Single-cell sequencing and its application in breast cancer.

Purpose: African-American (AA) breast cancer (BC) and colorectal cancer (CRC) patients experience higher mortality rates than Non-Hispanic Caucasian (CA) patients. These disparities are thought to be due to race/ethnicity-specific tumor molecular biology. Our initial gene expression investigations have identified a Beta B2-Crystallin (CRYBB2) gene that is preferentially over-expressed in CRC of AA patients. However, the precise role of CRYBB2 in aggressiveness of CRC and BC has not been examined. Therefore, we hypothesize that biological mechanism linked to CRYBB2 could contribute to the disparate progression of CRC and BC in AA patients. Experimental Design: Gene expression studies were performed using Affymetrix Human Genome U133 Plus 2.0 arrays and mRNA samples extracted from 10 fresh frozen Stage III CRCs (5 from AA and 5 from CA) that were previously analyzed for the microsatellite instability (MS) status. Established CRC (HT29 and SW480) and BC (MBA-MD 231) cell lines were used to examine the phenotypic expression of CRYBB2. Also, paired lesions of human CRC (hCRC) and human mice CRC (hmCRC) xenograft were assessed for CRYBB2 by western blot and immunofluorescence (IF). To study the involvement of CRYBB2 in CRC and BC biology, siRNA mediated gene silencing was used to inhibit CRYBB2 expression. The apoptosis of CRC and BC cells after knockdown of CRYBB2 was analyzed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. The epithelial-mesenchymal transition (EMT) of CRC and BC cells was analyzed by western blot for E-cadherin, MM9 and vimentin expression. Western blot analysis was performed on lysates of CRC and BC cells to assess the effect of CRYBB2 knockdown for expression/ activation of the AKT, and mitogen-activated protein kinase pathway proteins. Results: Our microarray analyses have suggested that several key genes involved in tumor metastasis (CRYBB2, VSNL-1and PFN2) are up-regulated in MS-CRCs of AA but down regulated in MS-CRCs of CA patients. The phenotypic expression of CRYBB2 was found to be higher in CRC tissues as compared to matching normal tissues. Over-expression of CRYBB2 correlated with metastatic CRC and N-cadherin expression and the expression of CRYBB2 was substantially increased in metastatic lesion of CRC and BC xenografts as compared to the primary tumors. Knockdown of CRYBB2 induced apoptosis in CRC and BC cells. In addition, Knockdown of CRYBB2 suppressed EMT signatures, such as decreasing the expression of MMP9, vimentin (mesenchymal markers) and increasing the expression of E-cadherin (epithelial marker). Furthermore, depletion of CRYBB2 decreased the phosphorylation of AKT (p-AKT) in CRC and BC cells. CRYBB2 was mainly localized in cytoplasm of BC and CRC cells. Conclusions: Our data suggest that CRYBB2 inhibits apoptosis, activates EMT process and AKT phosphorylation by which it contributes the aggressiveness of CRC and BC. It may serve as a promising biomarker for identifying CRC and BC patients at high risk for metastases. It may also be useful as a therapeutic target to inhibit the growth and metastasis of CRC and BC. These studies are supported by NIH/NCI funds (R21-CA171251 and U54- CA 118948). Citation Format: Harvey L. Bumpers, Venkat Katkoori, Dongquan Chen, Upender Manne. The role of BetaB2-crystallin in progression of colorectal and breast cancers in African Americans. [abstract]. In: Proceedings of the Sixth AACR Conference: The Science of Cancer Health Disparities; Dec 6–9, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2014;23(11 Suppl):Abstract nr C56. doi:10.1158/1538-7755.DISP13-C56

