Development Of Human Breast Cancer Research Articles

OPTIMIZATION OF EXPERIMENTAL MODEL SYSTEMS FOR EVALUATING RECIPROCAL INFLUENCE OF BIFIDOBACTERIUM ANIMALIS AND HUMAN BREAST CANCER CELLS IN VITRO.

The development of human breast cancer (BC) is known to be closely related to disturbances in the mammary gland microbiota. Bacteria of the genus Bifidobacterium are an important component of normal breast microbiota and exert antitumor activity. The molecular-biological mechanisms of interaction between BC cells and microbiota members remain poorly studied yet. The aim of this study was to develop and optimize an experimental model system for the co-cultivation of BC cells with Bifidobacterium animalis in vitro. Human ВС cells of the MCF-7, T47D, and MDA-MB-231 lines, as well as live and heat-inactivated bacteria of Bifidobacterium animalis subsp. lactis (B. animalis) were used as research objects. The growth kinetics and viability of B. animalis in the presence of different ВС cell lines and without them were determined by both the turbidimetry method and seeding on an elective nutrient medium. Glucose consumption and lactate production by bifidobacteria were assessed by biochemical methods. The viability of BC cells was determined by a standard colorimetric method. The growth kinetics of B. animalis in the complete DMEM nutrient medium showed standard patterns. The indicators of glucose consumption and lactate production of B. animalis confirm its physiological metabolic activity under the growth conditions. The presence of BC cells in the model system did not affect the duration of the growth phases of the B. animalis cells' population but contributed to the increase in their counts. A significant decrease in the number of live BC cells of all studied lines was observed only after 48 h of co-cultivation with live B. animalis. To achieve similar suppression of the BC cell viability, 10-30-fold higher counts of heatinactivated bacteria were required compared to live ones. The optimal conditions for co-cultivation of human BC cells and living B. animalis cells in vitro have been identified.

Mouse Mammary Tumour Virus (MMTV) in Human Breast Cancer-The Value of Bradford Hill Criteria.

For many decades, the betaretrovirus, mouse mammary tumour virus (MMTV), has been a causal suspect for human breast cancer. In recent years, substantial new evidence has been developed. Based on this evidence, we hypothesise that MMTV has a causal role. We have used an extended version of the classic A. Bradford Hill causal criteria to assess the evidence. 1. Identification of MMTV in human breast cancers: The MMTV 9.9 kb genome in breast cancer cells has been identified. The MMTV genome in human breast cancer is up to 98% identical to MMTV in mice. 2. Epidemiology: The prevalence of MMTV positive human breast cancer is about 35 to 40% of breast cancers in Western countries and 15 to 20% in China and Japan. 3. Strength of the association between MMTV and human breast cancer: Consistency—MMTV env gene sequences are consistently five-fold higher in human breast cancer as compared to benign and normal breast controls. 4. Temporality (timing) of the association: MMTV has been identified in benign and normal breast tissues up to 10 years before the development of MMTV positive breast cancer in the same patient. 5. Exposure: Exposure of humans to MMTV leads to development of MMTV positive human breast cancer. 6. Experimental evidence: MMTVs can infect human breast cells in culture; MMTV proteins are capable of malignantly transforming normal human breast epithelial cells; MMTV is a likely cause of biliary cirrhosis, which suggests a link between MMTV and the disease in humans. 7. Coherence—analogy: The life cycle and biology of MMTV in humans is almost the same as in experimental and feral mice. 8. MMTV Transmission: MMTV has been identified in human sputum and human milk. Cereals contaminated with mouse fecal material may transmit MMTV. These are potential means of transmission. 9. Biological plausibility: Retroviruses are the established cause of human cancers. Human T cell leukaemia virus type I (HTLV-1) causes adult T cell leukaemia, and human immunodeficiency virus infection (HIV) is associated with lymphoma and Kaposi sarcoma. 10. Oncogenic mechanisms: MMTV oncogenesis in humans probably differs from mice and may involve the enzyme APOBEC3B. Conclusion: In our view, the evidence is compelling that MMTV has a probable causal role in a subset of approximately 40% of human breast cancers.

