microRNAs (miR-137 and - 496), potentially affecting Del-1 expression in breast cancer and examined their impact on Del-1 expression in a variety of breast cancer cell lines to identify their potential role in Del-1 expression and thereby breast cancer development or progression. . Methods: Del-1 mRNA and miR-137/– 496 levels were measured by qRT-PCR among breast epithelial (MCF10A) and cancer cells (MDA-MB-231, MCF7, SK-BR3 and T-47D). The effects of miR-137/– 496 on cell proliferation and invasion were detected using MTT, wound healing and Transwell assays. Furthermore, luciferase reporter assay was used to identify the direct regulation of Del-1 by miR-137 or – 496 in MDA-MB-231 cells. Plus, we analyzed the expressions of miR-137 or – 496 and Del-1 mRNA from 20 patients with triple negative early breast cancer. Results: miR-137 and – 496 levels were low in all breast cancer cell lines. As Del-1 mRNA expression was remarkably higher in MDA-MB-231 compared to the other breast cancer cell lines, further functional analyses were done with MDA-MB-231 representing triple negative breast cancer subtype. Both miR-137 and miR-496 were revealed to directly bind at the 3’-UTR of Del-1. Del-1 by Luciferase reporter assay and Del-1 expression was upregulated by inhibitors and reversed by both mimics of both miR-137 and miR-496. Furthermore, both miR-137 and miR-496 were also demonstrated to inhibit cell proliferation, migration and invasion of MDA-MB-231, suggesting that these miRNAs affect cancer progression via Del-1. MiR-137 and miR-496 were remarkably down-regulated in 7 out of 12 triple negative breast cancer tissues, in particular with high Ki67 and high histologic grade. Conclusions: Although Del-1 was recently introduced as a new biomarker for triple negative breast cancer, the mechanisms of Del-1 expression were barely identified. The current study firstly demonstrated that microRNA 137 and 496 are involved in Del-1 regulation by binding at Del-1 gene, affecting cancer progression by altering Del-1 expression.

Long non coding RNAs (lncRNAs) have been identified as regulators of the cell cycle, apoptosis, and DNA damage among other processes that if deregulated, may lead to cancer by acting as proto-oncogenes, tumor suppressor genes, and drivers of metastatic transformation. Using RNA sequencing we have identified 42 differentially expressed lncRNAs from a healthy cohort of parous vs. nulliparous women. After bioinformatics and RT-qPCR analysis, we have focused on a vaguely studied lncRNA called BC200 that is highly expressed in the nulliparous postmenopausal breast tissue. It is known that BC200 lncRNA is overexpressed in invasive and pre-invasive breast cancer; however, its functional role in the initiation and progression of breast cancer is poorly understood. In the present work we provide insight on the role of BC200 in the context of luminal and triple negative breast cancer (TNBC). We have confirmed that BC200 is highly expressed in breast cancer tissue and in widely used breast cancer cell lines such as MCF7, T47D, MDAMB231, and Hs578T. Using a lentiviral system we successfully obtained cell lines which stably express BC200. Overexpression of BC200 increases proliferation, migration, and invasion potential in vitro in the cell lines tested, specifically luminal T47D and TNBC MDAMB231. Xenograft studies performed in the mammary fat pad of female SCID mice confirm the role of BC200 as a tumor promoter. Tumors in mice injected with MDAMB231 cells overexpressing BC200 were 4.5 times bigger than tumors in the control group in only 6 weeks when injecting 1 million cells. Moreover, we have determined, using reverse transcriptase PCR targeting genes less than 200 kb from the start site of BC200, that when BC200 is overexpressed, CALM2 is downregulated in both T47D and MDAMB231 cell lines. CALM2 or Calmodulin is a calcium binding protein that plays a role in signaling pathways, cell cycle progression, proliferation, and apoptosis. Mutations in CALM2 are associated with increased risk of breast cancer. Our positive results on Cis regulation are being expanded using chromatin isolation by RNA immunoprecipitation to determine BC200's genome wide regulation. These results demonstrate the participation of BC200 lncRNA in the progression of breast cancer. Notably, BC200 regulates nearby genes that have an implication in cancer progression. BC200, identified in the normal breast tissue of nulliparous women, not only plays a key role in breast cancer progression but also provides a new insight in the preventive role of pregnancy by the downregulation of the expression of this lncRNA in the normal parous breast. [This work was supported by the NCI (National Cancer Institute) Core Grant CA06927 to Fox Chase Cancer Center and generous support from Christian - Diane Martin, the Flyers Wives, and Joseph - Barbara Breitman to Dr. J. Russo, MD]. Citation Format: Barton M, Santucci-Pereira J, Su Y, Russo J. BC200 lncRNA is involved in the progression of triple negative 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 P1-08-06.

