Research Grants Awarded
The Epigenetic Impact Of Cancer Associated-Fibroblasts On Breast Neoplasm.
Career Catalyst Research
(1) BACKGROUND and RATIONALES: Silencing of tumor suppressor genes by DNA hypermethylation is a common event observed in breast cancer. Yet, the mechanistic cause(s) conveying such phenomenon remains unknown. However, recent studies have demonstrated that oncogenic signals from breast cancer-associated stromal fibroblasts (CAF) can dictate the disease progression and support the growth of the tumor, implicating that CAF might play a critical role in suppressing tumor suppressor genes by augmenting DNA methylation. This proposal, therefore, will utilize a laboratory co-culture model to explore ?whether? and ?how? the microenvironmental signals (from CAF) govern the silencing of tumor suppressor genes.
(2) HYPOTHESES: By augmenting DNA methylation, we hypothesize that CAF can confer epigenetic alterations in the non-cancerous breast epithelial cells. As a consequence of epigenetic perturbations, tumor suppressor genes and other genes that are critical in impeding breast tumorigenesis may become silenced. Moreover, the non-cancerous breast epithelial cells used in our laboratory model may be comparable to the ?histological normal appearance? epithelial cells adjacent to the breast tumor. We attempt to simulate how CAF interacts with the epithelial cells adjacent to the tumor core in patients, despite the fact that these cells may be judged as ?normal? cells by pathologists because of their normal appearance. Hence, we postulate that epigenetic impact from CAF observed in co-culture studies can be similarly observed in clinical specimens. Our model attempts to depict how the microenvironmental signals from CAF can convert these neighboring normal epithelial cells into a pre-malignant state and worsen the disease prognosis. If proven, such a paradigm will provide innovative molecular evidence in part explaining how CAF facilitates the invasion, metastasis and recurrence processes of this disease. However, the etiologic signals from CAF and govern promoter DNA hypermethylation in the normal epithelial cells remains unknown. By using pathway analysis software, we have deduced a hypothesis that DNA hypermethylation observed in our co-culture system is likely governed by the activated AKT pathway. We also hypothesize that additional signals upstream from AKT might be provoked from CAF. The overall signaling pathway cascades will be thoroughly studied in this project.
(3) SPECIFIC AIMS and DESIGN: To study the tumor microenvironment impact on breast cancer, laboratory co-culture system providing cell-to-cell contact is employed to simulate the physical and biochemical interactions between CAF and the normal breast epithelial cells adjacent to the primary tumor. MCF10A, an immortalized non-cancerous breast epithelial cell line, will be subjected to co-culture with CAF and the resultant cells will be analyzed for DNA methylation. The identified loci will be prioritized according to their known functions in relation to oncogenic activities, and their hypermethylation status will be further validated in independent replicates isolated from different batches of HMEC (Human Mammary Epithelial Cells). Moreover, CAF influenced-MCF10A cells will be evaluated for the gain of pre-neoplastic phenotypes in vitro including the rate of cell proliferation, susceptibility to chemotherapy drugs, and capability of invasion. On the other hand, we will decipher the signaling pathway cascades that govern the DNA hypermethylation. Using expression profiling analysis, we have identified a candidate signal pathway (AKT) whose activation may convey DNA hypermethylation. Furthermore, we will expand our study by seeking additional signaling pathway(s) that is (are) directly provoked from CAF and situated upstream of AKT activation. If identified, the pathway of interest will be abrogated by molecular targeting methods. The pathway-diminished cells will be subjected to co-culture and the resultant MCF10A will be further analyzed for AKT activation and DNA methylation. In addition, the in vitro finding regarding AKT activation and DNA hypermethylation will be confirmed in the clinical specimens.
(4) CLINICAL IMPACT: We attempt to provide a proof-of-principle demonstrating that micro-environment signals, provoked from breast cancer fibroblasts, can convey AKT activation in the neighboring non-cancerous epithelial cells. Activated AKT signaling pathway subsequently induces DNA methylation and tumor suppressor gene silencing. This phenomenon is novel and has not been reported in any human cancer. Since methylation is chemically and somatically stable, detection of hypermethylated genes in breast tissue may become a unique strategy for diagnosis. Multiple hypermethylated genes identified in this study can be incorporated into a test panel simultaneously analyzing multiple loci. These combinatorial tests will have higher sensitivity and specificity, as opposed to a test using a single gene. A PCR-based methylation screen test (MSP) used in our study is highly robust, and easy to perform with low cost. Together, these advantages provide the feasibility of adapting this research method into a clinical test. Moreover, these hypermethylated genes may correlate with transformation phenotypes. Therefore, they are potential candidates to be developed into promising predictors for poor prognosis. On the other hand, if signaling pathway cascades can be uncovered, molecular targeting by combating signal pathway(s) may impede the progression of malignancy. This strategy can be developed into a unique regimen to treat this disease, and reduce the mortality rate of women with breast cancer.