Overexpression of PSMC2 promotes the tumorigenesis and development of human breast cancer via regulating plasminogen activator urokinase (PLAU)

Emerging evidence has declared that Proteasome 26S subunit ATPase 2 (PSMC2) is involved in tumor progression. However, its role in breast cancer has not been investigated. Therefore, we sought to establish a correlation between breast cancer and PSMC2. PSMC2 expression in tissues was detected by immunohistochemistry. Loss-of-function study was used to evaluate the effects of PSMC2 knockdown in cell proliferation, apoptosis and migration. A gene microarray was performed to explore the potential downstream of PSMC2 with the help of Ingenuity Pathway Analysis (IPA). The effects of the PSMC2/PLAU axis on breast cancer were examined in vitro. Compared to para-cancer tissues, PSMC2 level was considerably elevated in breast cancer, which was significantly correlated with tumor grade. Knockdown of PSMC2 suppressed breast cancer progression in vitro and in vivo. The mechanistic research revealed that PSMC2 promotes the development and progression of human breast cancer through interacting with PLAU. Outcomes of our study showed that overexpression of PSMC2 provide tumorigenic and metastatic advantages in breast cancer, which may involve the regulation of PLAU. This study not only reveals a critical mechanism of breast cancer development, but also provides a promising therapeutic target for breast cancer treatment.

Abstract 2965: Generation of a human breast tissue mimetic organoid model

Abstract Purpose: Much effort has been focused on understanding breast cancer tumorigenesis. However, the regulation of human breast cancer development by gene mutations, hormone perturbation, external chemicals, and environmental cues is poorly understood at the molecular and cellular level. Although mouse models have been invaluable in advancing the knowledge of breast cancer tumorigenesis and progression, human breast ex vivo models comprising the breast tissue microenvironment are needed to help elucidate the underlying mechanisms of breast cancer risk factors. Here, we report a new human breast tissue mimetic organoid model. Methods: Fresh human breast tissue specimens from reduction mammaplasty reduction and prophylactic surgeries were cut and digested overnight. The breast microtissue containing stroma components were collected via centrifugation. They were then cultured in 3D using a matrix mixture containing Matrigel, collagen I, and specially formulated organoid medium. Using culture inserts, a “sandwich” method was utilized, in which we added 1 layer of matrix mixture, followed by 2 layers of organoids and matrix mixture, and a final layer of matrix mixture. Imaging analyses, including immunohistochemistry, immunofluorescence, and highly-multiplexed CODEX imaging, were used to study cellular marker expression in the organoids. Results: Our 3D culture system is distinct from previous models that reconstitute 3D mammary microstructures using isolated individual cell populations and is capable of growing patient-derived breast organoids for 3 weeks and longer. Via CODEX imaging we spatially localized more than 25 antigens for different cell types, allowing us to produce detailed characterizations of the organoid microarchitecture. Breast tissue microstructures with ducts and lobules along with stromal cell types normally found in the human breast are present. For the first time, we found that different stromal cell types grow alongside the mammary epithelium in the organoid system. The cultured breast organoids are hormone responsive and can be subcultured. We also found that BRCA1 mutant organoids are more sensitive to estrogen and progesterone treatment compared with BRCA1 wildtype, as indicated by organoid size changes. Furthermore, BRCA1 mutant organoids display unique gene expression profiles related to oxidative stress and DNA damage response. Conclusion: We have developed a 3D human breast tissue mimetic culture model that phenocopies the breast tissue microenvironment. This model will serve as the foundation for refining a breast organoid system that can faithfully recapitulate the physiology of human breast tissue. It will pave the way for a novel screening system that will test the pathogenic effects of breast cancer risk factors, providing a useful tool for elucidating the biological mechanisms of breast cancer tumorigenesis. Citation Format: Tian-Yu Lee, Nadya Nikulina, Srivarshini C. Mohan, Edward Ray, Armando Giuliano, Oliver Braubach, Xiaojiang Cui. Generation of a human breast tissue mimetic organoid model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2965.

