Zaidoun Salah

miRNA Signatures Associate with Pathogenesis and Progression of Osteosarcoma

Osteosarcoma remains a leading cause of cancer death in adolescents. Treatment paradigms and survival rates have not improved in two decades. Driving the lack of therapeutic inroads, the molecular etiology of osteosarcoma remains elusive. MicroRNAs (miRNAs) have demonstrated far-reaching effects on the cellular biology of development and cancer. Their role in osteosarcomagenesis remains largely unexplored. Here we identify for the first time an miRNA signature reflecting the pathogenesis of osteosarcoma from surgically procured samples from human patients. The signature includes high expression of miR-181a,miR-181b, and miR-181c as well as reduced expression of miR-16, miR-29b, and miR-142-5p. We also demonstrate that miR-181b and miR-29b exhibit restricted expression to distinct cell populations in the tumor tissue. Further, higher expression of miR-27a and miR-181c* in pre-treatment biopsy samples characterized patients who developed clinical metastatic disease. In addition, higher expression of miR-451 and miR-15b in pre-treatment samples correlated with subsequent positive response to chemotherapy. In vitro and in vivo functional validation in osteosarcoma cell lines confirmed the tumor suppressive role of miR-16 and the pro-metastatic role of miR-27a. Furthermore, predicted target genes for miR-16 and miR-27a were confirmed as down-regulated by real-time PCR. Affymetrix array profiling of cDNAs from the osteosarcoma specimens and controls were interrogated according to predicted targets of miR-16, miR142-5p, miR-29b, miR-181a/b, and miR-27a. This analysis revealed positive and negative correlations highlighting pathways of known importance to osteosarcoma, as well as novel genes. Thus, our findings establish a miRNA signature associated with pathogenesis of osteosarcoma as well as critical pre-treatment biomarkers of metastasis and responsiveness to therapy.

Oncogenic transformation induces tumor angiogenesis: a role for PAR1 activation

The formation of new blood vessels is a critical determinant of tumor progression. We find that Par1 gene expression plays a central role in blood vessel recruitment in animal models. By in vivo injection of either Matrigel plugs containing Par1‐expressing cells or of rat prostatic carcinoma cells transfected with tetracycline‐inducible Par1 expression vectors, we show that Par1 significantly enhances both angiogenesis and tumor growth. Several vascular endothelial growth factor (VEGF) splice forms are induced in cells expressing Par1. Activation of PAR1 markedly augments the expression of VEGF mRNAs and of functional VEGFs as determined by in vitro assays for endothelial tube alignment and bovine aortic endothelial cell proliferation. Because neutralizing anti‐VEGF antibodies potently inhibited Par1‐induced endothelial cell proliferation, we conclude that Par1‐induced angiogenesis requires VEGF. Specific inhibitors of protein kinase C (PKC), Src, and phosphatidylinositol 3‐kinase (PI3K) inhibit Par1‐induced VEGF expression, suggesting the participation of these kinases in the process. We also show that oncogenic transformation by genes known to be part of PAR1 signaling machinery is sufficient to increase VEGF expression in NIH 3T3 cells. These data support the novel notion that initiation of cell signaling either by activating PAR1 or by the activated forms of oncogenes is sufficient to induce VEGF and hence angiogenesis. Yin, Y.‐J., Salah, Z., Maoz, M., Cohen Even Ram, S., Ochayon, S., Neufeld, G., Katzav, S., Bar‐Shavit, R. Oncogenic transformation induces tumor angiogenesis: a role for PAR1 activation. FASEB J. 17, 163–174 (2003)

WWOX: Its genomics, partners, and functions

The WW domain‐containing oxidoreductase (WWOX) spans one of the most active common fragile sites (CFSs) involved in cancer, FRA16D. WWOX encodes a 46‐kDa protein that contains two N‐terminal WW domains and a central short‐chain dehydrogenase/reductase (SDR) domain. Through its WW domain, Wwox interacts with its partners and modulates their functions. Our data indicate that Wwox suppresses the transactivation function of several transcription factors implied in neoplasia by sequestering them in the cytoplasm. Work from our laboratory and other research groups have demonstrated that Wwox participates in a number of cellular processes including growth, differentiation, apoptosis, and tumor suppression. Targeted deletion of the Wwox gene in mice causes increased spontaneous and chemically induced tumor incidence supporting bona fide tumor suppressor function of WWOX. Moreover, generation of the Wwox‐deficient mice uncovers, at least in part, some of the physiological in vivo functions of the WWOX gene. This review focuses on recent progress that elucidates Wwox functions in biology and pathology. J. Cell. Biochem. 108: 737–745, 2009.

