Eighty-five percent of all tumors are carcinomas, which are tumors that form in layers of cells that line surfaces. Such cell layers are known as an epithelium. When carcinomas form, they undergo an “epithelial-mesenchymal” transformation” or EMT. EMT means that cells go from being closely aligned and tightly bound to each other in a an organized layer to cells that have little to do with each other and grow in unorganized clumps. Is there a molecule that unites the carcinomas and if so is this molecule a potential drug target for cancer treatments?
Researchers at the University of Texas MD Anderson Cancer Center have identified a protein that seems to play a pivotal role in EMT. This protein, FOXC2, may lay at the nexus of why some carcinomas resist chemotherapy and grow uncontrollably and spread. FOXC2 could, conceivably represent a novel drug target for chemotherapy.
Sendurai Mani, assistant professor of Translational Molecular Pathology and co-director of the Metastasis Research Center at MD Anderson, said, “We found that FOXC2 lies at the crossroads of the cellular properties of cancer stem cells and cells that have undergone EMT, a process of cellular change associated with generating cancer stem cells.”
Cancer stem cells are fewer in number than other tumor cells, yet research has tied them to cancer progression and resistance to treatment. Abnormal activation of EMT can actually create cancer stem cells, according to Mani.
Mani continued, “There are multiple molecular pathways that activate EMT. We found many of these pathways also activate FOXC2 expression to launch this transition, making FOXC2 a potentially efficient check point to block EMT from occurring. ” Mani’s research group used experiments with cultured cells and mice to discover these concepts, but the next step will require assessing the levels of FOXC2 expression in human tumors samples.
In the meantime, these new data from Mani’s research team may have profound implication for the treatment of particular types of carcinomas that have proven to be remarkably stubborn. Breast cancers, for example, are typically carcinomas of the mammary gland ductal system. A specific group of breasts cancers are very notoriously resistant to treatment, and FOXC2 seems to be at the center of such breast cancers.
The anti-cancer drug sunitinib, which is marketed under the trade name Sutent, has been approved by the US Food and Drug Administration (US FDA) for three different types of cancers. In this study, sunitinib proved effective against these particularly stubborn types of breast cancer; the so-called “triple-negative, claudin-low” breast cancers.
Mani explained why such breast cancers are so resistant to treatment: “FOXC2 is a transcription factor, a protein that binds to DNA in the promoter region of genes to activate them. For a variety of reasons, transcription factors are hard to target with drugs.”
However, sunitinib seems to target these triple-negative breast cancers. When mice with triple-negative breast cancer were treated with sunitinib, the treated mice had smaller primary tumors, longer survival, and fewer incidences of metastasis. The cancer cells also showed a marked decreased in their ability to form “mammospheres,” or balls of cancer stem cells (this is an earmark of cancer stem cells). Thus sunitinib seem to attack cancer stem cells.
As it turns out, FOXC2 activates the expression of the platelet-derived growth factor receptor-beta (PDGFRc-beta). Activation of PDGFRc-beta drives cell proliferation in FOXC2-positive cells, and sunitinib inhibits PDGFRc-beta and inhibits cells that have active FOXC2 and undergoing EMT.
Triple-negative breast cancer cells lack receptors that are used by the most common anti-cancer drugs. These deficiencies are responsible for the resistance of these cancers to treatment. Such cancer cells also tend to under go EMT because they lack the protein claudin, which binds epithelial cells together. Without claudin, these cancer cells become extremely aggressive.
Since cells undergoing EMT are heavily expressing FOXC2, Mani and his colleagues used a small RNA molecule that makes a short hairpin and inhibits FOXC2 synthesis. Unfortunately, blocking FOXC2 had no effect on cell growth, but it did alter the physical appearance of the cells and reduced their expression of genes associated with EMT and increased the expression of E-cadherin, a protein necessary for epithelial cell organization. Breast cancer cells also became less invasive when FOXC2 was inhibited, and they down-regulated CD44 and CD24, which are markers of cancer stem cells.. Additionally, triple-negative breast cancer cells that had FOXC2 inhibited had a reduced ability to make mammospheres. Thus, FOXC2 expression is elevated in cancer stem cells, and inhibition of FOXC2 decreased the ability of the cancer stem cells to behave as cancer stem cells.
Mani’s group also approached these experiments from another approach by overexpressing FOXC2 in malignant mammary epithelial cells. This forced FOXC2 expression drove cells to undergo EMT and become much more aggressive and metastatic (the cancer spread to the liver, hind leg, lungs, and brain). Breast cancer cells without forced FOXC2 overexpression showed no tendency to metastasize.
Finally, Mani’s group examined metastatic mammary tumors that were highly aggressive when implanted into nude mice (mice that cannot reject transplants). Two of the tumors were claudin-negative and both of these tumors showed elevated FOXC2 expression. When FOXC2 expression was blocked by Mani’s hairpin RNA, the claudin-negative tumors became less aggressive and grew more as mesenchymal cells. The cells that underwent EMT also showed high levels of PDGF-RC-beta expression.
Mani said of these data: “We thought PDGF-B might be a drugable target in these FOXC2-expressing cells.” Mani’s group also showed that suppressing FOXC2 reduced the expression of PDGFRC-Beta. Thus, this small molecule might be an effective therapeutic strategy for treating these hard-to-treat breast cancers.
MD Anderson has filed a patent application connected to this study.
See Hollier B.G., Tinnirello A.A., Werden S.J., Evans K.W., Taube J.H., Sarkar T.R., Sphyris N., Shariati M., Kumar S.V., Battula V.L., Herschkowitz J.I., Guerra R., Chang J.T., Miura N., Rosen J.M., and Mani S.A.,. FOXC2 expression links epithelial-mesenchymal transition and stem cell properties in breast cancer. Cancer Research. e-Pub 2/2013.