Combination treatment with immunogenic and anti-PD-L1 polymer-drug conjugates of advanced tumors in a transgenic MMTV-PyMT mouse model of breast cancer

Immune checkpoint blockade has revolutionized the treatment of tumors with immunogenic microenvironments. However, low response rate and acquired resistance are still major challenges. Herein we used a more clinically relevant model of transgenic MMTV-PyMT tumor that more closely mimics the development of human breast cancer in an immunocompetent background to investigate a polymer-based chemo-immunotherapy. We have found that tumors acquired an increased degree of immune suppression during progression, rendering them unresponsive to anti-PD-L1 therapy. To treat large tumors at their advanced stage, we applied a combination strategy consisting of two polymer-drug conjugates that could induce immunogenic cell death (ICD) and disrupt the PD-L1/PD-1 interaction, respectively. Although ICD-inducing conjugate remodeled tumor immune microenvironment by facilitating significant CD8+ T cell infiltration, advanced tumor adapted the immune suppressive mechanism of elevating PD-L1 expression on both cancer cells and myeloid cells thereafter to enable continued tumor growth. Concurrent treatment of PD-L1 blocking conjugate not only abrogated the PD-L1 expression from the two disparate cellular sources, but also considerably reduced the number of immunosuppressive myeloid cells, thereby leading to a significant shrinkage of advanced tumors. Our data provide evidence that combinatory strategy of ICD–inducing and PD-L–blocking modalities could reverse immune suppression and establish a basis for the rational design of cancer immunotherapy.

Molecular mechanism study of BPAF-induced proliferation of ERα-negative SKBR-3 human breast cancer cells in vitro/in vivo

Bisphenol AF (BPAF) is a known estrogen disruptor of the ERα pathway. The aim of the present study was to characterize the proliferation effects of BPAF on ERα-negative SKBR-3 breast cancer cells with mechanistic insights. BPAF at low concentrations (0.001–0.1 μM) significantly induced the proliferation of SKBR-3 cells. In a SKBR-3 tumor model in BALB/c nude mice, BPAF at 100 mg/kg body weight/day also significantly promoted the growth of SKBR-3 tumors. Low concentrations of BPAF markedly increased the expression of G protein-coupled estrogen receptor (GPER1), c-Myc, CyclinD1 and c-Fos proteins, and enhanced phosphorylation of extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) in SKBR-3 cells. Further, BPAF significantly upregulated mRNA levels of related target genes in SKBR-3 cells and SKBR-3 tumor tissues in nude mice. The GPER1 inhibitor G15 and phosphatidylinositide 3-kinase (PI3K) inhibitor wortmannin (WM) inhibited phosphorylation of Erk and Akt. The specific signal inhibitors also markedly decreased the expression of target genes and weakened the cell proliferation induced by low-concentration BPAF. The findings showed that GPER1 could independently regulate BPAF-induced proliferation of SKBR-3 cells without requiring ERα. These results provide mechanistic insights into the effects of BPAF regarding ERα-negative human breast cancer development.

Long non-coding antisense RNA HYOU1-AS is essential to human breast cancer development through competitive binding hnRNPA1 to promote HYOU1 expression

Triple negative breast cancer (TNBC) has poor prognosis due to lack of biomarker and therapeutic target. Emerging research has revealed long noncoding RNAs (lncRNAs) are involved in breast cancer progression, but their functions and regulatory mechanisms remain poorly understood, especially in TNBC. In this study, we performed lncRNA microarray analysis of five TNBC samples and their matched normal tissues, and discovered a number of differentially expressed lncRNAs. We identified an antisense lncRNA, HYOU1-AS, which is transcribed from the opposite strand of the hypoxia up-regulated 1 (HYOU1) gene, enriched in the nucleus and highly expressed in TNBC. HYOU1-AS knockdown could inhibit the proliferation and migration of the TNBC MDA-MB-231 cells, and reduce their xenograft tumor formation in nude mice. In mechanistic studies, we found that HYOU1-AS could promote the expression of HYOU1, a proliferative gene, through competitively binding to hnRNPA1, an RNA-binding protein, to relieve its post-transcriptional inhibition of the HYOU1 mRNA. Consistently, increased HYOU1 levels correlated with poor clinical outcomes of breast cancer patients based on our study of the TCGA database. Overall, our data indicated that the lncRNA HYOU1-AS promoted TNBC progression through upregulating HYOU1.

Regulatory effect of the MAFG‑AS1/miR‑150‑5p/MYB axis on the proliferation and migration of breast cancer cells.