Negative regulation of the Hippo pathway by E3 ubiquitin ligase ITCH is sufficient to promote tumorigenicity

The Hippo tumor suppressor pathway, originally defined in fruit flies, regulates cellular proliferation and survival and exerts profound effects on normal mammalian cell fate and tumorigenesis. The present understanding of Hippo pathway components and mechanisms remains incomplete in cancer. WW domain-containing proteins regulate diverse biological processes through interaction with proline-tyrosine (PPxY)-containing targets. In this study, we report that the E3 ubiquitin ligase ITCH regulates stability of LATS1, a serine/threonine kinase in the Hippo pathway, through protein-protein interaction of the PPxY motifs of LATS1 with the WW domains of ITCH. Ubiquitination of LATS1 catalyzed by ITCH stimulated the proteasomal degradation of LATS1. Furthermore, ITCH-mediated degradation of LATS1 was associated with enhanced cell growth, induction of epithelial-mesenchymal transition, and increased tumorigenicity. Conversely, ITCH depletion increased LATS1 levels, enhancing FAS-induced apoptosis and reducing proliferation, survival, and migration. These phenotypes were rescued when both ITCH and LATS1 were depleted. Together, our results reveal a novel functional link between ITCH and the Hippo pathway, deepening their critical roles in tumorigenesis.

Differential expression of Protease activated receptor 1 (Par1) and pY397FAK in benign and malignant human ovarian tissue samples

Protease activated receptors (PAR) form a family of G‐protein coupled receptors (GPCR) encoding their own ligands and uniquely activated via proteolytic cleavage. Although proteases in general have been implicated in the remodeling of the extracellular tumor microenvironment, the role of cell surface receptors activated by proteolysis is now emerging. In our present study we investigated the expression pattern of protease activated receptor 1 hPar1 in ovarian carcinoma tissue samples. Abundant hPar1 mRNA and protein were detected in “low malignant potential” and in invasive carcinomas, regardless of the histological subtype. In contrast, no hPar1 expression was detected on the cell surface of normal ovarian epithelium. The differential expression pattern of hPar1 was shown by in situ hybridization, immunohistochemistry and semi‐quantitative RT‐PCR analyses. In early stages of ovarian carcinoma (Ia), the contra lateral normal ovary showed strong PAR1 expression as opposed to the lack of expression in the ovarian epithelium obtained from normal individuals. In parallel, we analyzed the expression pattern of αvβ5 integrin and of activated focal adhesion kinase (FAK), a major focal contact protein, in these tissues. Although abundant expression of αvβ5 integrin was observed in all tissues specimens examined, regardless of either normal or malignant, the level of activated FAK was differentially expressed. Phosphorylated FAK was seen in invasive ovarian carcinoma, but not in the normal ovarian epithelium. The abundant hPar1 levels in pathological malignant ovarian carcinoma is likely to transmit signals leading to the phosphorylation of FAK and thereby alterations in the integrin functional state. Altogether our data suggest that hPar1 and FAK cooperate to promote ovarian cancer malignancy.

Frequent Attenuation of the WWOX Tumor Suppressor in Osteosarcoma Is Associated with Increased Tumorigenicity and Aberrant RUNX2 Expression

The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor that is deleted or attenuated in most human tumors. Wwox-deficient mice develop osteosarcoma (OS), an aggressive bone tumor with poor prognosis that often metastasizes to lung. On the basis of these observations, we examined the status of WWOX in human OS specimens and cell lines. In human OS clinical samples, WWOX expression was absent or reduced in 58% of tumors examined (P < 0.0001). Compared with the primary tumors, WWOX levels frequently increased in tumors resected following chemotherapy. In contrast, tumor metastases to lung often exhibited reduced WWOX levels relative to the primary tumor. In human OS cell lines having reduced WWOX expression, ectopic expression of WWOX inhibited proliferation and attenuated invasion in vitro, and suppressed tumorigenicity in nude mice. Expression of WWOX was associated with reduced RUNX2 expression in OS cell lines, whereas RUNX2 levels were elevated in femurs of Wwox-deficient mice. Furthermore, WWOX reconstitution in HOS cells was associated with downregulation of RUNX2 levels and RUNX2 target genes, consistent with the ability of WWOX to suppress RUNX2 transactivation activity. In clinical samples, RUNX2 was expressed in the majority of primary tumors and undetectable in most tumors resected following chemotherapy, whereas most metastases were RUNX2 positive. Our results deepen the evidence of a tumor suppressor role for WWOX in OS, furthering its prognostic and therapeutic significance in this disease.

