Carl W. Miller

Cyr61 Is Overexpressed in Gliomas and Involved in Integrin-Linked Kinase-Mediated Akt and β-Catenin-TCF/Lef Signaling Pathways

Cyr61 is a member of the CCN family of growth factors; these proteins are secreted and can act as ligands of distinct integrins. We show that Cyr61 can enhance tumorigenicity of glioma cells acting through activated integrin-linked kinase (ILK) to stimulate β-catenin-TCF/Lef and Akt signaling pathways. Overexpression of Cyr61 occurred in highly tumorigenic glioma cell lines and in 68% of the most malignant glioblastoma multiforme brain tumors. Forced expression of Cyr61 in U343 glioma cells accelerated their growth in liquid culture, enhanced their anchorage-independent proliferation in soft agar, and significantly increased their ability to form large, vascularized tumors in nude mice. Overexpression of Cyr61 in the U343 cells led to the up-regulation of distinct integrins, including β1 and ανβ3, which have been shown to interact with Cyr61 and ILK. The activity of ILK was increased dramatically in these cells. Overexpression of Cyr61 also resulted in the phosphorylation of glycogen synthase kinase-3β and accumulation and nuclear translocation of β-catenin, leading to activation of the β-catenin-TCF/Lef-1 signaling pathway. Furthermore, forced expression of Cyr61 in the glioma cells activated phosphatidylinositol 3′-kinase pathway, resulting in prominent phosphorylation of Akt and the antiapoptotic protein Bad. Cyr61 appears to stimulate several signaling pathways in the development of gliomas.

Breast Cancer Cyr61 IS OVEREXPRESSED, ESTROGEN-INDUCIBLE, AND ASSOCIATED WITH MORE ADVANCED DISEASE

To identify genes involved in breast cancer, polymerase chain reaction-selected cDNA subtraction was utilized to construct a breast cancer-subtracted library. Differential screening of the library isolated the growth factor-inducible immediate-early geneCyr61, a secreted, cysteine-rich, heparin binding protein that promotes endothelial cell adhesion, migration, and neovascularization. Northern analysis revealed that Cyr61was expressed highly in the invasive breast cancer cell lines MDA-MB-231, T47D, and MDA-MB-157; very low levels were found in the less tumorigenic MCF-7 and BT-20 breast cancer cells and barely detectable amounts were expressed in the normal breast cells, MCF-12A. Univariate analysis showed a significant or borderline significant association between Cyr61 expression and stage, tumor size, lymph node positivity, age, and estrogen receptor levels. Interestingly, expression of Cyr61 mRNA increased 8- to 12-fold in MCF-12A and 3- to 5-fold in MCF-7 cells after 24- and 48-h exposure to estrogen, respectively. Induction of Cyr61mRNA was blocked by tamoxifen and ICI182,780, inhibitors of the estrogen receptor. Stable expression of Cyr61 cDNA under the regulation of a constitutive promoter in MCF-7 cells enhanced anchorage-independent cell growth in soft agar and significantly increased tumorigenicity and vascularization of these tumors in nude mice. Moreover, overexpression of Cyr61 in MCF-12A normal breast cells induced their tumor formation and vascularization in nude mice. In summary, these results suggest that Cyr61 may play a role in the progression of breast cancer and may be involved in estrogen-mediated tumor development.

Alterations of thep53, Rb andMDM2 genes in osteosarcoms

Molecular defects affecting tumor-suppressor genes are an important step in the genesis of sarcomas. For example, inheritance of a defectiveRb orp53 gene predisposes the carrier to develop osteosarcoma, among other malignancies. In this study, we have assessed the occurrence ofp53, Rb andMDM2 alterations in the same samples of osteosarcomas, along with representative samples of various other sarcomas. Point mutations of thep53 gene were found in 13 of 42 osteosarcomas and 1 of 8 leiomyosarcomas, and gross rearrangement of thep53 gene was demonstrated in 5 of 37 osteosarcomas. The retinoblastoma susceptibility gene (Rb) was either rearranged or deleted in 7 of 37 osteosarcomas, 1 of 7 soft-tissue sarcomas and 1 of 4 Ewing sarcomas. Remarkably, 5 of the osteosarcomas havingRb alterations also hadp53 mutations. Amplification and overexpression of theMDM2 oncogene may lead to increasedMDM2-p53 binding resulting in inactivation ofp53 function. A two- to threefold increase in the copy number ofMDM2 was detected in 7 of 37 samples, 5 of which were osteosarcomas. Amplification of theMDM2 gene occurred independently ofp53 mutation; one sample having threefold amplification ofMDM2 also had ap53 mutation. In summary, 34 alterations of thep53, Rb andMDM2 genes were found in 26 of 42 (62%) osteosarcomas.