Long noncoding RNA (lncRNA) MAF BZIP transcription factor G antisense RNA 1 (MAFG-AS1) has been demonstrated to serve an important role in the progression of various types of cancer, whereas its role in breast cancer has not been fully elucidated. The present study aimed to explore the potential role and underlying mechanism of MAFG-AS1 in breast cancer. To achieve this, the expression of MAFG-AS1, microRNA (miR)-150-5p and MYB was detected by reverse transcription-quantitative PCR. The binding between miR-150-5p and MAFG-AS1 or MYB was verified using a luciferase reporter assay. Cell proliferation was analyzed by MTS, apoptosis and cell cycle were detected by Annexin V/propidium iodide, and cell migration was analyzed by wound healing assay. The results demonstrated that the expression levels of MAFG-AS1 were significantly upregulated in breast cancer tissues and cells compared with those in normal breast tissues and cells. High MAFG-AS1 expression promoted the proliferation, migration and epithelial-mesenchymal transition of breast cancer cells. By contrast, miR-150-5p expression was reduced in breast cancer tissues compared with that in healthy breast tissues, and low expression of miR-150-5p was associated with poor overall survival in patients with breast cancer. Bioinformatics and luciferase assay revealed that MAFG-AS1 served as a sponge of miR-150-5p, and that miR-150-5p bound to MYB. The functional rescue assay results demonstrated that MAFG-AS1 knockdown suppressed the proliferation and migration of breast cancer cells by regulating miR-150-5p, which in turn targeted MYB. In conclusion, the results of the present study demonstrated that MAFG-AS1 functioned as a novel oncogenic lncRNA in the development of human breast cancer via regulating the miR-150-5p/MYB axis, which suggested that MAFG-AS1 may be a novel biomarker for the diagnosis and prognosis of human breast cancer.

Expression and promoter methylation status of OPCML andits functions in the inhibition ofcell proliferation, migration, and invasion in breast cancer.

Opioid binding protein/cell adhesion molecule-like (OPCML) has been demonstrated to be a tumor suppressor gene, as it has been shown in previous studies to play a tumor-suppressive role in a variety of cancers. However, the role of OPCML in breast tumorigenesis remains unclear. In this study, we analyzed OPCML expression in breast cancers and adjacent non-tumor tissue samples and examined its molecular function in the breast cancer-derived cell lines MDA-MB-231 and MCF7. We found that OPCML was downregulated in most breast cancer samples but that this protein was expressed in most adjacent non-tumor samples. The loss or downregulation of OPCML is associated with hypermethylation of its promoter. Methylation of the OPCML promoter was detected in all breast cancer cell lines and primary tumors but was not detected in surgical margin tissues and normal breast tissues. Furthermore, functional assays showed that ectopic OPCML expression could inhibit breast tumor cell proliferation in vivo and in vitro and further suppresses tumor cell migration and invasion. Our results show that OPCML exerts its tumor-suppressive functions in human breast cancer cells. Moreover, the promoter-specific hypermethylation of OPCML plays an important role in human breast cancer development.

VWCE Functions as a Tumor Suppressor in Breast Cancer Cells.

Breast cancer remains a leading cause of cancer-related death, for which the majority of deaths result from metastases. Von Willebrand factor C and EGF domain (VWCE) is a member of the Von Willebrand factor (VWF) gene family; however, its function, regulatory mechanism, and clinical value in breast cancer remain unclear. In the present study, we sought to elucidate the role of VWCE in breast cancer metastasis. We examined the expression of VWCE in breast cancer tissues and normal control tissues of 50 breast cancer patients. We found that VWCE expression was downregulated in breast cancer cells and tissues compared to normal breast epithelial cells or the adjacent normal tissues. To explore the role of VWCE in human breast cancer development, we introduced a VWCE-overexpressing or control lentiviral vector into the breast cancer MDA-MB-453 and MDA-MB-231 lines in vitro. The overexpression of VWCE inhibited the proliferation, migration, invasion, and chemoresistance of the breast cancer cell lines. More importantly, the forced expression of VWCE suppressed tumor formation and metastasis in nude mice. iTRAQ-based quantitative proteomic analysis revealed that VWCE overexpression induced a 10-fold decrease in the level of WD-repeat domain 1 (WDR1) protein expression. Rescue experiments further verified that WDR1 was a downstream molecule of VWCE, and WDR1 overexpression reversed the above effects of VWCE overexpression on tumor growth. Therefore, VWCE may represent a novel tumor suppressor, for which its deregulation promotes breast cancer progression via the upregulation of WDR1.

Chronic Stress Exposure Suppresses Mammary Tumor Growth and Reduces Circulating Exosome TGF-β Content via β-Adrenergic Receptor Signaling in MMTV-PyMT Mice.

