The Secretome of Human Neonatal Mesenchymal Stem Cells Modulates Doxorubicin-Induced Cytotoxicity: Impact in Non-Tumor Cells

Doxorubicin (Dox) is one of the most widely used treatments for breast cancer, although limited by the well-documented cardiotoxicity and other off-target effects. Mesenchymal stem cell (MSC) secretome has shown immunomodulatory and regenerative properties, further potentiated under 3D conditions. This work aimed to uncover the effect of the MSC-derived secretome from 3D (CM3D) or 2D (CM2D) cultures, in human malignant breast cells (MDA-MB-231), non-tumor breast epithelial cells (MCF10A) and differentiated AC16 cardiomyocytes, co-treated with Dox. A comprehensive proteomic analysis of CM3D/CM2D was also performed to unravel the underlying mechanism. CM3D/CM2D co-incubation with Dox revealed no significant differences in MDA-MB-231 viability when compared to Dox alone, whereas MCF10A and AC16 viability was consistently improved in Dox+CM3D-treated cells. Moreover, neither CM2D nor CM3D affected Dox anti-migratory and anti-invasive effects in MDA-MB-231. Notably, Ge-LC-MS/MS proteomic analysis revealed that CM3D displayed protective features that might be linked to the regulation of cell proliferation (CAPN1, CST1, LAMC2, RANBP3), migration (CCN3, MMP8, PDCD5), invasion (TIMP1/2), oxidative stress (COX6B1, AIFM1, CD9, GSR) and inflammation (CCN3, ANXA5, CDH13, GDF15). Overall, CM3D decreased Dox-induced cytotoxicity in non-tumor cells, without compromising Dox chemotherapeutic profile in malignant cells, suggesting its potential use as a chemotherapy adjuvant to reduce off-target side effects.

Endotoxin Triggers Tumor Initiation Events in Nontumorigenic Breast Epithelial Cells and Enhances Invasion-Related Phenotype in Pretumorigenic and Tumorigenic Breast Epithelial Cells

Inflammation is associated with the development of several cancers, including breast cancer. However, the molecular mechanisms driving breast cancer initiation or enhancement by inflammation are yet to be deciphered. Hence, we opted to investigate the role of inflammation in initiating and enhancing tumor-like phenotypes in nontumorigenic, pretumorigenic, and tumorigenic breast epithelial cells. Noncytotoxic endotoxin (ET) concentrations capable of inducing an inflammatory phenotype were determined for the different cell lines. Results showed that short-term ET exposure upregulated matrix metalloproteinase-9 (MMP-9) activity in nontumorigenic mammary epithelial cells of mouse (SCp2) and human origins (HMT-3522 S1; S1) and upregulated inflammatory mediators including nitric oxide (NO) and interleukin 1-β in tumorigenic human breast cells (MDA-MB-231), all in a dose-dependent manner. Long-term ET treatment, but not short-term, triggered the migration of SCp2 cells, and proliferation and migration of tumorigenic human breast cells MCF-7 and MDA-MB-231. Both short- and long-term ET exposures preferentially enhanced the invasion of pretumorigenic S1-connexin 43 knockout (Cx43-KO S1) cells compared to their nontumorigenic S1 counterparts. Moreover, both ET exposures disrupted lumen formation and apicolateral distribution of β-catenin in 3D cultures of S1 cells. In conclusion, ET treatment at concentrations that elicited inflammatory phenotype triggered tumor initiation events in nontumorigenic and pretumorigenic breast cells, and increased tumorigenicity of breast cancer cells. Our findings highlight the role of inflammation in enhancing migration, invasion, and loss of normal 3D morphology and suggest that such inflammatory insults can “add injury” to pretumorigenic and tumorigenic breast epithelial cells.

