One of the current goals in cancer research is to discover and validate novel molecular targets that may be useful for diagnostic and therapeutic purposes in fighting this disease. The PRL phosphatases (PRL-1, PRL-2, and PRL-3) are low molecular weight protein tyrosine phosphatases with unknown biological function(s) that have gained attention from cancer researchers in the past couple of years, mainly due to reports that these phosphatases may play important roles in tumor progression and metastasis. Motivated by the particular urgent need for molecular targets in pancreatic cancer this work was undertaken to determine what role PRL proteins played in pancreatic cancer biology and to determine if targeting PRLs would be effective in treating this disease. In this dissertation, it was found that both PRL-1 and PRL-2, but not PRL-3 are upregulated in pancreatic adenocarcinomas, suggesting that some cancer cells are dependent upon their activity for continued proliferation and survival. To validate this hypothesis, siRNAs were used in cell-based assays to evaluate the biological consequences of PRL-1 and/or PRL-2 inhibition. It was found that perturbations in PRL phosphatase signaling result in reduced proliferation, migration and especially the ability to grow in soft agar. Oligonucleotide microarray analysis revealed that many Erk and/or Akt dependent stress and growth factor inducible genes were differentially regulated between pancreatic cancer cells treated with PRL-targeting siRNA and their non-targeting siRNA treated counterparts. Subsequently, PRL knockdown was found to alter serum induced as well as amino acid deprivation induced Akt and Erk phosphorylation in multiple pancreatic cancer cell lines, suggesting that PRLs function upstream of these key pathways. Interestingly, we show that PRL proteins in cell free assays exhibit higher activity on doubly phosphorylated phosphatidylinositol substrates than tyrosine-phosphorylated peptides, suggesting that the biological substrate(s) might include non-protein molecules. These data support the hypothesis that PRL-1 and PRL-2 might play important biological roles in pancreatic cancer cells and further studies should be undertaken to determine the usefulness of these phosphatases as potential molecular biomarkers and targets.

Indiana University-Purdue University Indianapolis (IUPUI) / Phosphatases of regenerating liver, consisting of PRL-1, PRL-2 and PRL-3, belong to a novel protein tyrosine phosphatases subfamily, whose overexpression promotes cell proliferation, migration and invasion and contributes to tumorigenesis and metastasis. However, although great efforts have been made to uncover the biological function of PRLs, limited knowledge is available on the underlying mechanism of PRLs’ actions, therapeutic value by targeting PRLs, as well as the physiological function of PRLs in vivo. To answer these questions, we first screened a phage display library and identified p115 RhoGAP as a novel PRL-1 binding partner. Mechanistically, we demonstrated that PRL-1 activates RhoA and ERK1/2 by decreasing the association between active RhoA with GAP domain of p115 RhoGAP, and displacing MEKK1 from the SH3 domain of p115 RhoGAP, respectively, leading to enhanced cell proliferation and migration. Secondly, structure-based virtual screening was employed to discover small molecule inhibitors blocking PRL-1 trimer formation which has been suggested to play an important role for PRL-1 mediated oncogenesis. We identified Cmpd-43 as a novel PRL-1 trimer disruptor. Structural study demonstrated the binding mode of PRL-1 with the trimer disruptor. Most importantly, cellular data revealed that Cmpd-43 inhibited PRL-1 induced cell proliferation and migration in breast cancer cell line MDA-MB-231 and lung cancer cell line H1299. Finally, in order to investigate the physiological function of PRLs, we generated mouse knockout models for Prl-1, Prl-2 and Prl-3. Although mice deficient for Prl-1 and Prl-3 were normally developed, Prl-2-null mice displayed growth retardation, impaired male reproductive ability and insufficient hematopoiesis. To further investigate the in vivo function of Prl-1, we generated Prl-1-/-/Prl-2+/- and Prl-1+/-/Prl-2-/- mice. Similar to Prl-2 deficient male mice, Prl-1-/-/Prl-2+/- males also have impaired spermatogenesis and reproductivity. More strikingly, Prl-1+/-/Prl-2-/- mice are completely infertile, suggesting that, in addition to PRL-2, PRL-1 also plays an important role in maintaining normal testis function. In summary, these studies demonstrated for the first time that PRL-1 activates ERK1/2 and RhoA through the novel interaction with p115 RhoGAP, targeting PRL-1 trimer interface is a novel anti-cancer therapeutic treatment and both PRL-1 and PRL-2 contribute to spermatogenesis and male mice reproductivity.