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Dec 5, 2011

Role Found for mRNA Translation Regulator in Two Cancers

  • Scientists have identified a role for a protein that controls mRNA translation in the reprogramming of cells that drives and supports tumor progression. In vitro and in vivo studies by a team of investigators at the Hospital del Mar Research Institute’s (IMIM) Cancer Research Programme in Barcelona showed that cytoplasmic polyadenylation element binding protein 4 (CPEB4) is selectively produced in pancreatic cancer and glioblastoma, but not healthy pancreatic or brain tissue. Their work confirmed that in cancer cells, CPEB4 activates the translation of hundreds of mRNAs including tissue plasminogen activator (tPA), which are involved in tumor-related processes. In contrast, these same RNAs are silenced in healthy cells that don’t produce CPEB4.

    Moreover, report Pilar Navarro, M.D., and colleagues, knocking down CPEB4 in pancreatic cancer cells reduced their cancer-forming capabilities and invasive potential, led to the growth of smaller tumors in vivo, and reduced tumor vasculature. Describing their findings in Nature Medicine, the authors suggest their findings may lead to the development of diagnostic or therapeutic approaches against pancreatic cancer, glioblastoma, and potentially other cancers. Their paper is titled “Key contribution of CPEB4-mediated translational control to cancer progression.”

    Gene reprogramming is a critical process in the development of cancer, and leads to the abnormal control of genes that are related to cell proliferation, apoptosis, metabolism, and invasion. Integral to this is the post-transcriptional regulation of specific mRNA subpopulations, which has been found to contribute to the broad expression changes of the genes involved in turning cells cancerous. The control of translation is mediated by elements in the 3’ untranslated regions (UTRs) of mRNAS. One of these, cytoplasmic polyadenylation element (CPE), binds to activating CPE binding proteins (CPEBs) that promote extension of the polyadenine tail and hence activate mRNA translation.

    CPEBs can, in fact, act as either translational repressors or activators that regulate mitotic and meiotic cell cycles and senescence, the authors continue. To investigate whether CPEB-mediated mRNA regulation is specifically involved in cancer, they looked more closely at CBEP expression in pancreatic ductal adenocarcinoma (PDA). Initial analyses provided a clear indication that CPEB4 was linked with tumoral phenotype, as five out of seven PDA cell lines tested demonstrating high levels of CPEB4 mRNA and protein, when compared with the nontumorigenic pancreatic cell line HPDE.

    Evaluation of CPEB4 levels in 190 human pancreas samples confirmed these findings. In normal pancreas CPEB4 was restricted to the islets and occasionally to ductal cells. However, the protein was expressed in nearly 20% of ducts in the inflammatory atrophic pancreas, and in an increasing number of ductal cells as the disease progressed: in moderately differentiated or well-differentiated PDAs CPEB4 was expressed in nearly 90% of ductal cells.

    Notably, transfecting a pancreatic tumor cell line using short hairpin RNAs (shRNAs) that target CPEB4 led to a marked reduction in invasiveness and colony formation, even though there was no effect on proliferation rate. To test this in vivo, the investigators subcutaneously injected either parental RWP-1 pancreatic cancer cells, or RWP-1 cells transfected with one of two different shRNAs against CPEB4, into nude mice. The results showed that compared with tumor growth in animals injected with RWP-1 parental cells, the two different shCPEB4 effectively downregulated CPEB4, and led to a more than 50% reduction in tumor weight and up to an 80% reduction in tumor volume after three weeks. CPEB4 knockdown also reduced the ability of pancreatic tumor cells to invade and form tumors in organs other than the pancreas.

    Histopathological analysis of tumors that developed after subcutaneous and intraperitoneal injection of cancer cells showed that tumors developing from shCPEB4 cells had a reduced prolifera­tion rate and decreased microvessel density compared with parental RWP-1 cells and those transfected with a control shRNA. Reduced cell proliferation and tumoral angiogenesis were also observed in tumors developing from shCPEB4-transfected Capan-1 pancreatic cancer cells. “These results indicate that, although CPEB4 is not required in a cell-autonomous manner for cellular proliferation, reduced amounts of CPEB4 affect both the proliferation rate of tumors and their archi­tecture by reducing vascularization and increasing stroma formation, with the consequence of reduced tumor growth and invasion during in vivo pancreatic tumorigenesis,” the authors write.

    The team also wanted to see whether overexpression of CPEB4 in PDA resulted in changes in the translational activation of mRNAS encoding protumoral factors. RNA immunoprecipitation (RIP) of CPEB4 from RWP-1 cell extracts and analysis identified 842 mRNAS that were significantly associated with CPEB4. Interestingly, gene ontology analysis of the CPEB4-associated mRNAs found a significant enrichment of mRNAs involved in tumor-relevant cellular functions, including cell cycle, transcriptional regulation, apoptosis, and DNA damage. “The most significant enrichment was for mRNAS encoding general translation factors and ribosomal proteins, which are differentially regulated and are key mediators of cellular transformation,” the team remarks.

    They looked more closely at  one of the most enriched CPEB4-associated mRNAs, tPA (which is known to be overexpressed in pancreatic tumors), to see if it was differentially regulated in PDA. Interestingly, while the tPA protein is not present in normal pancreatic acinar or ductal cells, levels of its mRNA were high in both these types of cells. In contrast, levels of both tPA mRNA and tPA protein were high in the pancreatic cancer cells. This indicated that tPA mRNA is stored in normal pancreatic ducts, but is only translationally activated in PDA.

    A potential role for CPEB4 in this activation was suggested by the finding that in normal pancreas, tPA mRNA had a short poly(A) tail, whereas in ductal tumors and in PDA cell lines the poly(A) tail was elongated. More evidence was generated by the demonstration that downregulating CPEB4 in RWP-1 cells by transfection with a shCPEB4 resulted in tPA mRNA with a shorter poly(A) tail, and also led to reduced tPA protein expression, without affecting tPA mRNA levels. Effecting ectopic expression of tPA in xenografted CPEB4-downregulated RWP-1 cells resulted in an increased number of tumors. “This observa­tion suggests that although CPEB4 binds to hundreds of mRNAs, tPA mRNA translation is a key con­tributor to the decreased in vivo tumorigenicity that occurs after CPEB4 downregulation.”

    Finally, to verify whether a role for CPEB4 in propagating a tumorigenic phenotype could be identified in other cancers, the team carried out a similar set of experiments using glioblastoma cells. Again, they found that CPEB4 was absent from normal astrocytes but was elevated in high-grade gliomas, and that downregulation of CPEB4 in a glioblastoma cell line resulted in reduced tumor size, cellular proliferation, and microvessel density. “Thus, the contribution of CPEB4 to tumor malignancy seems to extend to other tumor types, suggesting that translational control by CPEB4 may be a general mechanism to control gene expression during carcinogenesis...These findings may therefore hold diagnostic and therapeutic implications for PDA, glioblastoma and possibly other tumor types.”

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