Transformation Of Normal Mammary Cells Research Articles

Abstract 4326: Tissue density can create a carcinogenic microenvironment for normal mammary epithelial cells

Abstract Background Breast cancer is the most diagnosed cancer and the fourth leading cause of cancer related mortality among females. Mammographic density (MD) is defined by the amount of stromal and epithelial tissues present in the breast tissue. Fifty percent of women have high MD and are 4-6 times more likely to develop breast cancer in their lifetime compared to women with low MD. Additionally, mammograms often miss abnormal lesions in high MD breast leading to late-stage diagnosis. The stromal microenvironment is characterized by extracellular matrix (ECM) reorganization and stiffness, and increase in ECM proteins including collagen I and fibronectin, which drives proliferation, tumor cell survival and migration. Expression of fibronectin and collagen are significantly higher in women with high MD compared to women with low MD. However, the mechanisms underlying the initiation and progression of breast cancer associated with MD remains largely unknown. Therefore, understanding the molecular pathways involved in the unique transformation of normal mammary cells to cancer cells in a dense microenvironment is an important step in finding diagnostic tools and therapeutic targets for prevention and treatment of breast cancer. We hypothesize that normal mammary epithelial cells behave like cancer cells in an ECM protein-rich microenvironment by activating pathways associated with tumor growth and survival and contributing to the initiation and progression of breast cancer. Methods We conducted an in vitro study, determining the effects of different concentrations of ECM proteins on the activation of pro-carcinogenic pathways including the MAPK pathway and the PI3 Kinase pathway using Western blotting analysis. This study helps us understand if there is a difference in the expression of ERK1/2 signaling and/or PI3 Kinase signaling in normal mammary cells (hTERT) seeded on culture dish coated with ECM-proteins (collagen and fibronectin), compared to a non-coated control. We also examined morphological changes that can occur in a breast cancer cell line (HCC1806) that are seeded on ECM-rich coating dishes compared to non-coated dishes. Results We observed changes in the morphology of breast cancer cell lines after cells were seeded on increasing concentration of extracellular matrix coatings using light microscopy. We also observed an increase in the expression of PI3 Kinase in normal mammary cell lines compared to control after cells were stressed using hypoxia. Conclusion Collagen and fibronectin-rich microenvironment can induce desmoplastic-like phenotype and behavior for normal mammary cells, which highlights the possible first steps in transformation of normal mammary cells to desmoplastic cells. Given that high MD is one of the major risk factors for breast cancer development, it is critical to decipher the molecular mechanisms associated with the high risk. Citation Format: Shayan Nazari, Pinku Mukherjee. Tissue density can create a carcinogenic microenvironment for normal mammary epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4326. doi:10.1158/1538-7445.AM2017-4326

Adipose-derived mesenchymal stem cells (ASCs) may favour breast cancer recurrence via HGF/c-Met signaling

Adipose tissue is a reservoir of Mesenchymal Stem Cells (Adipose-derived Mesenchymal Stem Cells, ASCs), endowed with regenerative properties. Fat graft was proposed for breast reconstruction in post-surgery cancer patients achieving good aesthetic results and tissues regeneration. However, recent findings highlight a potential tumorigenic role that ASCs may have in cancer recurrence, raising some concerns about their safety in clinical application. To address this issue, we established a model where autologous ASCs were combined with primary normal or cancer cells from breast of human donors, in order to evaluate potential effects of their interactions, in vitro and in vivo. Surprisingly, we found that ASCs are not tumorigenic per sè, as they are not able to induce a neoplastic transformation of normal mammary cells, however they could exhacerbate tumorigenic behaviour of c-Met-expressing breast cancer cells, creating an inflammatory microenvironment which sustained tumor growth and angiogenesis. Pharmacological c-Met inhibition showed that a HGF/c-Met crosstalk between ASCs and breast cancer cells enhanced tumor cells migration, acquiring a metastatic signature, and sustained tumor self-renewal. The master role of HGF/c-Met pathway in cancer recurrence was further confirmed by c-Met immunostaining in primary breast cancer from human donors, revealing a strong positivity in patients displaying a recurrent pathology after fat grafts and a weak/moderate staining in patients without signs of recurrence. Altogether our findings, for the first time, suggest c-Met expression, as predictive to evaluate risk of cancer recurrence after autologous fat graft in post-surgery breast cancer patients, increasing the safety of fat graft in clinical application.

Decreased eIF3e/Int6 expression causes epithelial-to-mesenchymal transition in breast epithelial cells

eIF3e/Int6 is a component of the multi-subunit eIF3 complex, which binds directly to the 40S ribosome to facilitate ribosome recruitment to mRNA and hence protein synthesis. Reduced expression of eIF3e/Int6 has been found in up to 37% of human breast cancers, and expression of a truncated mutant version of the mouse eIF3e/Int6 protein leads to malignant transformation of normal mammary cells. These findings suggest that eIF3e/Int6 is a tumor suppressor; however, a recent study has reported that a reduction of eIF3e/Int6 expression in breast cancer cells leads to reduced translation of oncogenes, suggesting that eIF3e/Int6 may in fact have an oncogenic role in breast cancer. To gain a better understanding of the role of eIF3e/Int6 in breast cancer, we have examined the effects of decreased eIF3e/Int6 expression in an immortalized breast epithelial cell line, MCF-10A. Surprisingly, we find that decreased expression of eIF3e/Int6 causes breast epithelial cells to undergo epithelial-to-mesenchymal transition (EMT). We show that EMT induced by a decrease in eIF3e/Int6 expression imparts invasive and migratory properties to breast epithelial cells, suggesting that regulation of EMT by eIF3e/Int6 may have an important role in breast cancer metastasis. Furthermore, we show that reduced eIF3e/Int6 expression in breast epithelial cells causes a specific increase in the expression of the key EMT regulators Snail1 and Zeb2, which occurs at both the transcriptional and post-transcriptional levels. Together, our data indicate a novel role of eIF3e/Int6 in the regulation of EMT in breast epithelial cells and support a tumor suppressor role of eIF3e/Int6.

