Breast Cancer-secreted MicroRNAs in the Pre-metastatic Niche

Institution: Beckman Research Institute of the City of Hope
Investigator(s): Shizhen Emily Wang, Ph.D. -
Award Cycle: 2011 (Cycle 17) Grant #: 17IB-0054 Award: $252,000
Award Type: IDEA
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease



Initial Award Abstract (2011)

Metastasis, the spread of cancer to distant organs from the original tumor site, is the most advanced stage and leading cause of death in breast cancer (BC). Patients with metastatic BC (MBC) have a median survival of only 1–2 years, and lack efficient treatment options. Therefore, there is urgent need for early diagnosis or reliable prediction of metastasis, and for novel therapies that target early steps involved in cancer spread in order to prevent metastasis.

Recently discovered small RNA molecules, called micrRNAs (miRNAs), will be investigated in this study for their role in these diagnostic and therapeutic applications. We will identify cancer secreted miRNAs whose levels in blood are linked to metastasis in BC patients. These miRNAs are valid candidates for blood-based biomarkers that can predict BC metastasis at a nonmetastatic stage. Because BC-secreted, metastasis-related miRNAs can travel to other organs through the blood, they may invade and alter the environment (pre-metastatic niche) in a distant healthy organ to make it favorable for the growth of BC cells, thereby enabling BC metastases to form in that organ. Therefore, blocking this function of BC-secreted, metastasis-related miRNAs may protect the distant organ and prevent metastasis of BC.

First, we will identify BC-secreted miRNAs associated with metastasis. Using an Ultra High Throughput Sequencing System (Solexa), we will compare the miRNAs secreted by BC cells with high and low/no metastatic potential, and will compare the blood miRNA pattern among BC patients who developed metastases after an initial, non-metastatic BC diagnosis and those who did not within a similar follow-up time. Metastasis-associated miRNAs identified in this aim may include both early diagnostic markers and therapeutic targets for MBC. Next, we will determine the function of BC-secreted, metastasis-associated miRNAs in the pre-metastatic niche. The biological functions and mechanisms of BC-secreted miRNAs will be investigated by a combination of computational tools and molecular biological approaches, such as the use of inhibitors and mimics of miRNAs. These functional studies will be carried out in various types of niche cells, including those important for new blood vessel formation, secretion of factors that encourage the growth of cancer cells, and immuno-modulation (such as inflammation). In addition, a mouse model will be used to directly examine the effects of these miRNAs on metastasis.

This study will provide proof-of-principle for predicting the risk of BC metastasis based on the miRNA profile of the patient’s blood. Identification of blood-based miRNAs associated with metastasis, as an immediate outcome of this study, will enhance our ability to target lethal metastasis at an earlier stage and reduce the incidence of metastasis and mortality. The study will also reveal novel therapeutic targets that may be pharmaceutically targeted in BC treatments to protect organs that are common metastatic sites from actions of cancer-secreted miRNAs and therefore, to prevent metastasis, increase survival and improve quality of life of BC patients.




Final Report (2013)

Metastasis, the spread of cancer to distant organs from the original tumor site, is the most advanced stage and leading cause of death in breast cancer (BC). Patients with metastatic BC have a median survival of only 1–2 years, and lack efficient treatment options. Therefore, there is urgent need for early diagnosis or reliable prediction of metastasis, and for novel therapies that target early steps involved in cancer spread in order to prevent metastasis. Recently discovered small RNA molecules, called microRNAs (miRNAs), will be investigated in this study for their role in these diagnostic and therapeutic applications. The goals of this study are to identify cancer-secreted miRNAs associated with BC progression to metastasis (Aim 1), and to determine the function of these miRNAs in the pre-metastatic niche (Aim 2).

During the funding period, we have completed the studies proposed in both Aims. By comparing the small RNA deep sequencing data in the pre-treatment sera of 42 stage II–III locally advanced and inflammatory BC patients, we found that two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with cancer progression to metastatic disease. Through quantitative PCR, we further confirmed that higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II–III BC patients. These results were recently published in the Journal of Translational Medicine. Interestingly, we observed that many breast cancer cells secrete higher levels of miR-122 compared to non-tumor mammary cells. Therefore, we chose miR-122 as a BC-secreted, metastasis-related miRNA for the subsequent functional studies. We found that overexpression of miR-122 significanly downregulated the expression of pyruvate kinase muscle (PKM), a gene critical for glucose metabolism. The molecular mechanism through which miR-122 regulates PKM was determined. Using biochemical assays, we showed that cancer-derived miR-122 indeed suppressed the enzymatic function of PKM and glucose utilization in the non-cancerous cell types that are frequently found in the tumor microenvironment. Notably, anti-miR-122 treatment significantly suppressed metastasis to lung and brain in an animal model.

Through these funded studies, we for the first time demonstrated that cancer, through secreting miRNAs, can adapt its habitat by suppressing the glucose utilization of normal cells residing there and therefore maximize the local glucose supply to cancer cells, a mechanism contributing to cancer growth and metastasis. We have also gained important insight into the role of BC-secreted, metastasis-related miRNAs, in particular miR-122, as potential biomarkers and therapeutic targets for BC treatment. Blocking the function of this special group of miRNAs may protect the distant organs and prevent metastasis of BC.




Progress Report 1 (2012)

We have completed the identification of BC-secreted miRNAs associated with metastasis. By comparing the small RNA deep sequencing data in the pre-treatment sera of 42 stage II–III locally advanced and inflammatory BC patients, we found that two circulating miRNAs, miR-375 and miR-122, exhibited strong correlations with relapse with metastatic disease. Through quantitative PCR, we further confirmed that higher levels of circulating miR-122 specifically predicted metastatic recurrence in stage II–III BC patients. These results were recently published in the Journal of Translational Medicine. Interestingly, we observed that the metastatic breast cancer cell line MDA-MB-231 secretes significantly higher level of exosome-encapsulated, miR-122, compared to the non-tumor MCF-10A mammary epithelial cells. Therefore, we chose miR-122 as a BC-secreted, metastasis-related miRNA for the subsequent functional studies.

We have also started to pursue the functions of selected miRNAs in the pre-metastatic niche. Focusing on miR-122 identified in Aim 1, we found that overexpression of miR-122 significantly downregulated the expression of pyruvate kinase muscle (PKM), a gene critical for glycolysis. The miR-122 binding site in the 3’UTR of PKM transcript was determined. Using biochemical assays, we showed that miR-122 indeed suppressed the enzymatic function of PKM and glycolysis.

In the future 6-month funding period, we will focus on the effect of BC-secreted miR-122 on various types of niche cells, such as endothelial cells, using cell line and animal models. Upon completion of the entire project, we will gain important insight into the role of BC-secreted, metastasis-related miRNAs as potential biomarkers and therapeutic targets.



De novo sequencing of circulating miRNAs identifies novel markers predicting clinical outcome of locally advanced breast cancer.
Periodical:Journal of Translational Medicine
Index Medicus: J Transl Med
Authors: Wu X, Somlo G, Yu Y, et al, and Wang SE
Yr: 2012 Vol: 10 Nbr: 1 Abs: Pg:42