Research Grants Awarded
From Cell Culture To Mouse Models - Determining The Role Of Klf8 Transcription Factor In Human Breast Cancer Metastasis
Breast cancer remains one of the leading causes of death in women due to the lack of approaches to efficiently prevent tumor metastasis. Our long-term goal is to understand the mechanisms underlying breast cancer metastasis so that novel anti-metastasis strategies can be designed to improve patient survival.
EMT (epithelial to mesenchymal transition) is a determining step for a cancer cell to progress from a non-invasive to invasive state. However, how EMT is regulated during breast cancer progression and how it contributes to breast cancer invasion and metastasis are largely unknown. The defining event for EMT is disruption of E-cadherin-mediated intercellular adhesion, which leads to loss of epithelial morphology and gain of a motile and invasive fibroblast-like phenotype. This process is associated with the functional loss of E-cadherin due largely to the repression of its transcription. Downregulation of E-cadherin has been well correlated with breast cancer metastasis and poor patient survival.
Our previous studies identified Krppel-like factor 8 (KLF8) transcription factor as a downstream effector of FAK (Mol Cell?03 11 p1503). FAK plays a crucial role in breast cancer metastasis. Recently, we found that KLF8 plays an important role in oncogenic transformation and is highly overexpressed in invasive human breast cancer cells (Oncogene?07 26 p456). However, the potentially important role of KLF8 in human breast cancer metastasis is completely unknown.
Our foundation data resulting from overexpression of KLF8 in both MCF-10A, a human mammary epithelial cell line, and MDCK, a dog kidney epithelial cell line, strongly suggest that KLF8 is a potent inducer of EMT and invasiveness and a novel repressor of E-cadherin in the cells. We also demonstrate that the upregulation of KLF8 plays a large part in the loss of E-cadherin in invasive human breast cancer cells such as MDA-MB-231 and the cell invasiveness and is tightly correlated with the loss of E-cadherin in tumor tissues of metastatic breast cancer patients. THIS WORK HAS BEEN JUST PUBLISHED IN CANCER RESEARCH (CANCER RES?07 67 P7184). Furthermore, we found that KLF8 strongly activates the transcription of the matrix metalloproteinase MMP9, a primary contributor to breast cancer invasion. Our results provide strong evidence that KLF8 may play a critical role in human breast cancer metastasis.
We therefore hypothesize that KLF8 promotes human breast cancer invasion and metastasis by initiating EMT through repression of E-cadherin expression and by regulating the expression and activation of MMP9.
To test this hypothesis, we will first DETERMINE THE MOLECULAR MECHANISMS FOR KLF8-INDUCED EMT AND INVASIVENESS (Aim1). Using lentiviral infection, we have just generated MCF-10A cells with inducible expression of KLF8 (MCF-10A-iKLF8 cells) and MDA-MB-231 variant (4175TGL, a lung-prone metastastic cell line expressing thymidine kinase-GFP-luciferase fusion protein) with inducible knockdown of KLF8 (4175TGL-KLF8ikd cells). We will test whether E-cadherin overexpression or MMP9 knockdown can prevent/reverse EMT and invasiveness induced by KLF8 in the MCF-10A-iKLF8 cells; whether E-cadherin knockdown or MMP9 overexpression can protect or rescue the invasive phenotype in 4175TGL-KLF8ikd cells when KLF8 is knocked down; and the molecular mechanisms by which KLF8 regulates MMP9 expression and the correlation of this regulation with the cell invasiveness. We will then STUDY THE ROLE OF KLF8 IN REGULATING MAMMARY ACINUS FORMATION IN SEMI-IN VIVO 3D CULTURE MODEL (Aim2). We have established the 3D culture procedures that work very well in the lab. We will test whether induced KLF8 expression prevents the 3D acinus formation and break down pre-formed acini in the MCF-10A-iKLF8 cells; whether induced KLF8 knockdown results in formation of relatively normal acini in the 4175TGL-KLF8ikd cells; and if E-cadherin and MMP9 play a role in KLF8-regulated acinus formation. Finally, we will DETERMINE THE ROLE AND MECHANISMS FOR KLF8 IN TUMOR METASTASIS IN VIVO USING A MOUSE MODEL OF HUMAN BREAST CANCER. Using the 4175TGL-KLF8ikd cells, nude mice and biofluorescence and bioluminescence imaging, we will evaluate the inhibitory effect of KLF8 knockdown on the lung metastasis after tail vein injection of the cells and mammary fat pad xenografts; characterize the circulating tumor cells (CTC) derived from the orthotopically xenografted cells; and determine the role of E-cadherin and MMP9 in KLF8-regulated metastasis.