Preclinical models of breast cancer have established mechanistic links between psychological stress and cancer progression. However, epidemiological evidence linking stress and cancer is equivocal. We tested the impact of stress exposure in female mice expressing the mouse mammary tumor virus polyoma middle-T antigen (MMTV-PyMT), a spontaneous model of mammary adenocarcinoma that mimics metastatic hormone receptor–positive human breast cancer development. MMTV-PyMT mice were socially isolated at 6 to 7 weeks of age during premalignant hyperplasia. To increase the potency of the stressor, singly housed mice were exposed to acute restraint stress (2 hours per day for 3 consecutive days) at 8 to 9 weeks of age during early carcinoma. Exposure to this dual stressor activated both major stress pathways, the sympathetic nervous system and hypothalamic-pituitary-adrenal axis throughout malignant transformation. Stressor exposure reduced mammary tumor burden in association with increased tumor cleaved caspase-3 expression, indicative of increased cell apoptosis. Stress exposure transiently increased tumor vascular endothelial growth factor and reduced tumor interleukin-6, but no other significant alterations in immune/inflammation-associated chemokines and cytokines or changes in myeloid cell populations were detected in tumors. No stress-induced change in second-harmonic generation-emitting collagen, indicative of a switch to a metastasis-promoting tumor extracellular matrix, was detected. Systemic indicators of slowed tumor progression included reduced myeloid-derived suppressor cell (MDSC) frequency in lung and spleen, and decreased transforming growth factor β (TGF-β) content in circulating exosomes, nanometer-sized particles associated with tumor progression. Chronic β-adrenergic receptor (β-AR) blockade with nadolol abrogated stress-induced alterations in tumor burden and cleaved caspase-3 expression, lung MDSC frequency, and exosomal TGF-β content. Despite the evidence for reduced tumor growth, metastatic lesions in the lung were not altered by stress exposure. Unexpectedly, β-blockade in nonstressed mice increased lung metastatic lesions and splenic MDSC frequency, suggesting that in MMTV-PyMT mice, β-AR activation also inhibits tumor progression in the absence of stress exposure. Together, these results suggest stress exposure can act through β-AR signaling to slow primary tumor growth in MMTV-PyMT mice.

Papillomavirus DNA is not Amplifiable from Bladder, Lung, or Mammary Gland Cancers in Dogs or Cats.

Simple SummaryPapillomaviruses (PVs) are well established to cause genital and oral cancer in humans. Additionally, some evidence suggests that these viruses may also have a role in the development of human lung, breast, and bladder cancer. Recent studies have revealed that PVs can influence the development of some cancers in cats and, less frequently, in dogs. In the present study, 47 lung, mammary gland, and bladder cancers in dogs and 25 cancers in cats were evaluated for evidence of any role PVs may have in cancer development. Histology did not reveal any lesions suggestive of PV infection, and no PV DNA was amplified from any cancer. Therefore, these findings suggest that PVs do not frequently infect these tissues and are therefore unlikely to be significant factors in the development of lung, mammary gland, or bladder cancer in either dogs or cats.Papillomaviruses (PVs) cause around 5% of all human cancers, including most cervical cancers and around a quarter of all oral cancers. Additionally, some studies have suggested that PVs could cause a proportion of human lung, breast, and bladder cancers. As PVs have been associated with skin cancer in cats and, more rarely, dogs, it was hypothesized that these viruses could also contribute to epithelial cancers of the lung, mammary gland, and bladder of dogs and cats. Formalin-fixed paraffin-embedded samples of 47 canine and 25 feline cancers were examined histologically for evidence of PV infection. Additionally, three sets of consensus PCR primers were used to amplify PV DNA from the samples. No histological evidence of PV infection was visible in any of the cancers. DNA from a bovine PV type was amplified from one sample, while two different samples were found to contain human PV DNA. However, these were considered to be contaminants, and no canine or feline PV types were amplified from any of the cancers. These results suggest that PVs do not frequently infect the lung, mammary gland, or bladder of dogs and cats and therefore are unlikely to be significant factors in the development of cancers in these tissues.

Novel olive oil phenolic (-)-oleocanthal (+)-xylitol-based solid dispersion formulations with potent oral anti-breast cancer activities.