While the majority of breast cancer consists of a special type of abnormal breast cells known as epithelial cells, the primary tumor mass is surrounded by another layer of supportive connective tissue. The surrounding tissue along with its secreted soluble proteins is collectively known as tumor microenvironment. Cells taken from the tumor microenvironment cannot directly cause a breast tumor in laboratory mice, but can assist the growth, metastasis, and recurrence of breast cancer. Within the tumor microenvironment, fibroblasts are the most active secretory cell type that can aid the progression of breast cancer. Therefore, we attempt to study the effect of fibroblasts on breast cancer. This effect is, perhaps, mediated by changing the genomic DNA structure in the targeted breast epithelial cells. The best studied DNA structure change is by adding a methyl molecule to the DNA bases, a type of process known as epigenetic alterations. Methylation is a very stable chemical modification without altering the DNA sequence. The main purpose of DNA methylation is to inhibit the production of its coded proteins which otherwise should be manufactured from the un-modified DNA. DNA methylation is an important physiological process to control tissue-specific expression. For example, during various developmental stages, some proteins are made in certain organs, but are shut down in others by DNA methylation. Likewise, many tumor suppressor genes are silenced by DNA methylation in human cancers, without somatic DNA mutations which require a lengthy process. Under normal circumstances, tumor suppressor genes produce essential proteins to protect an individual from acquiring cancer. However, its absence or reduced quantity may confer human malignancies including breast cancer. We hypothesize that CAF provoke epigenetic impact on breast epithelial cells (mainly by DNA methylation) to turn off the tumor suppressor genes. To address this issue, this study has set off a laboratory system to grow CAF along with the non-cancerous breast epithelial cells line (known as MCF10A) in a cell-to-cell contact manner. Three weeks later, the epithelial cells are segregated from the fibroblasts and analyzed for DNA methylation. The rationale for using MCF10A cells is the fact that they are comparable to the ?histologically normal appearance? epithelial cells adjacent to the tumor core. As such, our model can simulate how cancer fibroblasts convert the neighboring epithelial cells into pre-neoplastic or cancerous stage by methylating their tumor suppressor genes. If proven, our laboratory model may simulate how CAF promote the progression of breast cancer in patients. Thus far, we have identified at least six genes (including two tumor suppressor genes) that are methylated in MCF10A, as a consequence of interacting with CAF. This novel result, therefore, has provided the first proof-of-principle demonstrating that breast CAF can silence tumor suppresser genes by augmenting DNA methylation in the targeted epithelial cells. The epigenetic changes may drive the neighboring epithelial cells (close to CAF) towards a pre-cancerous stage, expand the tumor mass, and worsen the disease outcome. Such phenomenon has not been reported in human cancer. Moreover, we will evaluate if the CAF-influenced MCF10A cells gain any behavioral changes that aid breast prognosis, such as growing at a faster speed, resisting chemotherapy drugs, or invading to a distal site (a process preceding metastasis). If proven, methylated genes might be indicative of worsening breast cancer and can be developed into potential predictors (biomarkers) for poor prognosis. Furthermore, the methylation status of these six genes will be validated in clinical breast epithelial cells microdissected from the tissue adjacent to the CAF. As studies pertaining to CAF-induced DNA methylation has been undertaken, another goal of this project is to identify what signal(s) (released from CAF) is (are) triggering DNA methylation in MCF10A. Our pilot studies have implicated a candidate mechanistic cause that involves an excessive activation of a signaling pathway (known as AKT) which is otherwise set at a stand-by mode in normal cells. Although activation of AKT has been well studied in breast caner, the etiologic cause (from CAF) that governs abnormal AKT activation and DNA methylation remains unknown. Therefore, we will take on a statistical approach coupled to bioinformatics (using software GenMAPP and MAPPFinder) to identify the mechanistic cause (from CAF) that imposes AKT activation followed by DNA methylation (in MCF10A cells). If identified, a therapy regimen can be developed by combating this signaling pathway and subsequently abolishing DNA methylation in the targeted cells. Disrupting signaling pathway(s) may impede the invasion, metastasis, resistance to chemotherapy drugs, or disease recurrence. Altogether, the outcome of this innovative molecular therapy will reduce the mortality rate of women with breast cancer.