Human Protease-Activated Receptor 1 Expression in Malignant Epithelia: A Role in Invasiveness

While protease-activated receptors (PARs) play a traditional role in vascular biology, they emerge with surprisingly new assignments in tumor biology. PAR1 expression correlates with the invasion properties of breast carcinoma, whereas human PAR1 antisense reduces their ability to migrate through Matrigel. Part of the molecular mechanism of PAR1 invasion involves the formation of focal contact complexes on PAR1 activation. PAR1 induces angiogenesis in animal models in vivo and exhibits an oncogenic phenotype of enhanced ductal complexity when overexpressed in mouse mammary glands.

WWOX gene and gene product: tumor suppression through specific protein interactions

The WWOX gene, an archetypal fragile gene, encompasses a chromosomal fragile site at 16q23.2, and encodes the approximately 46-kDa Wwox protein, with WW domains that interact with a growing list of interesting proteins. If the function of a protein is defined by the company it keeps, then Wwox is involved in numerous important signal pathways for bone and germ-cell development, cellular and animal growth and death, transcriptional control and suppression of cancer development. Because alterations to genes at fragile sites are exquisitely sensitive to replication stress-induced DNA damage, there has been an ongoing scientific discussion questioning whether such gene expression alterations provide a selective advantage for clonal expansion of neoplastic cells, and a parallel discussion on why important genes would be present at sites that are susceptible to inactivation. We offer some answers through a description of known WWOX functions.

Targeted Ablation of the WW Domain-Containing Oxidoreductase Tumor Suppressor Leads to Impaired Steroidogenesis

The WW domain-containing oxidoreductase (WWOX) gene encodes a 46-kDa tumor suppressor. The Wwox protein contains two N-terminal WW domains that interact with several transcriptional activators containing proline-tyrosine motifs and a central short-chain dehydrogenase/reductase domain that has been suggested to play a role in steroid metabolism. Recently, we have shown that targeted deletion of the Wwox gene in mice leads to postnatal lethality and defects in bone growth. Here, we report that Wwox-deficient mice display impaired steroidogenesis. Mutant homozygous mice are born with gonadal abnormalities, including failure of Leydig cell development in testis and reduced theca cell proliferation in ovary. Furthermore, Wwox(-/-) mice displayed impaired gene expression of key steroidogenesis enzymes. Affymetrix microarray gene analysis revealed differentially expressed related genes in steroidogenesis in knockout mice testis and ovary as compared with control mice. These results demonstrate the essential requirement for the Wwox tumor suppressor in proper steroidogenesis.

Wwox inactivation enhances mammary tumorigenesis

Breast cancer is the leading cause of cancer-related death in women worldwide. Expression of the WWOX tumor suppressor is absent or reduced in a large proportion of breast tumors suggesting that loss of WWOX may contribute to breast tumorigenesis. Wwox-deficient mice die by 3–4 weeks of age precluding adult tumor analysis. To evaluate the effect of WWOX-altered expression on mammary tumor formation, the Wwox-heterozygous allele was back crossed onto the C3H mammary tumor-susceptible genetic background (WwoxC3H+/−) and incidence of mammary tumor formation was evaluated. Although 50% of the female WwoxC3H+/− mice developed mammary carcinomas, only 7% of WwoxC3H+/+ mice did. Intriguingly, mammary tumors in WwoxC3H+/− mice frequently lost WWOX protein expression suggesting a genetic predisposition toward mammary tumorigenesis. Immunohistochemical staining of hormone receptors revealed loss of estrogen receptor-α (ER) and progesterone receptor in the majority of these tumors. In vitro, depletion of WWOX in MCF7 ER-positive cells led to reduced ER expression and reduced sensitivity to tamoxifen and estrogen treatment and was associated with enhanced survival and anchorage-independent growth. Finally, cDNA array analyses of murine normal mammary epithelial cells and mammary tumors identified 163 significantly downreguated and 129 upregulated genes in the tumors. The majority of differentially expressed genes were part of pathways involved in cellular movement, cell-to-cell signaling and interaction, cellular development, cellular growth and proliferation and cell death. These changes in gene expression of mouse mammary tumors in WwoxC3H+/− mice resemble, at least in part, human breast cancer development. Our findings demonstrate the critical role that the WWOX tumor suppressor gene has in preventing tumorigenesis in breast cancer.