SummaryM2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC).Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-β enhancement, IL-17F secretion, and extracellular vesicle protein content modulation.We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.

Chromatin remodeling pathways are critical in the regulation of cancer-related genes and are currently being explored as potential targets for therapeutic intervention. The metastasis tumor antigen (MTA) family of proteins, MTA1, MTA2, and MTA3, are components of chromatin remodeling pathways with potential roles in breast cancer. Although all three MTA family proteins have been shown to be associated with metastatic progression of breast cancers, the expression characteristic of MTA1-3 proteins in a multistep breast cancer progression model remains unknown. Structural and functional studies have suggested that they are heterogeneous in the Mi-2/NuRD complex, exhibit tissue-specific patterns of expression, and impart unique properties to estrogen receptor-alpha (ERalpha) action. This led us to hypothesize that each member of the MTA family possesses a unique role and interacts with different pathways in the stepwise process of breast cancer development and progression. MTA family proteins were examined by immunohistochemistry in breast cancer processes ranging from normal duct, to premalignant lesions, to invasive carcinoma, and to metastasized tumors in PyV-mT transgenic mice, which represents a reliable model for multistage tumorigenesis of human breast cancer. We also determined the association of MTA proteins with the status of cell proliferation, ER, E-cadherin and cytoplasmic beta-catenin, and cancer-related coactivators, AIB1 and PELP1. The expression of all three MTA proteins was altered in primary breast tumors. Each MTA protein had a unique expression pattern during the primary breast tumor progression. Altered expression of MTA1 was observed in both premalignant lesion and malignant carcinoma, but an elevated nuclear expression was observed in ER-negative carcinomas. MTA3 was exclusively expressed in a subset of cells of ER-positive premalignant lesions but not in carcinomas. MTA2 expression seems to be unrelated to ER status. Loss of MTA3 expression and more nuclear localization of MTA1 occurred with loss of E-cadherin and decreased cytoplasmic beta-catenin, two molecules essential for epithelial cell adhesion and important tumor cell invasion. At the late stage of tumor formation, MTA1 is usually expressed in the center of tumors. Coincidentally, the distribution of MTA1-positive cells at this stage was complementary to that of AIB1 and PELP1, which were localized to the tumor periphery with relatively active cell proliferation, scattered ER-positive cells and a limited differentiation. In metastasized lung tumors, the expression pattern of MTA-protein expression was distinct from that in primary counterparts. The findings presented here support the notion that each member of the MTA family might potentially play a stepwise role in a cell type-specific manner during breast cancer progression to metastasis. On the basis of the noted temporal expression patterns of MTA proteins with ER status, cell adhesion-essential regulators (E-cadherin and cytoplasmic beta-catenin), and coactivators, we propose that MTA protein-related chromatin remodeling pathways interact with steroid receptors, growth factor receptors, and other transcriptional signaling pathways to orchestrate the governing of events in breast cancer progression and metastasis.