FAM83A is a Potential Biomarker for Breast Cancer Initiation

Background: Family with sequence similarity 83 member A (FAM83A) presents oncogenic properties in several cancers including breast cancer (BC). Recently, we reported FAM83A overexpression in normal breast tissues from women at high risk of breast cancer. We now hypothesize that FAM83A is a key factor in BC initiation. Methods: Immunohistochemical staining was used to evaluate FAM83A protein levels in both a normal breast tissue microarray (TMA, N=411) and a breast tumor TMA (N=349). EGFR staining and its correlation with FAM83A expression were also assessed. Lentivirus-mediated manipulation of FAM83A expression in primary and hTERT-immortalized breast epithelial cells was employed. Biological and molecular alterations upon FAM83A overexpression/downregulation and FAM83A’s interaction partners were investigated.Results: TMA analysis revealed a 1.5-fold increase in FAM83A expression level in BC cases as compared with normal breast tissues (p<0.0001). FAM83A protein expression was directly correlated with EGFR level in both normal and BC tissues. In in vitro assays, exogenous expression of FAM83A in either primary or immortalized breast epithelial cells promoted cell viability and proliferation. Additionally, Ingenuity Pathway Analysis (IPA) revealed that in normal cells FAM83A is involved in cellular morphology and metabolism. Mass spectrometry analysis identified DDX3X and LAMB3 as potential FAM83A interaction partners in primary cells, while we detected FAM83A interaction with cytoskeleton reorganization factors, including LIMA1, MYH10, PLEC, MYL6 in the immortalized cells.Conclusions: This study shows that FAM83A promotes metabolic activation in primary epithelial cells and survival in immortalized cells. These findings support its role in early breast oncogenesis.

Leptin Mediates Obesity-Induced DNA Damage in BRCA1 Breast Epithelial Cells

Background: Obesity is a risk factor for breast cancer development in women who carry a mutation in the DNA repair enzymes BRCA1 or BRCA2. Previously, we found that obesity was positively associated with DNA damage in breast epithelium from BRCA mutation carriers. Furthermore, factors secreted by obese breast adipose tissue stimulated DNA damage in BRCA mutant breast epithelial cells, suggesting a cross-talk between breast epithelial cells and the adipose tissue that surrounds them. We hypothesized that leptin, a hormone secreted in abundance by obese adipose tissue, may be a driver of DNA damage and/or decrease capacity for DNA repair in breast epithelial cells. If true, this would provide a molecular target for intervention to reduce the risk of tumor formation in this high-risk population of women. Methods: RNA-seq followed by Ingenuity Pathway Analysis (IPA) was conducted on primary breast epithelial organoids isolated from lean and obese BRCA mutation carriers. Breast adipose tissue obtained from lean and obese women were cultured as explants for 24 hours to produce lean and obese conditioned media (CM). The effect of leptin on DNA damage was assessed in a non-cancerous breast epithelial cell line (MCF10A) carrying a heterozygous BRCA1 mutation. Immunofluorescence staining of the DNA damage marker ƴH2AX was carried out after treatment with leptin (100ng-800ng), CM, or CM+leptin antibody, used to neutralize leptin. To test whether leptin affects DNA repair capacity, BRCA1+/- MCF10A cells were treated with leptin or vehicle and then irradiated (1Gy) to induce DNA damage. Resolution of damage was quantified at 0, 0.5, 4, 12, and 24 hrs post-irradiation. Results: IPA analysis identified leptin signaling as significantly upregulated in breast epithelial organoids from obese women compared with lean women. Both obese CM and leptin treatment induced DNA damage in BRCA1+/- MCF10A cells while lean CM did not have this effect. Neutralizing leptin in obese CM was sufficient to inhibit obese CM-mediated induction of DNA damage. No significant difference was observed between leptin or vehicle treatments on DNA repair capacity after irradiation of BRCA+/- MCF10A cells. Conclusions: These data identify leptin, an adipose-derived hormone, as a novel driver of DNA damage in breast epithelial cells. To date, no studies have elucidated the molecular mechanisms that explain the increased penetrance of breast cancer in obese BRCA mutation carriers compared to lean BRCA mutation carriers. This work suggests that leptin may be a mediator of the link between obesity and breast cancer development in this population. Further studies are warranted to determine if targeting the leptin signaling axis will be an effective risk reduction strategy in BRCA mutation carriers who have excess adiposity.

Oncogenic and Tumor Suppressive Components of the Cell Cycle in Breast Cancer Progression and Prognosis

Cancer, a disease of inappropriate cell proliferation, is strongly interconnected with the cell cycle. All cancers consist of an abnormal accumulation of neoplastic cells, which are propagated toward uncontrolled cell division and proliferation in response to mitogenic signals. Mitogenic stimuli include genetic and epigenetic changes in cell cycle regulatory genes and other genes which regulate the cell cycle. This suggests that multiple, distinct pathways of genetic alterations lead to cancer development. Products of both oncogenes (including cyclin-dependent kinase (CDKs) and cyclins) and tumor suppressor genes (including cyclin-dependent kinase inhibitors) regulate cell cycle machinery and promote or suppress cell cycle progression, respectively. The identification of cyclins and CDKs help to explain and understand the molecular mechanisms of cell cycle machinery. During breast cancer tumorigenesis, cyclins A, B, C, D1, and E; cyclin-dependent kinase (CDKs); and CDK-inhibitor proteins p16, p21, p27, and p53 are known to play significant roles in cell cycle control and are tightly regulated in normal breast epithelial cells. Following mitogenic stimuli, these components are deregulated, which promotes neoplastic transformation of breast epithelial cells. Multiple studies implicate the roles of both types of components—oncogenic CDKs and cyclins, along with tumor-suppressing cyclin-dependent inhibitors—in breast cancer initiation and progression. Numerous clinical studies have confirmed that there is a prognostic significance for screening for these described components, regarding patient outcomes and their responses to therapy. The aim of this review article is to summarize the roles of oncogenic and tumor-suppressive components of the cell cycle in breast cancer progression and prognosis.

