Using TCGA Data to Find a Novel Target for Triple-Negative Breast Cancer

Teagan Keating Kuruna

Triple-negative breast cancer (TNBC) is notoriously difficult to treat. These tumors are aggressive, more likely to metastasize, and result in poor outcomes for patients.  TNBC is disproportionately found in African-American, Hispanic, and young women. TNBC is difficult to treat because it lacks the HER2 amplification and the estrogen receptors (ER) and progesterone receptors (PR) present in 80 to 90 percent other breast cancer types. This means that the hormone and targeted therapies that are often successful in treating the majority of breast cancers are not effective in treating TNBC. Research into other molecular targets as treatment possibilities could result in improved clinical outcomes for this aggressive and deadly form of breast cancer.

A study published in Molecular Cancer Research in September 2014 used TCGA data to help illuminate a potential novel target for TNBC treatments and points to the mechanism by which doxycycline may be a useful treatment for TNBC.

Mdm2 (Two)-binding protein and its role in TNBC

In this study, researchers at the Vanderbilt University Medical Center explored the role of the Mdm2 (Two)-binding protein (MTBP) in TNBC. Because MTBP/Mtbp overexpression has been implicated in the development of two types of lymphomas, colorectal carcinoma, and multiple myeloma, researchers were interested in exploring whether its expression contributes to breast cancer. MTBP is a gene that encodes the MTBP protein, which, in cancer, assists tumor growth induced by the Myc oncogene. Myc regulates the transcription of other genes. Mutations, duplications, rearrangements, and translocations that result in overexpression of Myc are associated with a variety of tumors. Studies suggest that MTBP is also involved with cell-cycle progression and regulating cell proliferation.

Using patient survival and gene expression data for 844 TCGA breast cancer samples, and gene copy number alteration data for 913 samples obtained from cBioPortal, as well as human cell line cultures, researchers found:

      • MTBP is overexpressed in TNBC more often than in other breast cancer tumors, and correlates to decreased patient survival.
      • When compared to normal breast tissue and other breast cancer subtypes, MTBP mRNA and protein levels are highest in TNBC.
      • Knockdown of MTBP in cell lines induced apoptosis, reduced cell growth and soft agar colony formation in vitro, which was rescued by expression of shRNA-resistant Mtbp. This impaired tumor growth in vivo, including the growth of already established tumors.

Researchers posit that these findings show that MTBP is a significant contributor to breast cancer development. MTBP may then be a potential option for newly developed targeted therapies.

Analyzing TCGA data reveals differences in MTBP expression among breast cancer subtypes

Using data from 844 TCGA breast cancer samples, researchers found that MTBP expression was significantly elevated in breast cancer tumors compared to healthy breast tissue. Compared with patients whose tumors had relatively lower levels of MTBP, patients with elevated levels of MTBP had reduced survival. Nineteen percent of tumors showed amplified MTBP, which also correlated with decreased patient survival. After determining that elevated levels of MTBP were common in breast cancers, researchers subdivided the TCGA samples into clinically the relevant subgroups: ER-positive (ER+), HER2-positive (HER2+), and triple-negative (TN, which means its negative for ER and PR and lacks the HER2 amplification) to explore whether the subgroups showed differences in MTBP levels. TNBC showed significantly higher expression of MTBP than the ER+ or HER2+ subtypes as well as higher levels of MTBP mRNA.

Reducing MTBP levels inhibits TNBC cell proliferation and apoptosis

After learning the extent to which MTBP is overexpressed in TNBC, researchers wondered if reducing the levels of MTBP would change the tumor cells’ ability to proliferate. Using the knockdown process, an experimental technique used to impede expression of a gene, researchers investigated what happens when two TNBC breast cancer cells lines no longer express MTBP. Though the two cells lines were from molecularly-distinct TNBC subtypes, both overexpress MTBP. Once the gene was knocked down, both cell lines showed decreased TNBC cell proliferation.

Gene knockdown also appeared to impede agar colony formation, a laboratory test used to create an environment for anchorage-independent growth, which is generally considered to be an indicator for a tumor’s metastatic potential. Researchers also used doxycycline to try to reduce MTBP levels. Doxycycline, while also a useful antibiotic, blocks iNOS, a protein important to tumor cell growth. Testing this in three cell lines, researchers found that doxycycline reduced MTBP protein expression within 24 hours.

Experiments show the effect of doxycycline on MTBP in TNBC

Armed with this knowledge, researchers also wanted to investigate whether established TNBC tumor growth could be inhibited by doxycycline. Researchers injected mice with tumor cells that expressed doxycycline-inducible MTBP shRNA1. The mice were then provided water with or without doxycycline. The mice given water with doxycycline showed a statistically significant (70 percent) decrease in tumor volume compared to the mice not given doxycycline. The shrunken tumors also showed reduced levels of MTBP protein.

Simultaneously, researchers investigated whether MTBP knockdown would inhibit established TNBC tumor growth. Additional mice were injected with cells from tumors that showed doxycycline sensitivity. This time, however, the tumors were allowed to grow without intervention. On average, the tumors’ volume was 100-150 mmon/PopUps/popDefinition.aspx?id=CDR0000659791&version=Patient&language=English" onclick="javascript:popWindow('defbyid','CDR0000659791&version=Patient&language=English'); return false;">mm3 after 10 days.

After reaching this average volume, these mice were given doxycycline. The tumors exposed to doxycycline were significantly smaller after 72 hours than the untreated tumors. On average, the treated tumors’ volume was 188mm3, while the untreated tumors were 350mm3.

The decline in the growth rate of established tumors mirrored the rate of tumor growth in mice that had received doxycycline in their drinking water beginning on Day One. That is, giving mice doxycycline after a tumor was established slowed the tumor growth at a similar rate as tumor growth in mice given prophylactic doxycycline. Tumors exposed to doxycycline throughout the experiment and tumors exposed after 10 days of uninhibited growth were similar in size and weight at the end of the experiment. Both were significantly smaller than tumors never exposed to doxycycline. A paper published in Oncology Reports in May 2015 supports the researchers' findings about doxycycline and TNBC.

Determining MTBP's role in TNBC

This study identifies MTBP as critical for the growth and survival of TNBC cells. The link between MTBP overexpression and tumor cell growth and survival in B-cell lymphomas has been previously established. MTBP amplification occurs in colorectal cancer and multiple myeloma and TCGA data have shown that MTBP is amplified in many kinds of cancer.

This analysis also suggests that MTBP overexpression may be an indicator of metastatic potential. Since most breast cancer deaths are associated with metastasis, finding a target that could reduce the likelihood of metastasis could help reduce mortality rates. However, there have been other studies that conflict with these findings. Other research has shown that decreased MTBP expression increases cell migration, invasion, and metastasis. To reconcile these two possibilities, the Vanderbilt University Medical Center researchers propose that MTBP expression may be downregulated when cancer cells move throughout the body and upregulated once the cells find a new location and begin to proliferate again. Researchers also posit that MTBP could have a tissue-specific or mutation-specific function, though they note that data has shown that MTBP is key for TNBC cancer development and progression.

By using TCGA data, researchers found that MTBP is expressed at different levels in TNBC subtypes. These findings may have positive implications for further study and future treatments. The data used in this research, and all available TCGA data, can be found through the TCGA Data Portal and the cBioPortal.

Reference: Grieb, B.C., Chen, X., and Eischen, C.M. (2014) MTBP is overexpressed in triple-negative breast cancer and contributes to its growth and survival. Mol Cancer Res. doi: 10.1158/1541-7786.MCR-14-0069.