Introduction: We have previously found genomic gain on 17q22-24.2 to be a predictor of invasion when detected in duct carcinoma in situ (DCIS), and of nodal metastasis when detected in invasive duct carcinoma (IDC). Within this gene-rich amplicon we have identified a gene, Musashi homolog 2 (MSI2), which appears to play an important role in breast cancer progression. MSI2 is known to have a functional role in neural stem cell maintenance and the regulation of hematopoietic stem cells. More recently, this gene was shown to facilitate progression from the leukemic blast phase to the crisis phase. Methodology: The mRNA expression profile for MSI2 was determined by qRT-PCR in 9 breast cancer cell lines (MDA-MB-157, MDA-MB-231, MDA-MB-468, BT549, CAMA1, Hs578T, MCF7 and SKBR3) and a non-malignant mammary epithelial cell line (MCF10A). Western blots were used to confirm protein expression. MCF7 mammospheres and MSI2 overexpression clones were assessed for breast stem cell markers. Immunofluorescence was conducted at embryonic stages E10.5 to E15.5 to determine the role of MSI2 in murine mammary bud formation. MDA-MB-231 and MCF7 cells expressing MSI2-GFP and GFP clones were plated on transwells to examine cell migration and invasiveness. Proliferation was analyzed by the MTS assay. To understand the expression of MSI2 in patient tissue, tissue microarrays (TMAs) containing 232 breast cancers were analyzed by immunohistochemistry. Results: MSI2 was expressed in all breast cell lines tested. Immunohistochemical analyses of MSI2 in normal adult human breast tissue revealed expression in the basal luminal epithelial cells of the terminal duct lobular unit, with little expression in the stroma. In murine mammary line formation, E10.5 to E12.5 showed expression of MSI2 in both the mammary ductal epithelium and the mesenchyme. MSI2 expression diminished in mesenchyme and was restricted to the ductal epithelium by E14.5. MDA-MB-231 and MCF7 cells expressing MSI2-GFP demonstrated a 50% increase in cell migration and 30% more invasion compared to the GFP control. Correspondingly, knockdown clones showed the reverse effect. Increased MSI2 was coupled with an increase in the stem cell markers: CD24, CD44, Aldh1α1, CD133 and ABCG2 receptor. When the study was extended to patient TMAs, high MSI2 expression correlated with higher tumor grade. Conclusion: These experiments provide strong evidence that MSI2 plays an important role in normal breast development and breast cancer. MSI2 promotes migration and invasion and also appears to increase stem cell related properties which may explain the relationship to higher tumor grade. MSI2 is likely one of the drivers of the 17q22-24.2 amplicon in breast cancer and a better understanding of its role in breast cancer may lead the way to novel therapeutic strategies. Citation Format: Moustafa Abdalla, Ranju Nair, Nisha Kanwar, Nicholas Holzapfel, Juan C. Moreno, Susan J. Done. Characterization of the MSI2 gene on 17q22-24.2 and its role in Breast Cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1814. doi:10.1158/1538-7445.AM2013-1814

RUNX2, a master regulator of osteogenesis, is oncogenic in the lymphoid lineage; however, little is known about its role in epithelial cancers. Upregulation of RUNX2 in cell lines correlates with increased invasiveness and the capacity to form osteolytic disease in models of breast and prostate cancer. However, most studies have analysed the effects of this gene in a limited number of cell lines and its role in primary breast cancer has not been resolved. Using a human tumour tissue microarray, we show that high RUNX2 expression is significantly associated with oestrogen receptor (ER)/progesterone receptor (PR)/HER2-negative breast cancers and that patients with high RUNX2 expression have a poorer survival rate than those with negative or low expression. We confirm RUNX2 as a gene that has a potentially important functional role in triple-negative breast cancer. To investigate the role of this gene in breast cancer, we made a transgenic model in which Runx2 is specifically expressed in murine mammary epithelium under the control of the mouse mammary tumour virus (MMTV) promoter. We show that ectopic Runx2 perturbs normal development in pubertal and lactating animals, delaying ductal elongation and inhibiting lobular alveolar differentiation. We also show that the Runx2 transgene elicits age-related, pre-neoplastic changes in the mammary epithelium of older transgenic animals, suggesting that elevated RUNX2 expression renders such tissue more susceptible to oncogenic changes and providing further evidence that this gene might have an important, context-dependent role in breast cancer.

