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The human genome is now estimated to consist of just over 20,000 genes, no more than five times the number of genes of unicellular yeast. How then can the complexity of humans arise? The answer partially lies in RNA alternative splicing, a process that produces many forms of proteins from a single gene. These proteins can exert diverse functions that have profound biological consequences. De-regulation of alternative splicing is linked to disease, such as the development of cancer. In fact, a majority of tumor suppressors and oncogenes can be alternatively spliced. They include Ras, Src, Fas, BRCA1, p53, and Her2. Yet it remains largely unknown on how alternative splicing is regulated, and even less is known on the role of splice isoforms in oncogenic transformation.

The Cheng lab uses CD44 as a model to address two fundamental questions: 1) How does signaling regulate alternative splicing? 2) What is the role of alternatively spliced isoforms in the development of cancer? We hope that our research will provide a mechanistic paradigm on how regulated alternative splicing controls cancer pathogenesis.

CD44 can regulate cell proliferation, adhesion, migration, and invasion. CD44 is also a marker for the enrichment of cancer stem cells. These multiple functions of CD44 may be related to its different splice isoforms. The CD44 gene is composed of 10 constitutive exons and 10 variable exons residing between its constitutive exons . Through alternative splicing, cells can create a large number of CD44 isoforms with unique extracellular regions that are encoded by these variable exons. These variably expressed regions mediate growth factor stimulation, metalloprotease recruitment, and growth factor maturation. We discovered that alternative splicing of CD44 and Ras/MAPK signaling are coupled in a positive feedback loop. An isoform of CD44 (CD44v6) produced by alternative splicing augments the action of receptor tyrosine kinases, such as Met and EGFR, resulting in activation of the Ras/MAPK pathway. Activated Ras/MAPK signaling further stimulates CD44 alternative splicing that overproduces CD44 variants. The newly synthesized CD44v6 isoforms continue to act on receptor tyrosine kinases resulting in a positive feedback circuit that sustains Ras/MAPK signaling – a critical mechanism for oncogenic transformation. We further found that CD44v6 variants are important in cell cycle progression at the G1-S transition through sustaining Ras/MAPK signal. These studies reveal a mechanism that in part answers a long-standing question regarding how mitogenic signaling can be sustained for a sufficient time period to promote cell proliferation. In agreement with these findings, we recently discovered that CD44 alternative splicing is dynamically regulated during the progression of breast cancer. We are exploring the role of CD44 isoforms in tumorigenesis by combining cell culture and animal models of breast cancer.

Our investigation on mechanisms through which signaling regulates alternative splicing indicates that the splicing coactivator SRm160 regulates CD44 alternative splicing in a Ras-dependent manner. Furthermore, reduction of SRm160 by siRNA treatment inhibits alternative splicing of endogenous CD44 and also leads to decreased tumor cell invasiveness. These results indicate that Ras signaling stimulates CD44 alternative splicing through regulation of splicing factors. They support a link between regulation of alternative splicing and tumor cell invasiveness.