Breast cancer (BCa) and prostate cancer (PCa) are both hormone driven cancers with similar etiology and tumor microenvironment inflammation promotes BCa and PCa progression. Interleukin-1 (IL-1) is an inflammatory cytokine present in the tumor microenvironment and IL-1 is elevated in BCa and PCa patient tumor and/or blood serum and correlates with poor prognosis. We have shown that IL-1 represses the BCa and PCa therapeutic targets, Estrogen Receptor Alpha (ERa) and Androgen Receptor (AR), respectively, in PCa and BCa cell lines; yet the cells remain viable. Thus, IL-1 may promote treatment resistance and disease progression. The pro-survival protein Sequestome-1 (SQSMT1/p62) is also upregulated in both BCa and PCa cell lines exposed to IL-1, suggesting that BCa and PCa cells have evolved a shared response to IL-1 and hormone receptor loss. RNA sequencing of an IL-1-treated PCa cell line revealed an IL-1-modulated gene suite predicted to confer AR-independent tumorigenicity. We performed RT-qPCR in PCa cell lines for several select genes to confirm the RNA sequencing results and given the similar etiology and IL-1 regulation of ERa, AR, and p62 expression in BCa and PCa cell lines, we presumed that our select genes would be similarly regulated by IL-1 in BCa cells. However, outside of ERa, AR, and p62 expression, our select genes did not show similar IL-1 regulation in BCa cell lines, suggesting that IL-1 regulates a unique set of genes that could contribute to ERa-independent tumorigenicity in BCa cells. Therefore, to identify a BCa-specific IL-1-modulated gene suite, we performed RNA sequencing on an IL-1-treated ERα+ BCa cell line and compared gene expression changes with 1) basal gene expression in an ERa- BCa cell line and 2) our IL-1-modulated PCa-specific gene suite. Our bioinformatics analysis revealed pathways that are predicted to promote ERa-independent tumorigenicity in BCa cells and investigations are underway to demonstrate the functional significance of our gene expression data. Taken together, our studies provide insight into the mechanistic function of IL-1 in PCa and BCa resistance to hormone receptor-targeted therapies and tumor progression. Citation Format: Afshan Fathima Nawas, Mohammed Kanchwala, Shayna Thomas-Jardin, Vanessa Anunobi, Ally Wong, Chao Xing, Nikki Delk. Identification of Interleukin-1 (IL-1) induced gene expression pattern in breast cancer (BCa) cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3444.

Giant Cell Tumor of Bone (GCTB) is a common intermediate tumor, and the specific molecular mechanisms of this disease have not been fully elucidated. It exhibits strong heterogeneity in terms of targets, regulatory mechanisms, cell types, states, and subset distributions in the immune microenvironment. Traditional collective-level analyses cannot accurately reveal these differences. Single-cell sequencing technology is a technique that sequences the genome, transcriptome, and epigenome of diseases at the single-cell level. Single-cell sequencing can utilize a higher pixel resolution to reveal characteristic states of different cell subpopulations, contributing to broadening new perspectives for the study of GCTB heterogeneity. It holds significant value for the precise diagnosis of GCTB, identification of potential immunotherapy targets, and prognosis assessment. This study primarily reviews research related to GCTB, single-cell high-throughput sequencing technology, GCTB immune microenvironment, GCTB heterogeneity, and the construction of GCTB cell maps. It aims to provide theoretical reference for research on single-cell sequencing technology in GCTB and offers a theoretical basis for in-depth exploration of the mechanisms and treatment of GCTB.

: Fatty acid binding protein 5 (FABP5) is a small, 14-15 kDa, molecular weight intracellular fatty acid shuttle. FABP5 functions to shuttle long chain fatty acids from the extracellular environment to the nucleus. These fatty acids are then used as ligands for nuclear receptors and activate transcription. FABP5 has been shown to coordinate metabolic and inflammatory responses in adipocytes and may play a role in the development of obesity-mediated breast cancer. Both FABP5 and S100A7 proteins are over-expressed in the inflammatory skin disease, psoriasis. Additionally, there is evidence that these two proteins associate with one another. Previously reported by our lab, S100A7 enhances tumor progression in orthotopic breast cancer mouse models. Taking together the evidence of S100A7 in breast cancer progression and the support that FABP5 is involved in breast cancer development and progression. We have shown overexpression of FABP5 in mammary glands of mice overexpressing mS100a7a15, the mouse homolog of human S100A7. FABP5 was also shown to be expressed higher in S100A7 overexpressing triple negative breast cancer cells compared to vector control. Additionally, FABP5 has higher expression in a highly metastatic breast cancer cell line compared to a low metastatic breast cancer cell line. We determined FABP5 knockout in the host significantly decreases tumor volume in an orthotopic model of breast cancer. We showed that shRNA mediated FABP5 knockdown in triple negative breast cancer cells results in less migration compared to scrambled shRNA. Additional cell lines are being evaluated and used to validate these results. FABP5 expression was examined in a tumor microarray and found that FABP5 expression is correlated with higher tumor grade and triple negative breast cancer. Additionally, high FABP5 protein expression is associated with shorter disease free recurrence.