Alterations of the p15, p16,and p18 genes in osteosarcoma.

Activation of cyclin-dependent kinases (CDKs) by interaction with cyclins regulates progression through cell cycle checkpoints. This process is counterbalanced by CDK inhibitors (CDKIs), which can inhibit progression through the cell cycle. Because CDKI expression acts to inhibit cellular proliferation, CDKIs may have a role as tumor suppressors. One class of CDKIs, characterized by the presence of ankyrin repeats, has at least four members (p15INK4B), p16INK4, p18, and p19). Two of these, p15INK4B, p16INK4, have been mapped to chromosome 9p21, a region of frequent loss in a wide variety of cancers. Alterations of p16INK4 have been detected in various tumors and cell lines. We analyzed p15INK4B, p16INK4, and p18 alterations in 52 osteosarcomas (including 11 explants), and 23 other various sarcomas. Single-stranded conformation polymorphism analysis [polymerase chain reaction (PCR-SSCP)] of the coding regions of these CDKI genes detected a missense mutation of p16INK4 exon 1 in one soft tissue sarcoma. Southern blotting detected complete deletion of p15INK4B and p16INK4 genes in osteosarcomas from 2 patients and a soft tissue sarcoma from another individual. Loss of heterozygosity (LOH) at chromosome 9p21 was observed with a microsatellite probe closely linked to the INK4 genes in the latter case. Deletions of both p15INK4B and p16INK4 genes were detected in five of eight osteosarcoma cell lines. By contrast, no alterations of p18 were detected in any sample. Together these data suggest that alterations of the p15INK4B and p16INK4 genes, but not p18, may occur in approximately 5% of sarcomas. However, deletions of the p15INK4B and P16INK4 genes are frequent in osteosarcoma cell lines and probably have a role in tumor cell growth in culture. Notably, all seven detectable deletions involved both p15INK4B and p16INK4 genes, suggesting that both contribute individual tumor suppressor activity.

Mutations of the CHK2 gene are found in some osteosarcomas, but are rare in breast, lung, and ovarian tumors.

Checkpoint genes, activated in response to DNA damage and other stresses, are frequently targeted for alteration in cancer. Checkpoint kinase 2 (CHK2, CDS1, RAD53) is activated by ataxia telangiectasia mutated (ATM) in response to γ irradiation. Activated CHK2 stabilizes TP53, and acts on other cell cycle and stress regulators. These findings place CHK2 in the middle of a pathway frequently targeted in cancer. Because of this, and the observation that CHK2 mutations are inherited in some Li‐Fraumeni cancer syndrome families, we decided to examine the role of CHK2 mutations in sporadic cancers. Exploiting the genomic sequence of chromosome 22, we looked for mutations in the exons and intron junctions of the CHK2 gene in DNA samples from 170 patients (57 osteosarcomas, 25 other sarcomas, 35 nonsmall‐cell lung, 20 ovarian, and 33 breast cancers). Missense mutations affecting the forkhead and kinase domains were detected in four osteosarcomas and in one ovarian and one lung cancer. These findings of CHK2 gene mutations are consistent with osteosarcoma being a defining tumor of Li‐Fraumeni syndrome. The occurrence of CHK2 mutations in sporadic cancers emphasizes the importance of the stress pathway which includes TP53.

