REGULATION BY P38 AND NR2F1 SIGNALING OF EARLY DISSEMINATION AND DORMANCY OF BREAST CANCER CELLS

Project Details

Description

Training ProgramMy career goals are to expand my scientific training in the study of breast cancer, more specifically within the area of metastasis and dormancy. Dr. Aguirre-Ghiso is a world-recognized leader in the signaling mechanisms regulating metastasis and dormancy. Thus, this is a unique opportunity to develop my postdoctoral training. The proposed training also includes co-mentoring by Dr. John Condeelis, who is a world expert in the imaging and metastasis fields. This will allow me to train in state-of-the-art imaging methods applied to the area of breast cancer cell motility, invasion, and metastasis. The training plan will allow me to train also in fluorescence activated cell sorting, multiphoton live imaging microscopy, and magnetic cell separation. Also, I seek to learn specific techniques such as isolation and characterization of disseminated tumor cells (DTCs), circulating tumor cells using single cell profiling techniques, and molecular marker detection in situ. I expect that this multidisciplinary research program will strengthen my scientific development as an independent investigator and will eventually provide me the tools to develop my own research program in metastasis biology with an emphasis in dormancy mechanisms.Research PlanBackground: DTCs are present in secondary organs (i.e., bone marrow, BM) in 35% of breast cancer patients upon diagnosis. DTCs can give origin to metastasis and these cells are detected even in patients carrying pathologically defined pre-invasive lesions (i.e., ADH, DCIS). This suggests that dissemination can occur early during cancer progression. In addition, DTCs can remain dormant for several years before metastases develop. In the MMTV-Neu mouse model, DTCs are detected in the BM of animals bearing preinvasive lesions, and these DTCs also appear to be dormant. We reasoned that understanding how early dissemination is regulated may provide new insight into how to predict and treat disseminated disease. Our preliminary data show that the transcription factor NR2F1, which is a target of the tumor suppressor kinase p38, negatively regulates beta-catenin and also induces tumor cell dormancy. We also found that NR2F1 is lost in premalignant and overt Neu+ tumors, but it is re-expressed in dormant BM DTCs. Importantly, inhibition of p38 in MMTV-Neu mice bearing premalignant lesions resulted in further loss of NR2F1 expression, and this coincided with a robust induction of an EMT in premalignant MECs. These events led to early dissemination of these premalignant MECs and their accumulation and or expansion in the BM.Hypothesis: We hypothesize that loss of NR2F1 promotes an EMT and dissemination of premalignant MECs. We further hypothesize that its re-expression is required to maintain dormancy of early DTCs at distant sites. Specific Aims: SA1: To determine whether early dissemination of premalignant MMTV-Neu MECs is due to an EMT and whether p38 and NR2F1 block these events. We will use MMTV-Neu-CFP transgenic mice untreated (DMSO) or treated with SB203580. Using CFP live imaging, we will study invasion and dissemination of premalignant MECs and compare their behavior with those bearing overt MMTV-Neu-CFP tumors. To correlate p38 activation with EMT induction, mammary glands sections will be used for immunohistochemistry for p-ATF2, p-ERK, beta-catenin, E-cadherin, Snail, and NR2F1. SA1.2: We will grow premalignant Neu+ colonies in 3D culture and treat them with SB203580, RNAi to p38alpha or NR2F1 or control treatments. We will score outward colony invasion and EMT induction. As a gain-of-function approach, we will retrovirally overexpress an inducible NR2F1 cDNA in MMTV-Neu-CFP premalignant or progressed tumor cells and the same markers will be tested. We will quantify the correlation of NR2F1 and P-p38 levels and the expression of beta-catenin and E-cadherin in MMTV-Neu-CFP and human breast tumor tissues. SA2: To uncover the mechanisms responsible for maintaining the quiescence of DTCs at secondary sites and test the role of NR2F1 in these events. MMTV-Neu-CFP transgenic mice will be treated with SB203580 or DMSO as in SA1. DTCs from BM and lung will be quantified and tested for the levels of NR2F1, RARbeta, SOX9, p27 (NR2F1 targets), phospho-H3, and Ki67 (e.g., proliferation markers) using IF and/or Q-PCR. This will reveal an association between these genes and dormancy or proliferation of DTCs. Finally, using MMTVNeu-CFP tumors carrying an inducible NR2F1 shRNA, we will test if induction of the shRNA in DTCs interrupts their dormancy in the BM and accelerates lung metastasis development after primary tumor surgery. We will correlate these changes with the modulation of RARbeta, SOX9, and p27 levels.Impact: Although the 5-year survival rate of breast cancer patient has increased in the last 10 years, the most frightening enemy is still metastasis development. Metastatic relapses will ultimately kill cancer patients, and because our current knowledge about DTC biology is limited, further investigation is urgently needed. Therefore, we believe that this project is original and significant for finding ways to identify patients carrying early-disseminated disease and to block DTCs from growing into metastasis.

StatusFinished
Effective start/end date15/03/1214/03/15

Funding

  • Congressionally Directed Medical Research Programs: $336,223.00

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