Breast cancer is the second leading cancer type in women, accounting for 11.6% of all cancers. Recently, its incidence has increased in younger individuals. Estrogen receptors (ERs) play important roles in the development and progression of breast cancer by controlling hormone signaling. Therefore, targeting ERs is one of the most promising therapeutic strategies for treating ER-positive breast cancer. High heterogeneity contributes to the tumorigenicity, metastatic ability, and therapeutic resistance of breast cancer. However, drug discovery studies at the single-cell level are lacking. In the present study, we used single-cell sequencing data analysis, together with a network pharmacology approach, to determine the targets and molecular mechanisms of formononetin in ER-positive breast cancer. Comparative single-cell sequencing analysis identified 3899, 3395, 333, 398, 398, and 17 differentially expressed genes in stromal cells, epithelial cells, fibroblasts, neutrophils, eosinophils, and macrophages, respectively, of ER-positive breast cancer compared with normal breast tissues. Further network pharmacology analysis highlighted the importance of formononetin targets in biological functions and signaling pathways related to immune and inflammatory responses, metastatic ability, metabolism, cell proliferation, and gland development in different ER-positive breast cancer cell types. For the first time, we used a systems biology approach to investigate the targets of formononetin and its anti-ER-positive breast cancer mechanisms at the single-cell level.

Abnormal expression of CUT-like homeobox 2 gene (CUX2) has been highlighted as potential clinical biomarkers in human cancers. Notably, the function of CUX2 has been less elucidated in breast cancer (BC). We focused on the role of the CUX2 in tumorigenesis and progression of BC with the involvement of the lysine demethylase 5B (KDM5B)/sex determining region Y-box 17 (SOX17) axis. CUX2, KDM5B, and SOX17 expression levels in BC tissues and cells were tested by reverse transcription quantitative PCR and Western blotting. Later, the effects of CUX2, KDM5B, and SOX17 on the malignant behaviors of MDA-MB-231 and MCF-7 cells were analyzed by CCK-8, colony formation, and Transwell assays in vitro. The interactions of CUX2, KDM5B, and SOX17 were validated by online website prediction, ChIP assay, and dual luciferase reporter gene assay. The subcutaneous tumorigenesis in nude mice was conducted to observe the roles of CUX2, KDM5B, and SOX17 in BC tumor growth in vivo. CUX2 and KDM5B were highly expressed while SOX17 had low expression in BC. Inhibition of CUX2 suppressed BC cell malignant phenotypes. CUX2 promoted KDM5B expression through transcriptional activation, enabling its high expression in BC. KDM5B inhibited SOX17 expression through histone demethylation. Overexpression of KDM5B or downregulation of SOX17 reversed the inhibitory effect of CUX2 downregulation on the malignant behaviors of BC cells. Inhibition of CUX2 impeded BC cell growth in vivo through the KDM5B/SOX17 axis. This study highlights that suppression of CUX2 inhibits KDM5B to repress tumorigenesis and progression of BC through overexpressing SOX17.

CD44 is closely linked to breast cancer progression; however, the regulatory functions of microRNAs (miRs) in breast cancer have yet to be fully elucidated. In order to investigate the regulation of CD44 by miRs in breast cancer, the present study isolated CD44+ and CD44- breast cancer cells by flow cytometry, revealing that CD44+ cells were enriched in transplanted compared with those in primary breast cancers, and that their proliferation and stem-cell sphere formation ability were enhanced. A miRNA array assay indicated that miR-143 expression in CD44+ breast cancer cells was lower than that in CD44- cells. Furthermore, miR-143 was decreased in breast cancer tissues and cell lines compared with that in normal tissues and cells. Restoration of miR-143 expression in CD44+ breast cancer cells inhibited their proliferation and sphere formation. A luciferase reporter assay demonstrated that miR-143 directly tartgeted the 3'-untranslated region of CD44. In addition, miR-143 inhibited metastasis-associated features in breast cancer and reduced tumor growth in a mouse model of breast cancer. In conclusion, the results of the present study demonstrated that miR-143 inhibited the progression and stem-cell properties of breast cancer cells by targeting CD44.