P1-02-01: c-Jun N-Terminal Kinase 1 (JNK1) Inhibits Tumor Growth and Metastasis by Downregulating Epithelial to Mesenchymal Transition (EMT) and Stem Cell-Related Genes.

Abstract Murine and human mammary cancers often show dysregulation of important signaling pathways including the canonical Wingless-iNTegrated (Wnt) and avian ERythroBlastosis oncogene B (ErbB) pathways. Transgenic expression of Wnt ligands causes transformation of normal mammary cells in mice, and Wnt10b is frequently upregulated in human breast cancers. Overexpression of ErbB ligands and amplification of receptors have also been implicated in human breast cancer. JNK1 is a tumor suppressor in the skin and intestinal epithelium, and JNKs are known to integrate ErbB and Wnt pathways as well as others to control cell growth and differentiation. In a murine mammary cancer model where 4T1.2 cells were injected into the mammary gland, reducing JNK1 levels by expressing shRNA (shJNK1) resulted in increased tumor growth and lung metastasis compared to mice injected with control vector- (pSM2) expressing cells. A microarray analysis comparing gene expression between shJNK1 and control tumors revealed 2 and 2.5-fold increases in the ErbB pathway genes Nrg3 and ETV5, 2-fold increases in the Wnt genes Bcl-9 and Wnt10a, and a 1.6-fold increase in the EMT gene Twist1. RT-PCR analysis of in vitro grown 4T1.2 cell lines transfected with shJNK1 confirmed increased expression of Bcl-9 and ETV5, as well as the ETV5 target Cox-2. ErbB2 protein was also overexpressed. The shJNK1 cells showed upregulation of pERK in response to Heregulin1 (an Erbb2/3 ligand) and Fibroblast Growth Factor (FGF) 1, which further amplifies canonical Wnt signaling. In a p53-/− tumor model, ETV5, Bcl-9, and Cox-2 were still upregulated in jnk1-/−compared to wildtype tumor cells, indicating this effect is p53-independent. In the normal mammary gland, a 4-fold increase in ETV5 and a 5-fold increase in Twist1 were found in jnk1-/− mice compared to wildtype, further indicating that this effect is dependent on JNK1 alone. The ErbB and Wnt pathways are known to upregulate EMT and stem-cell related genes, however, the involvement of JNK1 in these effects is a novel hypothesis. Thus far, our data suggest that JNK1 deficiency targets these oncogenic pathways to contribute to a more aggressive tumor phenotype due to heightened EMT and “stem-cellness”. Further studies using cell sorting and differentiation assays will determine whether normal jnk1-/− glands contain a higher fraction of stem cells than wildtype glands. Inhibition of EMT genes and/or ETV5 in the p53-/−;jnk1-/− cancer cells will determine whether the tumor growth or metastasis phenotypes are dependent on these genes. EMT and stem-cell genes are frequently expressed in human breast cancer subtypes that exhibit low survival rates, and EMT is known to be linked to increased metastasis. Some of these sub-types, such as claudin-low tumors, currently have no molecularly-targeted treatments, therefore it is important to determine what proteins critically contribute to these phenotypes so that efficient and effective treatments can be developed. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-02-01.

Antisense Downregulation of a Mouse Mammary Tumor Virus Activated Protooncogene in Mouse Mammary Tumor Cells Reverses the Malignant Phenotype

Activation of the protooncogeneWnt-1 by insertion of the mouse mammary tumor virus (MMTV) is known to cause mammary tumors in mice.Wnt-1 expression in mammary glands has been postulated to confer direct local growth stimulation of mammary epithelial cells leading to their acquisition of a preneoplastic state.Wnt-1 expression also induces morphological alterations in cultured normal mammary cells. However, it has not been determined whether or not transformed mammary cells require continuousWnt-1 expression for their ability to form tumorsin vivo.To address this question, we constructed antisense and senseWnt-1 expression vectors containing a synthetic promoter composed of five high-affinity glucocorticoid response elements (GRE5). This promoter is at least 50-fold more inducible by dexamethasone than the promoter contained in the long terminal repeats of MMTV. The vectors were introduced into a mouse mammary tumor cell line (R/Sa-MT) that expresses high levels of endogenousWnt-1 mRNA and forms rapidly growing tumors when transplanted into syngeneic hosts. Of the 12 stably transfected cell lines established (9 with antisense and 3 with sense constructs), 2 antisense cell lines (R/Sa-MT/antisense) and 1 sense cell line (R/Sa-MT/sense) were examined for inducibility by dexamethasone of antisense and senseWnt-1 RNAs, changes in endogenousWnt-1 RNA expression, and changes in cell morphology. The growth patterns of the cellsin vitroandin vivowere also examined. Our results show that (1) the levels of the expression of endogenousWnt-1 mRNA and protein were reduced significantly (>80%) in those cells (R/Sa-MT/antisense) that expressed antisenseWnt-1 RNA at high levels following exposure to dexamethasone, compared to the R/Sa-MT/sense and R/Sa-MT control cells and (2) transplantation of the R/Sa-MT/antisense cells produced smaller tumors (≃0.2 cm in 16 weeks) compared to the tumors (≃2.0 cm in 8 weeks) that were produced by the R/Sa-MT/sense and R/Sa-MT cells. We therefore suggest thatWnt-1 expression is required not only for the transformation of normal mammary cells into tumor cells, but also for the maintenance of their tumorigenicity.