Homozygous losses of the <i>CDKN2A/ARF</i> locus in human mesothelioma

In MMs, we previously performed PCR-based deletion mapping studies of chromosome 9p in human MM cell lines, which enabled us to document frequent homozygous deletions of the CDKN2A/ARF locus. This locus encodes the tumor suppressor gene products p16Ink4a and p14Arf, whose inactivation affects adversely the pRb and p53 growth regulatory pathways, respectively. More recently, we used single nucleotide polymorphism (SNP) mapping arrays to perform DNA copy number analysis of four MM cell lines and one non-small cell lung carcinoma cell line to map the end-points of deletions of 9p. All five cell lines exhibited homozygous deletions encompassing thee CDKN2A/ARF and CDKN2B loci, the latter encoding the p15INK4b tumor suppressor. The DNA analysis profiles demarcated precisely two different, but overlapping, deletions in each MM cell line, i.e., a homozygous deletion embedded within a heterozygous deletion. Unlike the MM cell lines, the lung cancer cells showed two copies of a single deletion. In the latter cell line, allele analysis revealed that virtually all SNPs for chromosome 9 were homozygous, suggestive of uniparental disomy, i.e., loss of one parental copy of chromosome 9 and duplication of the remaining (defective) homologue. These findings demonstrate the utility of SNP-based mapping arrays for precise, high-resolution analysis of genomic imbalances in cancer cells.

MMs also exhibit frequent biallelic inactivation of the NF2 gene, which results in loss of expression of the NF2 protein product, merlin. We found that adenovirus-mediated expression of merlin in NF2-deficient tumor cells inhibited cell proliferation and arrested cells at the G1 phase which was accompanied by decreased expression of cyclin D1, inhibition of CDK4 activity, and dephosphorylation of CDK4 activity, and dephosphorylation of pRb. The effect of merlin on cell cycle progression was partially overridden by ectopic expression of cyclin D1. RNA interference experiments showed that silencing of the endogenous NF2 gene in NF2-positive cells results in upregulation of cyclin D1 and S-phase entry. Pak-stimulated cyclin D1 promoter activity was repressed by cotransfection of NF2, and Pak activity was inhibited by expression of merlin. Collectively, our data indicate that merlin exerts its antiproliferative tumor-suppressor effect, at least in part, via repression of Pak-induced cyclin D1 expression.

MM is a highly invasive form of cancer often characterized by massive local spreading. To investigate a possible correlation between merlin inactivation and MM invasiveness, we restored merlin expression in NF2-deficient MM cells. Re-expression of merlin markedly inhibited cell motility, spreading and invasiveness, properties connected with the malignant phenotype of MM cells. To test directly whether merlin inactivation promotes invasion in a non-malignant system, we used small interfering RNA to silence NF2 in mouse embryonic fibroblasts (MEFs) and found that downregulation of merlin resulted in enhanced cell spreading and invasion. To delineate signaling events connected with this phenotype, we investigated the effect of merlin expression on focal adhesion kinase (FAK), a key component of cellular pathways affecting migration and invasion. Expression of merlin attenuated FAK phosphorylation at the critical phosphorylation site Tyr397 and disrupted the interaction of FAK with its binding partners Src and p85, the regulatory subunit of phosphatidylinositol-3-kinase. In addition, NF2-null MM cells stably overexpressing FAK showed increased invasiveness, which decreased significantly when merlin expression was restored. Collectively, these findings suggest that merlin inactivation is a critical step in MM pathogenesis and is related, at least in part, with upregulatin of FAK activity.