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.

PurposeBreast cancer is the most frequent cancer in women with significant death rate. Morbidity is associated with drug resistance and metastasis. Development of novel drugs is unmet need. The aim of this study is to show potent anti-neoplastic activity of the UM171 compound on breast cancer cells and its mechanism of action.MethodsThe inhibitory effect of UM171 on several breast cancer (BC) cell lines was examined using MTT and colony-forming assays. Cell cycle and apoptosis assays were utilized to determine the effect of UM171 on BC cell proliferation and survival. Wound healing scratch and transwell migration assays were used to examine the migration of BC cell lines in culture. Xenograft of mouse model with 4T1 cells was used to determine inhibitory effect of UM171 in vivo. Q-RT-PCR and western blotting were used to determine the expression level of genes effected by UM171. Lentivirus-mediated shRNAs were used to knockdown the expression of KLF2 in BC cells.ResultsUM171 was previously identified as a potent agonist of human hematopoietic stem cell renewal and inhibitor of leukemia. In this study, UM171 was shown to inhibit the growth of multiple breast cancer cell lines in culture. UM171-mediated growth inhibition was associated with the induction of apoptosis, G2/M cell cycle arrest, lower colony-forming capacity, and reduced motility. In a xenotransplantation model of mouse triple-negative breast cancer 4T1 cells injected into syngeneic BALB/c mice, UM171 strongly inhibited tumor growth at a level comparable to control paclitaxel. UM171 increased the expression of the three PIM genes (PIM1-3) in breast cancer cells. Moreover, UM171 strongly induced the expression of the tumor suppressor gene KLF2 and cell cycle inhibitor P21CIP1. Accordingly, knockdown of KLF2 using lentivirus-mediated shRNA significantly attenuated the growth suppressor activity of UM171. As PIM1-3 act as oncogenes and are involved in breast cancer progression, induction of these kinases likely impedes the inhibitory effect of KLF2 induction by UM171. Accordingly, combination of UM171 with a PAN-PIM inhibitor LGH447 significantly reduced tumor growth in culture.ConclusionThese results suggested that UM171 inhibited breast cancer progression in part through activation of KLF2 and P21. Combination of UM171 with a PAN-PIM inhibitor offer a novel therapy for aggressive forms of breast cancer.

ABSTRACTTumor‐associated neutrophils (TANs) contribute to breast cancer (BC) progression, and aquaporin 9 (AQP9) plays a critical role in tumor development. However, the interactions between TANs and AQP9 in BC are poorly understood. Bioinformatics analyses and clinical samples revealed a positive correlation between neutrophil infiltration and AQP9 expression in BC. Treating BC cells with TAN‐conditioned media significantly elevated AQP9 expression compared with neutrophil‐conditioned and control media treatments. Immunohistochemical analysis revealed higher AQP9 protein expression in BC tissues than in adjacent normal tissues, and AQP9 expression was negatively correlated with recurrence‐free survival and overall survival in patients with BC. Functional studies demonstrated that AQP9 promoted BC cell proliferation but did not affect migration or invasion. AQP9 knockdown markedly inhibited the ability of TANs to enhance BC cell proliferation, migration, and invasion. Intravenous and intratumoral injection of TANs in mice increased tumor growth rate, weight, and volume compared with controls; moreover, histological examination revealed lung metastasis in two mice and bone involvement in one mouse out of six in the TAN treatment group. AQP9 knockdown significantly reduced the tumor growth rate. In BC cells, TAN treatment elevated STAT3 phosphorylation, and this effect was amplified by AQP9 overexpression. In conclusion, TANs promote BC progression by enhancing STAT3 phosphorylation through AQP9 upregulation. AQP9 is crucial for TAN‐mediated BC progression and is a potential target for immunotherapy in patients with BC.

