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CDMRP 2012 Investigator Vignette

Title: Identification and Function of Ets Target Genes Involved in Lung Cancer Progression

Investigator: Hasmeena Kathuria, M.D., Boston University Medical Campus

The award that I have with the Department of Defense is the Lung Cancer Promising Clinician Award. And I work with mouse models of lung cancer to try to understand how tumor cells exit the primary tumor and cause metastases.

The first model is called the K-ras mouse model. These mice develop tumors that continue to grow but don't metastasize. The other model I use has the K-ras oncogene overexpressed as well as loss of a tumor suppressor called LKB1. When you have both of these hits these mice now develop metastases. And they metastasize very similar to how human lung cancer metastasizes. They start with a primary tumor and then go onto the lymph nodes.

And what's nice is this LKB1 mutation correlates with human lung cancer so 20 to 30% of human lung cancer patients harbor this LKB1 mutation. So we now have a mouse model that really mimics what happens in human disease. And you can use these models to help advance the understanding of the molecular mechanisms of lung cancer progression.

I collaborate with Dr. Kwok-Kin Wong at Dana Farber, and together we have found that that there's several genes that are overexpressed in the mouse model that metastasizes. These genes are in a family that's called the Epithelial Mesenchymal Transition (EMT) transcription factors. These are proteins that when they're expressed at higher levels they allow these tumor cells, these epithelial tumor cells, that have very tight junctions and allows those cells to flatten, express what we call mesenchymal markers or markers of fibroblasts that allows those cells to escape the basement membrane and then metastasize.

One of these transcription factors is Twist, and Twist is thought to be the master regulator of EMT. What we hypothesized in this grant is that the Ets proteins, Ets proteins are a family of transcription factors thought to be very important in lung cancer and other cancer progression. And we hypothesize that these Ets proteins were regulating Twist expression to cause this process of EMT.

This gene expression microarray shows the differences in gene expression between the K-ras nonmetastatic tumor and the K-ras LKB1 metastatic tumor. And as you can see by the genes in red that there are several genes that are over-expressed in the mice harboring both mutations. And when we looked at those genes in detail we found that many of these overexpressed genes were genes thought to be important in EMT including Vimentin and N-Cadherin, Fibronectin, Twist, and SNAIL.

Using primary mouse tumors and lymph nodes from these mice, we compared expression by immunohistochemistry. Brown cells are positive, blue cells are negative, and what you can see here is that as these mice develop metastases there's more brown cells or increased expression of Twist compared to expression of Twist in those mice that don't metastasize.

These Ets proteins, which our hypothesis was that they regulate Twist, are also expressed at higher levels in the metastatic mouse model compared to these nonmetastatic mouse models as shown by the blue cells.

We confirmed by CHIP-binding assays and promoter studies that these Ets proteins indeed regulate Twist. And when we silence Ets1, we silence Twist expression both by mRNA expression and protein expression.

And we show here when we silence Ets1, we decrease migration and we decrease invasion so fewer cells escape out of the basement membrane.

And we looked at-does the same thing happen in human lung tumors? And again, this is immunohistochemistry showing that same pattern that when you have poorly differentiated tumors you have higher expression of Ets and Twist represented by the red arrows or brown cells compared to those tumors that are more moderately differentiated where there's all blue cells and no brown cells detected of both Twist and Ets1.

Similarly we showed that Ets1 regulates Twist expression and that when we silence Ets1 we silence Twist expression.

And so Ets1 is playing a role in regulating Twist which is then regulating epithelial mesenchymal transition. So the function that we're proposing is that it's a regulator of EMT through Twist expression.

So our next step is to-in the human specimens-validate that these genes and proteins are truly overexpressed in patients that develop metastases and then eventually to try to target those genes and proteins to see if we can prevent metastasis and target the downstream effectors of this process.