To further delineate the significance of NF2 inactivation in MM and identify tumor suppressor gene alterations that cooperate with NF2 inactivation in MM pathogenesis, we employed asbestos treatment, the main cause of MM, to induce tumors in Nf2 (+/-) knockout mice. Asbestos-exposed Nf2 (+/-) mice exhibited markedly accelerated formation of MMs compared with asbestos-treated wild-type (WT) littermates. Loss of the WT Nf2 allele, leading to biallelic inactivation, was observed in all nine asbestos-induced MMs from Nf2 mice and in 50% of MMs from asbestos-exposed WT mice. For a detailed comparison with the murine model DNA analyses were also done on a series of human MM samples. Remarkably, just as in human MMs, tumors from Nf2 (+/-) mice showed frequent homologous deletions of the Cdkn2alArf locus and adjacent Cdkn2b tumor suppressor gene, as well as reciprocal inactivation of Tp53 in a subset of tumors that retained the Arf locus. As in human disease counterpart, MMs from the Nf2 (+/-) mice also showed frequent activation of Akt kinase, which plays a central role in tumorigenesis and therapeutic resistance. This is noteworthy, because MMs are usually diagnosed at an advanced disease stage and are refractory to conventional therapy. Thus, this murine model of environmental carcinogenesis faithfully recapitulates many of the molecular features of human MM and has significant implications for the further characterization of MM pathogenesis and preclinical testing of novel therapeutic modalities.

The human CDKN2A locus encodes 2 distinct proteins, p16(INK4A) and p14(ARF) [mouse p19(Arf)], designated INK4A (inhibitor of cyclin dependent kinase 4) and ARF (alternative reading frame) here, that are translated from alternatively spliced mRNAs. Human ARF is implicated as a tumor suppressor gene, mainly in association with the simultaneous deletion of INK4A. However, questions have persisted as to whether loss of ARF alone is sufficient to drive tumorigenesis. We reported that mice deficient for Arf are susceptible to accelerated asbestos-induced malignant mesothelioma (MM). MMs arising in Arf (+/-) mice consistently exhibit biallelic inactivation of Arf, but, unexpectedly, do not acquire additional recurrent genetic alterations that we previously identified in asbestos-induced MMs arising in Nf2 (+/-) mice. Array-CGH analysis was used to detect a recurrent deletion at chromosome 4C6 in MMs from Arf (+/-) mice. A candidate gene in this region, Faf1 (FAS-associated factor 1), was further explored, because it encodes a protein implicated in tumor cell survival and in the pathogenesis of some human tumor types. We confirmed hemizygous loss of Faf1 and down-regulation of Faf1 protein in a series of MMs from Arf (+/-) mice, and we then showed that Faf1 regulates TNF-alpha-mediated NF-kappaB signaling, a pathway previously implicated in asbestos-induced oncogenesis of human mesothelial cells. Collectively, these data indicate that Arf inactivation has a significant role in driving MM pathogenesis, and implicate Faf1 as a key component in the TNF-alpha/NF-kappaB signaling node that has now been independently implicated in asbestos-induced oncogenesis.

As part of Fox Chase Cancer Center’s comprehensive initiative on cancer prevention, we are working with a local diagnostics company to validate a new diagnostic kit to detect MM. The objective is to perform validation studies to confirm the merits of the test and to establish an infrastructure for long-term (~10 years) studies in which blood would be collected annually from individuals at high risk of developing MM and tested with the diagnostic kit. Initial work by another group has suggested that the test has the ability to detect MM at an early stage, when the tumor may be managed surgically and the chances of a cure are greatly improved. A positive test would identify persons who need to undergo extensive medical evaluations to confirm the actual presence of a MM. Such early detection and immediate treatment could result in an improved survival outlook.

We recently performed a phosphotyrosine proteomic screen that identified multiple kinases that are aberrantly activated in a large proportion of MM cell lines. Some of these tyrosine kinases were found to be concomitantly activated in a large percentage of MM cell lines tested. We are currently testing the efficacy of combinatorial targeting of several of the kinases identified in our screen to identify which combinations are efficacious in preclinical models of MM. This work is funded by the Meso Foundation.

