Era of Hope 2011 Investigator Vignette

Title: SLUG Regulation of Cyclin D1 in Breast Cancer Cells

Investigator: Gautam Chaudhuri, PhD; Meharry Medical College

The focus of my research right now is on triple-negative breast cancer. Triple-negative breast cancer does not have those hormone receptors and they are usually drug resistant—conventional drug resistant. And it affects this proportionately to African Americans and I work in a School of Oncology that’s historically a black college, Meharry Medical College.

And we discovered that this SLUG protein is playing a central role in suppressing lots of those drugs utilizing enzymes. And this poster also describes one of those proteins.

All these projects are focusing on one suppressor protein called SLUG. And SLUG is a metastatic modulator protein so what it does, it regulates the expression of many genes which prevent the cell from becoming cancerous or metastatic.

So this poster is actually describing that—how this SLUG regulates a growth stimulated protein called Cyclin D1. Cyclin D1 actually takes the cell from the resting phase, non-dividing phase to the dividing phase. And in cancer cells, this protein is very critical. This protein goes up in cancer cells. That’s why they grow rapidly. So in this study what we established that if a cell overexpresses, SLUG—the cancer cell will produce lots of Cyclin D1 and that’s why they multiply very fast all right. If you inhibit SLUG, this protein will go down, and the cancer cell will die.

And this study figured out the molecular mechanism of that. So in this process, we discovered that there is a protein called UbcH5c and another name is UbE2D3, it’s an ubiquitinating enzyme. What it does it modifies Cyclin D, chemically modify it and put in ubiquitin on the peptide, so that they get tagged and get degraded. And what SLUG does, SLUG inhibits this enzyme so that Cyclin D1 level goes up and the cells becomes cancerous.

SLUG protein has these domain structures. It has a cluster of zinc fingers that which is bind to the DNA and the other domain is a repressor domain. And that’s the part that’s shown here. These domains—once the SLUG gets recruited to the E2-Box sequences. They recruit some co-factor. And this co-factor then deacetylizes histones and they make the gene heterochromatinized and that means it will not be transcribed. It will be completely packed and gene transcription will be stopped. That’s the mode of action that it does.

So here we are showing that some standard breast cancer lines. MDMB231 and BT549 cells. The red color is Cyclin D1, the green color is SLUG, and this is the part of the nucleus with TOPRO staining. These two cell lines do not express SLUG. and they have a moderate level of Cyclin D1, So we take these cells—468 and MCF7 cells and we topically expressed SLUG in those cells just to prove and verify our hypothesis that if SLUG was high can Cyclin D1 go high? And you can see that Cyclin D1 goes very high. If you compare this one to this one, and this one to this one, you can see that. And so Cyclin D1 level is proportional to the SLUG expression. That’s what we’re trying to establish.

But remember that SLUG is a silencer, a repressor protein; SLUG always do gene repression. And here we are showing that you if you increase SLUG, Cyclin D1 goes up. So it does not really fit, so there must be a mechanism by which SLUG is doing these things, how SLUG is stimulating a gene expression. So what we find out that Cyclin D1 is degraded by a protein, the ubiquitinating enzymes, called UBE2D3. Here you can see that—that when you over express SLUG—Cyclin D1 goes up. If you increase SLUG you can see that ubiquitin conjugation of Cyclin D1 goes down. So indeed our hypothesis is correct that SLUG is inhibiting the expression of this enzyme in the cancer cell. If SLUG is high we see ubiquitinating enzymes will go down, Cyclin D1 will not be ubiquitinated and it’ll not be degraded. So if Cyclin D1 is high, the cancer cell will multiply.

So another facet of that thing is that we find that if Cyclin D1 is high in a cancer cell, and the cancer cell is estrogen positive then by this mechanism estrogen receptor binds to Cyclin D1, and if it binds to Cyclin D1 even in the presence of the anti-estrogens like Tamoxifen. The purpose of the anti-estrogen is to inhibit the function of the estrogen and if you’re a Cyclin D1 high then anti-estrogen cannot work.

So we have shown that when you overexpress. SLUG, the cell becomes resistant to anti-estrogen. So that the two hypotheses that we established, first of all we discovered the mechanism of how SLUG is responsible for Cyclin D1 elevation and the correlating observation is that because of the SLUG, Cyclin D1 goes up and thus the cell becomes resistant to anti-estrogen like Tamoxifen.

So to carry it farther what we did. We discovered that this UB2ED3 enzyme is actually a tumor suppressor protein. That’s something new. People did not really describe that. And so if you overexpress UB2ED3 the cancer cell goes down. They die. They cannot grow. So one of the things we are exploring that—can we do some kind of chemical treatment which will stimulate UBE2D3 gene expression?

All these projects are focused on one thing—that because SLUG is preventing the drugs to work, so we are proposing to develop a molecular drug against SLUG. SLUG cannot really work in the presence of those drugs.

Like if you have lots of Cyclin D, then the cell becomes Tamoxifen resistant and estrogen resistant. And we are proposing that if you inhibit SLUG, Tamoxifen sensitivity will come back. And we have some preliminary data to support that.