Neoadjuvant chemotherapy (NACT) represents a standard option for breast cancer. Unfortunately, about 55–80% of breast cancer patients do not have a favorable response to chemotherapy. Highly specific tumor biomarker that can predict the pathological response to neoadjuvant chemotherapy is lacking. Stearoyl-CoA desaturase 5 (SCD5) is an integral membrane protein of the endoplasmic reticulum that participates in lipid metabolism. Previous studies on the role of SCD5 in human cancers drew different conclusions. Therefore, the role of SCD5 in breast cancer remains unclear. Our study aims to understand its expression signature, prognosis value and correlation with pathological response to NACT in breast cancer using bioinformatics from public databases. Analysis of samples from public databases showed that SCD5 expression was down-regulated in some human cancers including breast cancer, and low expression of SCD5 was associated with more aggressive breast cancer phenotypes. Survival analysis revealed that SCD5 expression was related to prognosis in breast cancer. Integrated analysis of multiple public datasets indicated that SCD5 expression signature was associated with pathological response to NACT, particularly in TNBC. Based on functional enrichment analysis, the most affected biological functions in high SCD5-expressing breast cancer tissues were involved in negative regulation of cell cycle. Moreover, a significantly negative correlation between SCD5 expression and several cell cycle regulators was noted. Taken together, SCD5 was involved in the development and progression of breast cancer and might be a predictive biomarker for response to NACT. In conclusion, SCD5 could serve as a predictive biomarker of pathological response to NACT and play a carcinostatic role in breast cancer. These results provided us with clues to better understand SCD5 from the perspective of bioinformatics and highlighted the clinical importance of SCD5 in breast cancer, especially triple negative breast cancer (TNBC).

BackgroundCyclin-dependent kinase 16 (CDK16) is an atypical PCTAIRE kinase, and its activity is dependent on the Cyclin Y (CCNY) family. Ccnys have been reported to regulate mammary stem cell activity and mammary gland development, and CCNY has been recognized as an oncoprotein in various cancers, including breast cancer. However, it remains unclear whether CDK16 has a role in breast cancer and whether it can be used as a therapeutic target for breast cancer.MethodsPublicly available breast cancer datasets analyses and Kaplan-Meier survival analyses were performed to reveal the expression and clinical relevance of atypical CDKs in breast cancer. CDK16 protein expression was further examined by immunohistochemical and immunoblot analyses of clinical samples. Cell proliferation was measured by colony formation and MTT analyses. Cell cycle and apoptosis were examined by fluorescence-activated cell sorting (FACS) analysis. Wound-healing and trans-well invasion assays were conducted to test cell migration ability. The functions of CDK16 on tumorigenesis and metastasis were evaluated by cell line-derived xenograft, patient-derived organoid/xenograft, lung metastasis and systemic metastasis mouse models. Transcriptomic analysis was performed to reveal the potential molecular mechanisms involved in the function of CDK16. Pharmacological inhibition of CDK16 was achieved by the small molecular inhibitor rebastinib to further assess the anti-tumor utility of targeting CDK16.ResultsCDK16 is highly expressed in breast cancer, particularly in triple-negative breast cancer (TNBC). The elevated CDK16 expression is correlated with poor outcomes in breast cancer patients. CDK16 can improve the proliferation and migration ability of TNBC cells in vitro, and promote tumor growth and metastasis of TNBC in vivo. Both genetic knockdown and pharmacological inhibition of CDK16 significantly suppress the tumor progression of TNBC. Mechanistically, CDK16 exerts its function by phosphorylating protein regulator of cytokinesis 1 (PRC1) to regulate spindle formation during mitosis.ConclusionCDK16 plays a critical role in TNBC and is a novel promising therapeutic target for TNBC.

Exploring the Potential of Breast Microbiota as Biomarker for Breast Cancer and Therapeutic Response