Epidemiological studies have compellingly documented the ability of the Mediterranean diet rich in extra-virgin olive oil to reduce the incidence of certain malignancies, and cardiovascular diseases, and slow the Alzheimer's disease progression. S-(-)-Oleocanthal (OC) was identified as the most bioactive olive oil phenolic with documented anti-inflammatory, anticancer, and anti-Alzheimer's activities. OC consumption causes irritating sensation at the oropharynx via activation of TRPA1. Accordingly, a taste-masked formulation of OC is needed for its future use as a nutraceutical while maintaining its bioactivity and unique chemistry. Therefore, the goal of this study was to prepare a taste-masked OC solid formulation with improved dissolution and pharmacodynamic profiles, by using (+)-xylitol as an inert carrier. Xylitol was hypothesized to serve as an ideal vehicle for the preparation of OC solid dispersions due to its low melting point and sweetness. The optimized OC-(+)-xylitol solid dispersion was physically and chemically characterized and showed effective taste masking and enhanced dissolution properties. Furthermore, OC-(+)-xylitol solid dispersion maintained potent in vivo anti-breast cancer activity. It effectively suppressed the human triple negative breast cancer development, growth, and recurrence after primary tumor surgical excision in nude mice orthotopic xenograft models. Collectively, these results suggest the OC-(+)-xylitol solid dispersion formulation as a potential nutraceutical for effective control and prevention of human triple negative breast cancer.

Targeting the phosphoinositide 3-kinase/AKT pathways by small molecules and natural compounds as a therapeutic approach for breast cancer cells.

The phosphoinositide 3-kinase/AKT/mTOR (PI3K/AkT/mTOR) pathway plays a pivotal role in the uncontrolled growth, migration and development of human breast cancer. The elevated expression of TGF-β1 increases the PI3K/AkT/mTOR activity in human breast cancer tissue and potentially motivates tumor metastasis and resistance to chemotherapy. Here, we investigated whether treatment with PI3K/AkT/mTOR dual inhibitor NVP-BEZ235 alone or in combination with caffeic acid phenyl ester (CAPE) could prevent TGF-β1 effects on breast cancer cells. MCF-7 human breast cancer cells were exposed to TGF-β1 for 14days and then were treated with/without NVP-BEZ235 and/or CAPE. Cell viability, apoptosis, CXCR4 surface expression and mRNA levels of CXCR4 and TWIST-1 were analyzed in all treated groups. We found that treatment of human breast cancer cells with acombination of NVP-BEZ235 and CAPE increased induction of cellular death. Although flow cytometry analysis demonstrated that NVP-BEZ235 alone treatment reduced CXCR4 expression while increasing CXCR4 mRNA level; when NVP-BEZ235 was combined with CAPE, inhibition of CXCR4 surface expression and enhancement of CXCR4 mRNA expression was diminished. In addition, TWIST-1 mRNA expression was down regulated in samples treated with both NVP-BEZ235 and CAPE. These altogether signified that NVP-BEZ235 in combination with CAPE showed improved therapeutic efficacy in breast cancer cells by decreasing apoptotic resistance and reduction of CXCR4 and TWIST-1 expression at mRNA level could be one of mechanism of action.

Abstract 1873: Water-pipe smoking induces epithelial-mesenchymal transition and enhances cell invasion of human breast cancer cells

Abstract Water-pipe smoking (WPS) has been recently recognized as an emerging risk factor for human health. The exact role of WPS in human breast cancer development and progression has not been investigated yet. We herein explored for the first time the impact of WPS on epithelial-mesenchymal transition (EMT) and cell invasion, which are considered crucial for cancer progression and metastasis in human carcinomas including breast. Our data revealed that WPS can initiate EMT in two human breast cancer cell lines, MCF-7 and BT20. WPS enhanced the cell invasion ability of both cell lines compared with their matched controls. We also found that WPS provoke a down- and up-regulation of two important regulator genes of cancer progression: E-cadherin and focal adhesion kinase (FAK). In addition, our data pointed out that WPS induces the activation of Erk1/Erk2 signaling pathways, which could be behind the stimulation of EMT and deregulation of E-cadherin and FAKgenes. Taken together, our results imply the significant effects of WPS on several cellular processes and genes involved in breast cancer progression and metastasis. Further studies are necessary to identify the clinical relevance and exact role of WPS in human carcinogenesis and metastasis. Key words: Water-pipe smoking, breast cancer, EMT, cell invasion, cell adhesion Citation Format: Ala-Eddin Al Moustafa. Water-pipe smoking induces epithelial-mesenchymal transition and enhances cell invasion of human breast cancer 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 1873.