Effect of mTOR inhibition in a mouse model of ovarian cancer

We previously showed that AKT kinase is frequently activated in ovarian carcinomas and was significantly associated with activation of mTOR (mammalian target of rapamycin), a downstream effector of AKT. Since mTOR plays a central role in regulating cell growth and cell cycle progression, we examined whether mTOR inhibition by RAD001 is therapeutically efficacious in the treatment of ovarian cancer as a single agent and in combination with cisplatin. RAD001 was found to markedly inhibit cell proliferation of human ovarian carcinoma cells with high AKT activity, but the effect was minimal in cells with low basal levels of AKT activity. RAD001 attenuated the expression of HIF-1α and VEGF, important factors in angiogenesis and tumor invasiveness. RAD001 also enhanced cisplatin-induced apoptosis in cells with high, but not low, AKT-mTOR activity. These results indicate that RAD001 could have therapeutic efficacy in human ovarian cancers with hyperactivated AKT-mTOR signaling.

We have also investigated AKT activation in other human types of human cancer, such as MM. We observed frequent activation (17 of 26, 65%) of AKT in human MM specimens. Consistent with reports implicating hepatocyte growth factor (HGF)/Met receptor signaling in MM, all nine human MM cell lines tested had HGF-inducible AKT activity. One of these cell lines had elevated AKT activity under serum-starvation conditions, which was associated with a homozygous deletion of PTEN. Treatment of this cell line with the mTOR inhibitor rapamycin resulted in growth arrest in G1 phase. Treatment of MM cells with the PI3K inhibitor LY294002 in combination with cisplatin had greater efficacy in inhibiting cell proliferation and inducing apoptosis than either agent alone. Collectively, these data indicate that MMs frequently express elevated AKT activity, which may be targeted pharmacologically to enhance chemotherapeutic efficacy.

We also examined whether RAD001 could inhibit tumorigenesis in TgMISIIR-TAg transgenic mice, which spontaneously develop ovarian carcinomas. RAD001 treatment markedly delayed tumor development. Tumor burden was reduced by nearly 85%. In addition, ascites formation together with peritoneal dissemination was detected in only 21% of RAD001-treated mice compared to 74% of placebo-treated animals. Approximately 30% of RAD001-treated mice developed early ovarian carcinoma confined within the ovary, whereas all placebo-treated mice developed advanced ovarian carcinoma. These preclinical findings suggest that mTOR inhibition, alone or in combination with other molecularly targeted drugs, could represent a promising chemopreventive strategy in women at high familial risk of ovarian cancer.

Finally, we continued to investigate the function of the adaptor molecule APPL1, a putative AKT2 interactor we identified in 1999, and more recently shown by others to bind to Rab5, a marker of signaling endosomes. We collaborated with D. Kaplan on a study showing that APPL1 associates with the neurotrophin receptor TrkA, which plays a critical role in the nervous system by recruiting signaling molecules that activate pathways required for the growth and survival of neurons. The C-terminus of APPL1 was found to directly engage the PDZ domain of GIPC1, a TrkA-interacting protein. In addition, the APPL1 PTB domain was shown to bind directly to TrkA, independently of GIPC1. Isolation of endosomal fractions by high-resolution centrifugation revealed that APPL1, GIPC1, and phosphorylated TrkA are enriched in the same fractions. Reduction of APPL1 or GIPC1 protein levels suppressed nerve growth factor (NGF)-dependent MEK, extracellular signal-regulated kinase, and Akt activation and neurite outgrowth in PC12 cells. Together, these results indicate that a population of endosomes bearing a complex of APPL1, GIPC1, and activated TrkA may transmit NGF signals. Another collaboration, with M. Farquhar, revealed that when PC12(615) cells are treated with either NGF or antibody agonists to activate TrkA, GIPC and APPL translocate from the cytoplasm and bind to incoming, endocytic vesicles carrying TrkA concentrated at the tips of the cell processes. GIPC, but not APPL, dissociates from these peripheral endosomes prior to or during their trafficking from the cell periphery to the juxtanuclear region. GIPC’s interaction with APPL was found to be essential for recruitment of GIPC to peripheral endosomes and for TrkA signaling, because a GIPC PDZ domain mutant that cannot bind APPL or APPL knockdown with small interfering RNA inhibits NGFinduced GIPC recruitment, mitogen-activated protein kinase activation, and neurite outgrowth.