The Role of microRNAs in Triple-Negative Breast Cancer

Institution: University of California, San Francisco
Investigator(s): Leonard Kusdra,  -
Award Cycle: 2010 (Cycle 16) Grant #: 16FB-0044 Award: $89,200
Award Type: Postdoctoral Fellowship
Research Priorities
Biology of the Breast Cell>Pathogenesis: understanding the disease

Initial Award Abstract (2010)

MicroRNAs are recently discovered, single-stranded RNA molecules of 18-24 nucleotides that regulate gene expression. It has been shown that a single microRNA can control the expression of hundreds of gene targets. Cancer is a disease where multiple genes are deregulated, so there has been much interest to study whether controlling the levels of microRNAs can restore normal gene expression to slow down the growth and spread of tumor cells. To date, there have been no studies to examine the role of microRNAs in the most aggressive subset of breast cancer, the triple negative breast cancer (TNBC). This subset represents about 20% of breast cancers at diagnosis with a corresponding high prognostic probability of metastasis. At present, no targeted treatments exist for TNBC and standard chemotherapy remains the only therapeutic option.

Thus, this project seeks to examine the role that microRNAs have in the biology of triple-negative breast cancer (TNBC) cell lines. First, we have found that TNBC tend to have high levels of a protein (oncogene) called Myc. For reasons not yet clear, increases in Myc appears to make cells sensitive to cell death by inhibition of another protein called CDK1, a regulator of cell division. This sensitivity to CDK1 inhibition in the context of MYC overexpression is termed “synthetic lethality.” We have also found that upon CDK1 inhibition, a single microRNA, called mir-19a is decreased. Thus, in this project we will further examine how mir-19a and other microRNAs regulate the sensitivity of triple-negative cells to cell death. Another aim is to investigate a group of about 30 microRNAs that appear to be increased in cells that are resistant to the therapeutic drug, Taxol. We will examine to what degree these microRNAs alter the sensitivity to Taxol. Since the recurrence rate of TNBC is very high, the microRNA profile in Taxol-resistant cells may provide additional therapeutic targets to overcome this resistance. Our final aim is to compare another group of 30 microRNAs that have been found to be increased in TNBC cell lines compared to the less aggressive estrogen receptor positive cell lines. We believe these microRNAs underlie the aggressiveness and invasiveness of TNBC relative to the more treatable ER-positive breast cancers.

Taken together, we anticipate that our studies will reveal individual or combinations of microRNAs that can be used to improve treatment for TNBC either as novel targets of treatment, or as a mean to overcome drug resistance typically seen in metastatic disease or recurrence.

Final Report (2012)

Breast cancer related deaths are the second highest cause of mortality in women after lung cancer. In particular, the ‘Triple-Negative’ subtype (lacking the three pathological surface markers: ER, PR and Her2) represents a major clinical challenge, in part because of the lack of clear, targeted therapeutic strategies that are available to combat this disease. Demographically, the disease appears in about 20-30 of women diagnosed with breast and primarily arises in younger African-American or Latina women. Triple-negative breast cancer (TNBC) usually has a higher rate of recurrence with the recurring tumor being more aggressive, less responsive, and more likely to metastasize; thus the prognosis for women with TNBC that has recurred within five years is quite poor. The particularly aggressive nature of TNBC along with the lack of targeted therapies against this subtype therefore makes this disease one in which more therapeutic strategies are urgently needed.

MicroRNAs are small RNA molecules that are expressed ubiquitously and have been shown to down-regulate (inhibit) genes by interacting with sequences that regulate expression of target genes. MicroRNAs have been the focus of intense interest in recent years because of their ability to inhibit expression of multiple genes and because numerous studies have found that tumorigenic tissues differentially express various microRNAs compared to normal tissue. Because microRNAs are small in size, they can more easily administered systemically to reach the target tissues. Conversely, microRNAs that are tumor promoting can easily be inhibited with small RNA molecules that inhibit microRNA activity. Indeed a recent report showed that systemic administration of a microRNA inhibitor target breast cancer cells and prevented early stages of metastasis in a mouse model. Thus, there has been intense interest in utilizing microRNAs as novel therapeutic strategies in treating breast cancer.