Abstract: Breast cancer is an aggressive disease with a significant morbidity and death rate among women worldwide. Despite the progress of diagnostic and therapy options for breast cancer in recent years, the prognosis and survival rates of breast cancer patients remain unsatisfactory. The aberrant growth and spread of tumor cells are the leading cause of death in these patients. More profound knowledge of molecular biology underlying breast cancer and a more accurate stratification are still necessary for more precise therapy. Further understanding of the disease's molecular mechanism and genetic aberrations may allow for the identification of more accurate prognostic and diagnostic markers and more effective treatments. Tumor protein D52 (TPD52) is an oncogene whose overexpression has been found in breast cancer. Overexpression of TPD52 has been linked to specific molecular subtypes of breast cancer, including luminal B and ERBB2-positive tumors. Besides, non-coding RNAs (ncRNAs) were found to play a significant role in breast cancer progression. ncRNAs play regulatory roles in cell behaviors, cancer pathogenesis, radiotherapy, and resistance to chemotherapy. Multiple ncRNAs could modulate the expression of TPD52 and regulate breast cancer cell proliferation, invasion, and metastasis. In this review, we summarized the functions of TPD52 in breast cancer cells.

In recent years, studies have shown that lncRNA plays an essential regulatory role in biological life activities. In disease and cancer research, the function of lncRNA is closely related to inflammatory response, tumor formation and cellular metabolism. Breast cancer is one of the most common malignant tumors in women. The research on the pathogenesis of breast cancer is the focus of current research. Although the regulatory mechanisms of some lncRNAs have been proven, the complexity of cancer regulation has led to incomplete research. The expression of LOXL1-AS1 and miR-143-3p was measured using qRT-PCR. Western blot was used to detect CDK, Cyclin D1, MMP-2, MMP-9, Bcl-2, Bax and Cleaved caspase-3 protein expression. MTT assay and transwell assay were applied to analyze cell proliferation, migration and invasion, respectively. Cell apoptosis rate of transfected cells was measured with flow cytometry. Luciferase reporter assay was applied to verify the relationship between LOXL1-AS1 and miR-143-3p. In this study, we found that LOXL1-AS1 expression was induced while miR-142-3p expression was decreased in breast cancer tissues and cells, implying that LOXL1-AS1 and miR-143-3p play an important role in cell progression of breast cancer. Further investigation showed that silencing LOXL1-AS1 inhibited proliferation, promoted cell apoptosis and decreased the capacity of cell migrated and invasive in breast cancer cells. The analysis of luciferase reporter assay determined that LOXL1-AS1 directly targeted miR-143-3p in breast cancer cells. The rescue experiments further proved that miR-143-3p reversed the inhibited effects of si- LOXL1-AS1 on breast cancer cells. In this study, we verified that LncRNA LOXL1-AS1 inhibited cell proliferation, migration and invasion as well as induced apoptosis in breast cancer via regulating miR-143-3p, providing a novel therapeutic target and improving understanding of the regulatory mechanism of cell progression in breast cancer.

Phosphorylated Rasal2 facilitates breast cancer progression.

Breast cancer, a complex and heterogeneous ailment impacting numerous women worldwide, persists as a prominent cause of cancer-related fatalities. MicroRNAs (miRNAs), small non-coding RNAs, have garnered significant attention for their involvement in breast cancer's progression. These molecules post-transcriptionally regulate gene expression, influencing crucial cellular processes including proliferation, differentiation, and apoptosis. This review provides an overview of the current research on the role of miRNAs in breast cancer. It discusses the role of miRNAs in breast cancer, including the different subtypes of breast cancer, their molecular characteristics, and the mechanisms by which miRNAs regulate gene expression in breast cancer cells. Additionally, the review highlights recent studies identifying specific miRNAs that are dysregulated in breast cancer and their potential use as diagnostic and prognostic biomarkers. Furthermore, the review explores the therapeutic potential of miRNAs in breast cancer treatment. Preclinical studies have shown the effectiveness of miRNA-based therapies, such as antagomir and miRNA mimic therapies, in inhibiting tumor growth and metastasis. Emerging areas, including the application of artificial intelligence (AI) to advance miRNA research and the "One Health" approach that integrates human and animal cancer insights, are also discussed. However, challenges remain before these therapies can be fully translated into clinical practice. In conclusion, this review emphasizes the significance of miRNAs in breast cancer research and their potential as innovative diagnostic and therapeutic tools. A deeper understanding of miRNA dysregulation in breast cancer is essential for their successful application in clinical settings. With continued research, miRNA-based approaches hold promise for improving patient outcomes in this devastating disease.

PER3 plays anticancer roles in the oncogenesis and progression of breast cancer via regulating MEK/ERK signaling pathway.