Breast cancers are often evaluated by their expression of estrogen, progesterone and HER2/Neu receptors. Triple negative breast cancers (TNBC), which do not have upregulation of any of these receptors, lack directed therapies and therefore, are associated with a worse prognosis. The lack of directed therapies for TNBC and high incidence of relapse and drug resistance found in all breast cancers underscores the need for identification of additional therapeutic targets. The analysis of gene expression data revealed that PIM1 and PIM2 kinases are upregulated in triple negative breast cancer. PIM kinases are a family of serine/threonine kinases that activate cell division, promote cell growth, and inhibit apoptosis. Due to PIM1/2’s effect on these cellular processes and PIM1/2 being upregulated in TNBC, we hypothesized that PIM1/2 play an important role in progression of triple negative breast cancer. Knockdown of PIM1 significantly inhibited the proliferation, migration, and invasion of TNBC cells, including MDA-231, BT-20, HCC-1806, and MDA-468 cells. To study the in vivo roles of PIM1/2 in breast cancer progression and metastasis, we crossed the highly metastatic MMTV-PyMT breast cancer mouse model with PIM1/2 knockout mice. Deletion of PIM1 or PIM2 alone significantly inhibited tumor growth and reduced lung metastasis in these mice. Dual deletion of PIM1 and PIM2 significantly reduced tumor growth and almost completely blocked lung metastasis in these mice. A major advantage of the MMTV-PyMT model over xenograft models is that the immune system is intact in these mice, and that enabled us to perform immune cell profiling on the tumors. Tumor associated macrophages (TAMs) have been shown to promote tumor metastasis and are a poor prognostic marker for TNBC. Deletion of PIM1 alone and combined PIM1 and PIM2 deletion resulted in a significant reduction in the number of TAMs, indicating PIM1/2 play a significant role in regulating the immune milieu of these tumors. To understand the underlying mechanism by which PIM1 and PIM2 contribute to these processes, we performed biochemical analysis. Western blot analysis of PIM1 knockdown in MDA-231 cells showed a decrease in phosphorylation of CXCR4, S6 ribosomal protein, and 4EBP1 and increased expression of cell cycle regulator p27. Additionally, we performed RNA-seq analysis to identify which pathways were affected by PIM1/2 deletion in MMTV-PyMT tumors. GSEA analysis showed a significant enrichment of genes related to inflammatory response, extracellular matrix, EMT and metastasis in PyMT tumors and those were significantly reduced in PIM1/2 double knockout tumors. These results suggest that PIM1 and PIM2 play a significant role in the progression and metastasis of triple negative breast cancer and indicate that PIM1/2 inhibition could be a useful therapeutic approach for TNBC. Citation Format: Patrick Faughnan, Golam Mohi, Chandrajeet Singh. The roles of PIM1 and PIM2 kinases in the progression and metastasis of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 149.

BackgroundWith the rapid development of the high throughput detection techniques, tumor-related Omics data has become an important source for studying the mechanism of tumor progression including breast cancer, one of the major malignancies worldwide. A previous study has shown that the G2 and S phase-expressed-1 (GTSE1) can act as an oncogene in several human cancers. However, its functional roles in breast cancer remain elusive.MethodIn this study, we analyzed breast cancer data downloaded from The Cancer Genome Atlas (TCGA) databases and other online database including the Oncomine, bc-GenExMiner and PROGgeneV2 database to identify the molecules contributing to the progression of breast cancer. The GTSE1 expression levels were investigated using qRT-PCR, immunoblotting and IHC. The biological function of GTSE1 in the growth, migration and invasion of breast cancer was examined in MDA-MB-231, MDA-MB-468 and MCF7 cell lines. The in vitro cell proliferative, migratory and invasive abilities were evaluated by MTS, colony formation and transwell assay, respectively. The role of GTSE1 in the growth and metastasis of breast cancer were revealed by in vivo investigation using BALB/c nude mice.ResultsWe showed that the expression level of GTSE1 was upregulated in breast cancer specimens and cell lines, especially in triple negative breast cancer (TNBC) and p53 mutated breast cancer cell lines. Importantly, high GTSE1 expression was positively correlated with histological grade and poor survival. We demonstrated that GTSE1 could promote breast cancer cell growth by activating the AKT pathway and enhance metastasis by regulating the Epithelial-Mesenchymal transition (EMT) pathway. Furthermore, it could cause multidrug resistance in breast cancer cells. Interestingly, we found that GTSE1 could regulate the p53 function to alter the cell cycle distribution dependent on the mutation state of p53.ConclusionOur results reveal that GTSE1 played a key role in the progression of breast cancer, indicating that GTSE1 could serve as a novel biomarker to aid in the assessment of the prognosis of breast cancer.