Abstract 4508: Cyclophilin A as a molecular switch regulating PRLr-Jak2 complex-mediated signaling, mammary tumorigenesis, and metastasis

Abstract Prolactin (PRL) and its receptor (PRLr) have been implicated in the development and progression of human breast cancer. PRL activates its receptor and induces activation of proximal Janus kinase 2 (Jak2). Jak2 associates with PRLr, phosphorylates c-terminus of the PRLr, which leads to Stat5 recruitment and activation. Cyclophilin A (CypA) is a peptidyl-prolyl isomerase (PPI), which is constitutively bound to the PRLr that catalyze the cis-trans interconversion of proline imide bonds. Treatment with Cyclosporine (CsA) inhibited CypA binding to the PRLr and blocked PRLr-driven activation of Jak2/Stat5. Recently, Waters et al., utilizing Fluorescence Resonance Energy Transfer (FRET) with transfectants expressing CFP and YFP-tagged forms of the growth hormone receptor (GHR) showed that GHR activation induced a rotational movement in C-terminus of the GHR, resulting in a loss of baseline FRET signal. Like the GHR, PRL stimulation of transfectants expressing CFP/YFP-tagged PRLr constructs resulted in a loss of FRET efficiency. In contrast, treatment with NIM811 (a non-immunosuppressive form of CsA) or siRNA knockdown of CypA resulted in a return of FRET signal in the presence of PRL. These studies reveal that ligand stimulation of the PRLr results in a conformational change as measured by FRET signal of the receptor that is reversed by CypA inhibition or knockdown, implicating CypA as the mediator of this conformational change and ligand-induced signaling. To further assess the consequences of CypA inhibition or knockdown on the PRLr/Jak2 mediated signaling/functions, analyses of phospho-tyrosine residues that are believed to be important for interactions/signaling were investigated in breast cancer cells. It was found that NIM811 inhibition or CypA shRNA knockdown significantly reduced prolactin-stimulated phosphorylation of PRLr/Jak2 intermediates in ER+/PR+ T47D cells in a time dependent manner. A microarray analysis revealed that NIM811 inhibited approximately 66% of the top 50 PRL induced genes. NIM811 inhibited ER-/+, and HER2+ breast cancer cell proliferation, survival, migration and anchorage-independent growth. Subsequent pre-clinical testing of NIM811 in relevant mouse mammary cancer models has found that NIM811 treatment of a TNBC xenograft inhibited primary tumor growth, outgrowth of macro-metastasis and induced central tumor necrosis. Furthermore, loss of CypA (by constitutive genetic deletion) in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. Overall, CypA modulates conformational change in the C-terminus of the PRLr through its PPI activity, and alters PRLr/Jak2 complex signaling/functions in breast cancer and mammary epithelium, identifying this isomerase as a novel target for therapeutic intervention as a chemo-preventive and as an inhibitor of metastasis. Citation Format: Shawn Hakim, Shannon E. Hedrick, Justin M. Craig, Charles V. Clevenger. Cyclophilin A as a molecular switch regulating PRLr-Jak2 complex-mediated signaling, mammary tumorigenesis, and metastasis [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 4508.

Quantitative Phase-Contrast Imaging for Distinction between Different States of Human Breast Cancer Cells

We present label-free phase imaging to differentiate human breast cancer cells for breast cancer diagnosis. Low-coherent quantitative phase microscopy (QPM) is exploited to provide speckle-free interference images of live cells adhered to a plate, from which the phase information of the individual cells can be extracted. The phase measurement performance of QPM is validated by the phase-derived optical thickness of a sub-wavelength featured sample, consistent with the results when using conventional atomic force microscopy (AFM). The phase sensitivity is recorded as ~44 mrad, corresponding to ~2.3 nm depth fluctuation during measurement. Phase-contrast imaging is performed with three breast epithelial cell lines: non-tumorous (MCF-10A), weakly malignant (MCF-7), and highly malignant (MDA-MB-231), which mimic an in vivo environments in development of human breast cancer. Our results indicate that MCF-10A and MDA-MB-231 exhibited significantly lower phase-shifts than those of MCF-7, but no difference in phase was observed between MCF-10A and MDA-MB-231. We expect that the use of the QPM approach would be beneficial for clinicians to quantitatively evaluate the breast cancer states from the unmodified specimen of breast biopsy.

