One of the most formidable weapons in a cancer’s arsenal is the ability to spread to other organs.
This process, known as metastasis, allows cells to detach from a tumor, travel through the bloodstream, and establish secondary tumors in distant organs. Once a tumor has acquired metastatic potential, it becomes refractory to most treatments and, as a result, about 90 percent of cancer-related deaths are due to metastasis.
In the face of this daunting challenge, researchers at Vanderbilt University Medical Center and Washington University School of Medicine in St. Louis have enlisted powerful new tools to study the early march to metastasis in colon cancer. Their findings implicate a previously uncharacterized protein called PLAC8.
This study, which appears in the current issue of the Journal of Clinical Investigation, characterizes PLAC8 in the context of development and cancer, elegantly demonstrating how cancer is reminiscent of development gone awry.
Very little was known about PLAC8 prior to this study and no one had looked at the endogenous protein or its role in cancer. “Now people will have the tools to be able to look at the protein,” said senior author Robert Coffey, M.D., Ingram Professor of Cancer Research at Vanderbilt.
The new tools developed in this study will enable researchers to study PLAC8 in other physiological systems, such as brown fat differentiation, obesity and innate immunity, where PLAC8 has been implicated.
Coffey’s lab became interested in PLAC8 when it was identified as the most differentially expressed gene in a new 3D colon cancer cell culture system the lab developed.
Co-first author Cunxi Li, M.D., Ph.D., was surprised to discover that the colon cancer cell line, HCA-7, formed either smooth hollow balls or clumps with protrusions when cultured in Type I collagen. The first formed benign tumors and the second formed invasive tumors when injected into nude mice.
PLAC8 was upregulated in the aggressive HCA-7 variant.
If excess PLAC8 could make cells more aggressive, perhaps it had a role in colon cancer. The Coffey lab discovered that in certain human colon cancers, PLAC8 was mis-localized from the cell membrane to the cytoplasm.
Cancers often reactivate developmental pathways to drive aggressive cell behavior.
With this in mind, Haiting Ma, Ph.D., a former graduate student in the Solnica-Krezel and Coffey labs, examined the plac8 gene in zebrafish, a classic developmental biology model system.
“We asked whether we could overexpress this gene at high level in zebrafish embryos, similar to what happens in colon cancer, and let the embryos tell us what this protein might be doing,” said Lilianna Solnica-Krezel, Ph.D., professor and head of the Department of Developmental Biology at Washington University.
When Ma overexpressed Plac8, the fish embryos exhibited abnormal shape and reduced cell movements, defects similar to zebrafish defective in E-cadherin protein. Indeed, Haiting later discovered that embryos overexpressing Plac8 had reduced E-cadherin levels.
E-cadherin is a protein expressed in epithelia, and its downregulation is a hallmark of the process known as epithelial-to-mesenchymal transition (EMT).
EMT is characterized by signature events, where certain epithelial genes, such as E-cadherin are down-regulated and mesenchymal genes, such as N-cadherin and Vimentin are up-regulated. This loss of epithelial features and acquisition of mesenchymal characteristics allows a cell to detach from a tumor and become invasive.
Haiting did not observe classical EMT changes, suggesting that PLAC8 EMT did not follow the canonical EMT pattern. Li turned back to colon cancer cells and saw that PLAC8 overexpression in HCA-7 cells also caused atypical EMT. Instead of decreased E-cadherin mRNA, E-cadherin protein moved from the plasma membrane to the cytoplasm and a different cadherin, P-cadherin, was expressed.
Working together, Li and Ma discovered that the mechanism by which PLAC8 induced EMT was through elevated activated levels of a protein called ERK2. ERK2 activity is negatively regulated by DUSP6. Li and Ma found that PLAC8 interacts with DUSP6 and turns it off, keeping ERK2 active.
Turning back to human colon cancer, the Coffey lab employed a new immunofluorescence technology called MultiOmyx that can use up to 60 different antibodies to stain a single tissue section. MultiOmyx resulted from the lab’s collaboration with GE Global Research, supported by National Institutes of Health (NIH) grant CA174377.
Using this technology, the researchers demonstrated that the same noncanonical EMT markers PLAC8, P-cadherin, and vimentin could be found at the leading edge of a human CRC tumor, the site where EMT and invasion typically occur.
“It was very exciting to combine developmental studies in zebrafish with a novel 3D culture system to uncover the function of a new cancer-related protein,” Ma said.
Other NIH grants that supported the research included CA046413, CA095103, CA119925, CA009582, CA068485 and GM055101.
— by Emily Poulin
