Joseph R. Testa, PhD
Office Phone: 215-728-2610
Molecular Biology of Malignant Mesothelioma (MM)Altomare, Gallagher, Hoelzle, Jianming Pei, Poulikakos, Xiao & Zhang, in collaboration with Carbone, Jhanwar, Kane, Mossman & Pass
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.Top
AKT Function and Oncogenic ActivityAltomare, Liu, Mabuchi, Jianming Pei, Rao, Yinfei Tan, Timakhov, Wang & You, in collaboration with Knepper, Denise Connolly, Di Cristofano, Farquhar, Thomas Hamilton, Kaplan, Andres Klein-Szanto, Ozols & Schilder
The Testa laboratory has had a longstanding interest in AKT kinases and AKT-related oncogenic signaling. We cloned AKT2 in 1992 and documented recurrent amplification and overexpression of AKT2 in various human cancers. The latter discovery provided the first evidence of recurrent genetic alterations in a signaling pathway that plays a central role in tumorigenesis and drug resistance. Genes encoding multiple components of this pathway, including PI3-kinase and PTEN, are now known to be mutated in many human cancers, leading to hyperactivation of AKT.
The oncogene v-akt was isolated from a retrovirus that induced naturally occurring thymic lymphomas in AKR mice. We hypothesized that constitutive activation of Akt2 could serve as a first hit for the clonal expansion of malignant T-cells by promoting cell survival and genomic instability, leading to chromosome alterations. Furthermore, genes that cooperate with Akt2 to promote malignant transformation may reside at chromosome translocation or inversion junctions found in spontaneous thymic lymphomas from transgenic mice expressing constitutively active Akt2 specifically in T cells. Our cytogenetic analysis revealed that thymic tumors from multiple founder lines exhibited either of two recurrent chromosomal rearrangements, a specific inversion of chromosome 6 and a reciprocal translocation between chromosomes 14 and 15. Fluorescence in situ hybridization, array CGH, and PCR analyses were used to delineate the inv(6) and t(14;15) breakpoints. Both rearrangements involved T-cell receptor loci, Tcrb and Tcra, respectively. The inv(6) results in robust upregulation of the homeobox/transcription factor gene Dlx5 because of its relocation near the Tcrb enhancer. The t(14;15) places the Tcra enhancer in the vicinity of the Myc proto-oncogene, resulting in upregulated Myc expression. These findings suggest that activation of the Akt pathway can act as the initial hit to promote cell survival and genomic instability, whereas the acquisition of T-cell-specific overexpression of Dlx5 or Myc leads to lymphomagenesis.
The identification of target genes of Dlx5 can facilitate our understanding of oncogenic mechanisms driven by overexpression of Dlx5. We demonstrated by luciferase assay that the Myc promoter is specifically activated by overexpression of Dlx5 and that two Dlx5 binding sites in the Myc promoter are important for transcriptional activation of Myc. We also showed that Dlx5 binds to the Myc promoter both in vitro and in vivo and that transfection of a Dlx5 expression plasmid promotes the expression of Myc in a dose-dependent manner in mammalian cells. Furthermore, overexpression of Dlx5 results in increased cell proliferation by up-regulating Myc. Knockdown of DLX5 in human lung cancer cells overexpressing DLX5 resulted in decreased expression of MYC and reduced cell proliferation, which was rescued by overexpression of MYC. Because DLX5 has a restricted pattern of expression in adult tissues, it may serve as a potential therapeutic target for the treatment of cancers that overexpress DLX5. This is clinically significant, because the MYC oncogene is aberrantly expressed in many human cancers and regulates transcription of numerous target genes involved in tumorigenesis.
In other recent work, we found upregulation of DLX5 in cell lines derived from several human tumor types, including ovarian cancer. We validated its upregulation in 20% of primary ovarian cancer specimens. Stable knockdown of DLX5 by lentivirus-mediated transduction of short hairpin RNA (shRNA) resulted in reduced proliferation of ovarian cancer cells due to inhibition of cell cycle progression in connection with the downregulation of cyclins A, B1, D1, D2 and E, and decreased phosphorylation of AKT. Cell proliferation resumed following introduction of a DLX5 cDNA harboring wobbled mutations at the shRNA-targeting sites. Cell proliferation was also rescued by transduction of a constitutively active form of AKT. Intriguingly, downregulation of IRS-2 and MET contributed to the suppression of AKT signaling. Moreover, DLX5 was found to directly bind to the IRS-2 promoter and augmented its transcription. Knockdown of DLX5 in xenografts of human ovarian cancer cells resulted in markedly diminished tumor size. In addition, DLX5 was found to cooperate with HRAS in the transformation of human ovarian surface epithelial cells. Together, these data suggest that DLX5 plays a significant role in the pathogenesis of some ovarian cancers by enhancing IRS-2/AKT signaling.
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. We showed that mTOR inhibition by the rapalog RAD001 delayed tumor onset and progression in a transgenic mouse model of ovarian cancer. In addition, 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. It also enhanced cisplatin-induced apoptosis in cells with high, but not low, AKT/mTOR activity. These results indicated that RAD001 could have therapeutic efficacy in human ovarian cancers that exhibit hyper-activation of AKT/mTOR signaling.
To test this notion further, we examined the role of mTOR as a therapeutic target in clear cell carcinoma of the ovary, which is regarded as an aggressive, chemoresistant histologic subtype. Immunohistochemical analysis showed that mTOR was more frequently activated in primary ovarian clear cell carcinomas than in ovarian serous adenocarcinomas (86.6% versus 50%). The growth-inhibitory effect of mTOR inhibition by RAD001 was examined using two pairs of cisplatin-sensitive parental and cisplatin-resistant clear cell carcinoma cell lines. Increased expression of activated AKT and activated mTOR was observed in cisplatin-resistant cells compared with parental sensitive cells. Importantly, the cisplatin-resistant cells showed greater sensitivity to RAD001 than did parental cells. These findings indicate that mTOR is frequently activated in clear cell carcinoma of the ovary and can be a promising therapeutic target in the management of this disease. Moreover, mTOR inhibition by RAD001 may be efficacious as a second-line treatment of recurrent disease in patients previously treated with cisplatin.
We also used preclinical cancer models with precisely defined genetic lesions to elucidate the efficacy of targeting Akt with the drug GSK690693. We tested the in vivo effects of GSK690693 in Lck-MyrAkt2 transgenic mice that develop lymphomas, heterozygous Pten (+/-) knockout mice that exhibit endometrial tumors, and TgMISIIR-TAg-DR26 mice that develop ovarian carcinomas, all of which exhibit hyperactivation of Akt. GSK690693 exhibited efficacy irrespective of the mechanism of Akt activation involved. Interestingly, GSK690693 was most effective in delaying tumor progression in Lck-MyrAkt2 mice expressing a membrane-bound, constitutively active form of Akt. Both tumors and primary cell cultures treated with GSK690693 showed downregulation of the Akt pathway, increased apoptosis, and primarily decreased cell proliferation. These results suggest that GSK690693 or other Akt inhibitors hold promise therapeutically in human cancers with hyperactivated AKT and/or a dependence on AKT signaling for tumor progression.Top