Downregulation of MMSET impairs breast cancer proliferation and metastasis through inhibiting Wnt/β-catenin signaling.

BackgroundRecently, the biggest challenge in the treatment of breast cancer is the metastasis of breast cancer cells. Multiple myeloma SET protein (MMSET), a histone lysine methyltransferase, overexpressed in various human cancers, was reported to be associated with carcinogenesis of human cancers.MethodsExpression of MMSET in breast cancer cell lines and tissues was quantified by real-time PCR and Western blotting. Immunohistochemistry was employed to analyze MMSET expression in 163 clinicopathologically characterized breast cancer cases. Cell functional assays such as MTT assay, colony formation, BrdU assay, flow cytometry, wound healing, Transwell assay, and 3D culture were used to investigate the effect of MMSET in the development and metastasis of human breast cancer. Effects of MMSET on Wnt/β-catenin signaling pathway were further studied by using Western blotting analysis.ResultsOur results showed that MMSET expression was markedly overexpressed in breast cancer cells and clinical specimens and was significantly correlated with patients’ clinicopatho-logic characteristics and prognosis. Moreover, silencing endogenous MMSET significantly inhibited the proliferation, migration, and metastasis of breast cancer cells through inhibiting the Wnt/β-catenin pathway.ConclusionThis study found that the downregulated expression of MMSET impaired proliferation and metastasis of human breast cancer through inhibiting Wnt/β-catenin signaling pathway. Notably, our results indicated that MMSET could be a useful biomarker for the prognosis of breast cancer.

CircRNA‑CER mediates malignant progression of breast cancer through targeting the miR‑136/MMP13 axis.

Chondrocyte extracellular matrix‑related circular RNAs(circRNA‑CER) have been demonstrated to be involved in various diseases. However, its role in the development of human breast cancer is not clearly understood. The aims of the present study were to assess circRNA‑CER expression in paired cancer tissue and adjacent non‑tumor tissue from 24patients with breast cancer, and to explore the roles and mechanisms by which circRNA‑CER mediates the malignant progression of breast cancer cells. The results revealed that circRNA‑CER and matrix metalloproteinase13 (MMP13) were upregulated, whereas miR‑136 was downregulated in breast cancer tissues compared with adjacent non‑tumor tissues. Invitro silencing of circRNA‑CER using small interfering RNA (siRNA) had inhibitory effects on MCF‑7 breast cancer cell proliferation and migration, and similar results were obtained following overexpression of microRNA (miR)‑136 in MCF‑7 cells by transfection with miR‑136 mimics. The subsequent mechanistic study revealed that the expression levels of MMP13 were significantly lower in MCF‑7 cells following transfection with miR‑136 mimics, and silencing of circRNA‑CER enhanced miR‑136 and decreased MMP13 expression levels. Furthermore, silencing of miR‑136 by transfection with miR‑136 inhibitors resulted in an increase in MCF‑7 cell proliferation and migration. miR‑136 inhibitor‑derived biological effects were reversed by co‑transfection of cells with miR‑136 inhibitors and circRNA‑CER siRNA. Taken together, the present results suggested that circRNA‑CER may serve an important role in the progression of breast cancer by regulating the activity of the miR‑136/MMP13 axis, and may be a potential biomarker for the prediction and treatment of breast cancer.

Transcriptional regulation of normal human mammary cell heterogeneity and its perturbation in breast cancer.

The mammary gland in adult women consists of biologically distinct cell types that differ in their surface phenotypes. Isolation and molecular characterization of these subpopulations of mammary cells have provided extensive insights into their different transcriptional programs and regulation. This information is now serving as a baseline for interpreting the heterogeneous features of human breast cancers. Examination of breast cancer mutational profiles further indicates that most have undergone a complex evolutionary process even before being detected. The consequent intra‐tumoral as well as inter‐tumoral heterogeneity of these cancers thus poses major challenges to deriving information from early and hence likely pervasive changes in potential therapeutic interest. Recently described reproducible and efficient methods for generating human breast cancers de novo in immunodeficient mice transplanted with genetically altered primary cells now offer a promising alternative to investigate initial stages of human breast cancer development. In this review, we summarize current knowledge about key transcriptional regulatory processes operative in these partially characterized subpopulations of normal human mammary cells and effects of disrupting these processes in experimentally produced human breast cancers.