We performed “global” microRNA profiling on a cohort of breast cancer biopsy samples (n=40) showed that 18 microRNAs were enriched in samples from patients with TNBC. A similar analysis of a panel of breast cancer cell lines representing all breast cancer subtypes showed 34 microRNAs to be enriched in the basal subtype of breast cancer, a subtype that shares many features with TNBC. Among these microRNAs, nine microRNAs showed overlap in being enriched in TN breast tumor samples and cell lines of the basal subtype, providing strong evidence that these microRNAs were particularly important in TNBC. Interestingly, most of the nine members consisted of a microRNA gene cluster called mir-17-92, a cluster that has been shown to be upregulated by the MYC transcription factor. Work in our laboratory found that MYC was elevated in TNBC, consistent with the mir-17-92 cluster being elevated in the TNBC/basal subtype. Additionally, an additional myc-regulated microRNA, mir-9 was also found in the 9 overlapping microRNAs, a result this is consistent with MYC levels/activity being elevated in TNBC. In a cell culture system where MYC could be conditionally turned on, we found that levels of members of the mir-17-92 cluster, in addition to mir-9, were elevated in response to MYC upregulation, providing further evidence that the MYC-mir-17-92 and MYC-mir-9 gene regulation circuit was important in TNBC.

In further studies, members of the mir-17-92 cluster have been found to bind and repress the expression of BIM, a protein that promotes cell death. Mir-9 has also been predicted to target BIM. This was particularly interesting since previous work in our lab showed that BIM upregulation was important in cell death mediated by inhibition of a key cell cycle regulator, CDK1 specifically in TNBC. To determine whether mir-9 did indeed inhibit the expression of BIM, we used molecular biology and cell based techniques to show that mir-9 did indeed bind to the regulatory regions of BIM that controlled BIM expression. In addition, when mir-9 was overexpressed in various breast cancer cell lines, the expression level of BIM decreased, providing evidence that mir-9 was an important regulator of BIM expression.

To further investigate what role the mir-9-BIM genetic regulatory network had in TNBC, we inhibited the activity of mir-9. We hypothesized that inhibiting mir-9 would lead to upregulation of BIM and would 1) be toxic to cells in and of itself or 2) to render them more sensitive to inhibitors that require BIM upregulation, such as CDK inhibitors. Indeed, strong inhibition of mir-9 activity led to cell death in three TN breast cancer cell lines compared to control cells where mir-9 was not inhibited. Addition of CDK inhibitor treatment synergized with mir-9 inhibition in inducing cell death at a level much high than either CDK inhibition or mir-9 inhibition alone. Thus mir-9 appears to be part of a basal cell-survival machinery that keeps TNBC cells viable and perhaps to counteract the pro-cell death killing effects of MYC. In addition, mir-9 may represent an attractive target to use in killing TNBC as a standalone therapeutic strategy or as a means of sensitizing cells to anti-cancer drugs.

MYC pathway activation in triple-negative breast cancer is synthetic lethal with CDK inhibition.
Periodical:Journal of Experimental Medicine
Index Medicus: J Exp Med
Authors: Horiuchi D, Kusdra L, Huskey NE, et al. and Goga A
Yr: 212 Vol: 209 Nbr: 4 Abs: Pg:679-96

Chemical-genetic analysis of cyclin dependent kinase 2 function reveals an important role in cellular transformation by multiple oncogenic pathways.
Periodical:Proceedings of the National Academy of Sciences of the United States of America
Index Medicus: Proc Nat Acad Sci, U S A
Authors: Horiuchi D, Huskey NE, Kusdra L et al and Goga A
Yr: 2012 Vol: 109 Nbr: 17 Abs: Pg:E1019-27