eIF2α integrates proteotoxic signals both from ER and cytoplasm: Hsp70-Bag3 module regulates HRI-dependent phosphorylation of eIF2α

The major heat shock protein Hsp70 has been implicated in many stages of cancer development. These effects are mediated by a scaffold protein Bag3 that binds to Hsp70 and links it to components of multiple cancer-related signaling pathways. Accordingly, the Hsp70-Bag3 complex has been targeted by small molecules, which showed strong anti-cancer effects. Here, our initial question was how JG-98, an allosteric inhibitor of Hsp70 that blocks its interaction with Bag3, causes cell death. Breast epithelial cells MCF10A transformed with a single oncogene Her2 showed higher sensitivity to JG-98 then parental MCF10A cells. RNA expression analysis showed that this enhanced sensitivity correlated with higher induction of the UPR genes. Indeed, depletion of the pro-apoptotic UPR responsive transcription factor CHOP significantly protected cells from JG-98. Surprisingly, only the eIF2α-associated branch of the UPR was activated by JG-98, suggesting that the response was not related to the ER proteotoxicity. Indeed, it was dependent on activation of a distinct cytoplasmic eIF2α kinase HRI. HRI-dependent phosphorylation of eIF2α was also activated by the cytoplasmic proteotoxicity via Hsp70-Bag3 complex, which directly associates with HRI. Dissociation of Hsp70-Bag3 complex led to Bag3-dependent degradation of HRI via autophagy. Therefore, eIF2α integrates proteotoxicity signals from both ER and cytoplasm, and the cytoplasmic response mediates cytotoxicity of the Hsp70-Bag3 inhibitors.

Obese Adipose Tissue as a Driver of Breast Cancer Growth and Development: Update and Emerging Evidence

Obesity is an established risk factor for breast cancer growth and progression. A number of advances have been made in recent years revealing new insights into this link. Early events in breast cancer development involve the neoplastic transformation of breast epithelial cells to cancer cells. In obesity, breast adipose tissue undergoes significant hormonal and inflammatory changes that create a mitogenic microenvironment. Many factors that are produced in obesity have also been shown to promote tumorigenesis. Given that breast epithelial cells are surrounded by adipose tissue, the crosstalk between the adipose compartment and breast epithelial cells is hypothesized to be a significant player in the initiation and progression of breast cancer in individuals with excess adiposity. The present review examines this crosstalk with a focus on obese breast adipose-derived estrogen, inflammatory mediators and adipokines, and how they are mechanistically linked to breast cancer risk and growth through stimulation of oxidative stress, DNA damage, and pro-oncogenic transcriptional programs. Pharmacological and lifestyle strategies targeting these factors and their downstream effects are evaluated for feasibility and efficacy in decreasing the risk of obesity-induced breast epithelial cell transformation and consequently, breast cancer development.

Mechanistic Targets and Nutritionally Relevant Intervention Strategies to Break Obesity–Breast Cancer Links

The worldwide prevalence of overweight and obesity has tripled since 1975. In the United States, the percentage of adults who are obese exceeds 42.5%. Individuals with obesity often display multiple metabolic perturbations, such as insulin resistance and persistent inflammation, which can suppress the immune system. These alterations in homeostatic mechanisms underlie the clinical parameters of metabolic syndrome, an established risk factor for many cancers, including breast cancer. Within the growth-promoting, proinflammatory milieu of the obese state, crosstalk between adipocytes, immune cells and breast epithelial cells occurs via obesity-associated hormones, angiogenic factors, cytokines, and other mediators that can enhance breast cancer risk and/or progression. This review synthesizes evidence on the biological mechanisms underlying obesity-breast cancer links, with emphasis on emerging mechanism-based interventions in the context of nutrition, using modifiable elements of diet alone or paired with physical activity, to reduce the burden of obesity on breast cancer.