This proposal is innovative and unique. Our identification of KLF8 as a potent inducer of EMT and invasiveness and E-cadherin and MMP9 as its targets is novel and significant. Results derived from this work will increase our understanding of molecular mechanisms responsible for breast cancer metastasis and make KLF8 a potentially novel target for early detection, treatment and prevention of breast cancer metastasis.
METASTASIS REMAINS THE MAIN CAUSE OF DEATH FROM HUMAN BREAST CANCER. One out of every 8 women in their lifetime will develop breast cancer. Each year in the US, metastatic breast cancer kills 1 woman every 13 minutes and every 3 minutes a new case of invasive breast cancer is found, despite significant advances made in the realms of cancer prevention, detection, and management of primary breast tumors. This is primarily due to the failure of effective detection and management of breast cancer metastases. Consequently, the chance for a patient to live for 5 more years after diagnosis falls from > 90% for localized diseases to < 20% once metastasis has occurred. Hence, the only way to improve the patient survival is to prevent the cancer from metastasis and understanding the molecular mechanisms responsible for the metastasis is the key.
BREAST CANCER METASTASIS starts from the primary tumor that originates from the mammary gland epithelial cells. From there, the cancer cells find their ways to invade the surrounding matrix tissues (dissemination) and to enter the blood or lymph streams (intravasation), to survive the streams as circulating tumor cells (CTC), to exit the circulation through the vessel walls (extravasation), and to form secondary tumors in distant vital organs such as the lungs and bones (colonization). This whole process is known as tumor metastasis. In the beginning of dissemination, the invasive tumor cells leave the primary tumors by a mechanism known as EPITHELIAL TO MESENCHYMAL TRANSITION (EMT) that disrupts the interaction between the tumor cells and provides the cells with enhanced ability to migrate away from the localized tumor and to invade through the surrounding tissues. The dissemination, intravasation and extravasation require the invasive tumor cells to create such an extracellular environment that allow enzymes, called proteases such as MMP (Matrix metalloproteinase) and PA (plasminogen activator) proteins, to degrade the matrix proteins particularly in the vessel?s basement membranes. During the travel in the circulation, the CTCs usually minimize or stop proliferating activity and must survive death threats and destruction by circulating killer leukocytes. It is believed that the CTCs that make their way out of the circulation can be dormant for up to 20 years after colonizing in the distant organs before onset of the secondary tumor growth. The secondary tumor growth requires the dormant cells to resume proliferation and angiogenesis. Given the multi-step nature of the metastatic process, opportunities exist for early diagnostic, prognostic and therapeutic targeting of the metastatic cells before clinical problems arise. Better understanding of the molecular mechanisms behind each of the steps is the key to secure the opportunities.
Since EMT occurs in the very early phase of tumor metastasis, targeting EMT inducing proteins should be an effective approach for metastasis prevention. The defining event for EMT is disruption of cell-cell interaction through a linker protein called E-CADHERIN. The loss of E-cadherin leads to separation of tumor cells from one another that gain drastically increased ability to migrate and invade (i.e., to spread). Loss of E-cadherin along with elevated MMP9 (a protein degrading enzyme known to be a primary contributor to breast cancer metastasis) levels has been found in the vast majority of invasive breast cancer, strongly suggesting the possibility that EMT is critical to breast cancer metastasis.
We have found that KRšPPEL-LIKE FACTOR 8 (KLF8), a protein serving as a switch that controls the production of other proteins, potently induces EMT and invasion in human breast cells by inhibiting the production of E-cadherin and increasing the production of MMP9. We have also found that in invasive breast cancer cells and tumor tissues collected from human patients, KLF8 levels are significantly elevated and this elevation plays a large part in the loss of E-cadherin and gain of MMP9. These novel findings prompt us to further determine the in vivo role and molecular mechanisms for KLF8 in cancer metastasis using in vivo models that truely mimic pathophysiology of human breast cancer metastasis. IF SUCH IN VIVO ROLE FOR KLF8 IS VERIFIED, KLF8 WOULD BECOME A NOVEL MOLECULAR TARGET AGAINST BREAST CANCER METASTASIS.
We have assembled all the tools necessary for the proposed studies. This project aims at extending our findings from in vitro cell culture and human tumors to in vivo studies of human breast cancer metastasis, with the ultimate goal of translating the lab discoveries into clinical application to treat and even prevent breast cancer metastasis and improve the patient survival. Completion of this project will not only allow the fellow to gain much more knowledge and skills of basic to translational breast cancer research useful for his commitment to becoming a career breast cancer research expert, but also make significant contribution to the Susan G. Komen?s goal to reduce breast cancer incidence and mortality within the next decade.