Conditional transgenic expression of PIM1 kinase in prostate induces inflammation-dependent neoplasia.

The Pim proteins are a family of highly homologous protein serine/threonine kinases that have been found to be overexpressed in cancer. Elevated levels of Pim1 kinase were first discovered in human leukemia and lymphomas. However, more recently Pim1 was found to be increased in solid tumors, including pancreatic and prostate cancers, and has been proposed as a prognostic marker. Although the Pim kinases have been identified as oncogenes in transgenic models, they have weak transforming abilities on their own. However, they have been shown to greatly enhance the ability of other genes or chemical carcinogens to induce tumors. To explore the role of Pim1 in prostate cancer, we generated conditional Pim1 transgenic mice, expressed Pim1 in prostate epithelium, and analyzed the contribution of PIM1 to neoplastic initiation and progression. Accordingly, we explored the effect of PIM1 overexpression in 3 different settings: upon hormone treatment, during aging, and in combination with the absence of one Pten allele. We have found that Pim1 overexpression increased the severity of mouse prostate intraepithelial neoplasias (mPIN) moderately in all three settings. Furthermore, Pim1 overexpression, in combination with the hormone treatment, increased inflammation surrounding target tissues leading to pyelonephritis in transgenic animals. Analysis of senescence induced in these prostatic lesions showed that the lesions induced in the presence of inflammation exhibited different behavior than those induced in the absence of inflammation. While high grade prostate preneoplastic lesions, mPIN grades III and IV, in the presence of inflammation did not show any senescence markers and demonstrated high levels of Ki67 staining, untreated animals without inflammation showed senescence markers and had low levels of Ki67 staining in similar high grade lesions. Our data suggest that Pim1 might contribute to progression rather than initiation in prostate neoplasia.

The essential role of PIM kinases in sarcoma growth and bone invasion.

PIM kinases are a family of serine/threonine kinases composed of three different isoforms (PIM1, PIM 2 and PIM 3) that are highly homologous. Their expression is mediated by the JAK/STAT signalling pathway, providing survival and cell cycle transition signals. PIM kinases are heavily targeted for anticancer drug discovery. However, very little is known about the relative contribution of the different isoforms to the tumourigenesis process in vivo, and how their individual inhibition might affect tumour growth. Taking advantage of genetically modified mice, we explored whether the inhibition of specific isoforms is required to prevent sarcomas induced by 3-methylcholanthrene carcinogenic treatment. We found that absence of Pim2 and Pim3 greatly reduced sarcoma growth to a similar extent to the absence of all three isoforms. This model of sarcoma generally produces bone invasion by the tumour cells. Lack of Pim2 and Pim3 reduced tumour-induced bone invasion by 70%, which is comparable with the reduction of tumour-induced bone invasion in the absence of all three isoforms. Similar results were obtained in mouse embryonic fibroblasts (MEFs) derived from these knockout (KO) mice, where double Pim2/3 KO MEFs already showed reduced proliferation and were resistant to oncogenic transformation by the RAS oncogene. Our data also suggest an important role of Gsk3ß phosphorylation in the process of tumourigenesis.

Spinophilin acts as a tumor suppressor by regulating Rb phosphorylation.

The scaffold protein Spinophilin (SPN) is a regulatory subunit of phosphatase1a located at 17q21.33. This region is frequently associated with microsatellite instability and LOH containing a relatively high density of known tumor suppressor genes, including BRCA1. Several linkage studies have suggested the existence of an unknown tumor suppressor gene distal to BRCA1. Spn may be this gene, but the mechanism through which this gene makes its contribution to cancer has not been described. In this study, we aimed to determine how loss of Spn may contribute to tumorigenesis. We explored the contribution of SPN to PP1a-mediated Rb regulation. We found that the loss of Spn downregulated PPP1CA and PP1a activity, resulting in a high level of phosphorylated Rb and increased ARF and p53 activity. However, in the absence of p53, reduced levels of SPN enhanced the tumorigenic potential of the cells. Furthermore, the ectopic expression of SPN in human tumor cells greatly reduced cell growth. Taken together, our results demonstrate that the loss of Spn induces a proliferative response by increasing Rb phosphorylation, which, in turn, activates p53, thereby neutralizing the proliferative response. We suggest that Spn may be the tumor suppressor gene located at 17q21.33 acting through Rb regulation.

Spinophilin loss contributes to tumorigenesis in vivo.

The scaffold protein spinophilin (SPN, PPP1R9B) is a regulatory subunit of phosphatase-1a located at 17q21.31. This region is frequently associated with microsatellite instability and LOH and contains a relatively high density of known tumor suppressor genes (such as BRCA1), putative tumor suppressor genes and several unidentified candidate tumor suppressor genes located distal to BRCA1. Spn is located distal to BRCA1, and we have previously shown that the loss of Spn contributes to human tumorigenesis in the absence of p53 function. In this work, we explore the role of Spn as putative tumor suppressor in in vivo models using genetically modified mice. Spn-knockout mice had decreased lifespan with increased cellular proliferation in tissues such as the mammary ducts and early appearance of tumors, such as lymphoma. Furthermore, the combined loss of Spn and mutant p53 activity led to increased mammary carcinomas, confirming the functional relationship between p53 and Spn. We suggest that Spn may be a novel tumor suppressor located at 17q21.

Exploring the gain of function contribution of AKT to mammary tumorigenesis in mouse models.

Elevated expression of AKT has been noted in a significant percentage of primary human breast cancers, mainly as a consequence of the PTEN/PI3K pathway deregulation. To investigate the mechanistic basis of the AKT gain of function-dependent mechanisms of breast tumorigenesis, we explored the phenotype induced by activated AKT transgenes in a quantitative manner. We generated several transgenic mice lines expressing different levels of constitutively active AKT in the mammary gland. We thoroughly analyzed the preneoplastic and neoplastic mammary lesions of these mice and correlated the process of tumorigenesis to AKT levels. Finally, we analyzed the impact that a possible senescent checkpoint might have in the tumor promotion inhibition observed, crossing these lines to mammary specific p53(R172H) mutant expression, and to p27 knock-out mice. We analyzed the benign, premalignant and malignant lesions extensively by pathology and at molecular level analysing the expression of proteins involved in the PI3K/AKT pathway and in cellular senescence. Our findings revealed an increased preneoplastic phenotype depending upon AKT signaling which was not altered by p27 or p53 loss. However, p53 inactivation by R172H point mutation combined with myrAKT transgenic expression significantly increased the percentage and size of mammary carcinoma observed, but was not sufficient to promote full penetrance of the tumorigenic phenotype. Molecular analysis suggest that tumors from double myrAKT;p53(R172H) mice result from acceleration of initiated p53(R172H) tumors and not from bypass of AKT-induced oncogenic senescence. Our work suggests that tumors are not the consequence of the bypass of senescence in MIN. We also show that AKT-induced oncogenic senescence is dependent of pRb but not of p53. Finally, our work also suggests that the cooperation observed between mutant p53 and activated AKT is due to AKT-induced acceleration of mutant p53-induced tumors. Finally, our work shows that levels of activated AKT are not essential in the induction of benign or premalignant tumors, or in the cooperation of AKT with other tumorigenic signal such as mutant p53, once AKT pathway is activated, the relative level of activity seems not to determine the phenotype.