FOXA1: Growth inhibitor and a favorable prognostic factor in human breast cancer

The transcription factor Forkhead‐box A1 (Foxa1), a member of the FOX class of transcription factors, has been implicated in the pathogenesis of lung, esophageal and prostate cancers. We have recently identified transcriptional activation of p27 by FOXA1. In this study, we analyzed the activities and expression pattern of FOXA1 in breast cancer. Forced expression of FOXA1 inhibited clonal growth of breast cancer cell lines, and FOXA1 levels inversely correlated with growth stimuli. In the estrogen receptor (ER)‐positive MCF‐7 cells, FOXA1 increased p27 promoter activity and inhibited the ER pathway activity. Analysis of FOXA1 expression in breast tissue arrays revealed significantly higher expression in pure ductal carcinomas in situ compared to invasive ductal carcinomas (IDC); and in IDC, high expression of FOXA1 was associated with favorable prognostic factors. Yet, FOXA1 expression was noted in a subset of the ER‐negative tumors. Taken together, our findings suggest a growth inhibitory role for FOXA1, and identify it as a novel, potential prognostic factor in breast cancer.

Epigenetic Silencing of the Candidate Tumor Suppressor Gene Per1 in Non–Small Cell Lung Cancer

Purpose: Epigenetic events are a critical factor contributing to cancer development. The purpose of this study was to identify tumor suppressor genes silenced by DNA methylation and histone deacetylation in non–small cell lung cancer (NSCLC). Experimental Design: We used microarray analysis to screen for tumor suppressor genes. Results: We identified Per1, a core circadian gene, as a candidate tumor suppressor in lung cancer. Although Per1 levels were high in normal lung, its expression was low in a large panel of NSCLC patient samples and cell lines. Forced expression of Per1 in NSCLC cell lines led to significant growth reduction and loss of clonogenic survival. Recent studies showed that epigenetic regulation, particularly histone H3 acetylation, is essential for circadian function. Using bisulfite sequencing and chromatin immunoprecipitation, we found that DNA hypermethylation and histone H3 acetylation are potential mechanisms for silencing Per1 expression NSCLC. Conclusions: These results support the hypothesis that disruption of circadian rhythms plays an important role in lung tumorigenesis. Moreover, our findings suggest a novel link between circadian epigenetic regulation and cancer development.

Simian virus 40 is present in human lymphomas and normal blood

Many independent studies have demonstrated Simian virus 40 (SV40) in normal and neoplastic human tissues. Clonal integration of virus in the DNA of several thyroid and bone tumors suggests a direct role for SV40 in some cancers. However, in most cases the role of SV40 remains unclear. This study determined the presence of SV40, by amplification followed by hybridization, in 266 normal and neoplastic blood and lymphoid samples. Amplification detected SV40 in 14% of non-autoimmune deficiency syndrome (AIDS) lymphomas, 28% of AIDS related lymphoma and 16% of peripheral blood lymphocytes from non-cancerous patients. No SV40 was detected in leukemia samples. Direct Southern blotting of SV40+ samples detected no virus, consistent with less than one viral genome in ten cells. Sequence analysis of SV40 in blood and lymphoid samples found sequences distinct from laboratory strains of SV40. The presence of limited quantities of SV40 in a small proportion of both normal and neoplastic tissues is suggestive of an adventitious presence with no apparent direct role in blood and lymphoid cancers.

Mutations in the mitotic check point gene, MAD1L1, in human cancers

Aneuploidy is a characteristic of the majority of human cancers, and recent studies suggest that defects of mitotic checkpoints play a role in carcinogenesis. MAD1L1 is a checkpoint gene, and its dysfunction is associated with chromosomal instability. Rare mutations of this gene have been reported in colon and lung cancers. We examined a total of 44 cell lines (hematopoietic, prostate, osteosarcoma, breast, glioblastoma and lung) and 133 fresh cancer cells (hematopoietic, prostate, breast and glioblastoma) for alterations of MAD1L1 by RT–PCR–SSCP and nucleotide sequencing. Eight mutations consisting of missense, nonsense and frameshift mutations were found, together with a number of nucleotide polymorphisms. All the alterations in cell lines were heterozygous. Frequency of mutations was relatively high in prostate cancer (2/7 cell lines and 2/33 tumor specimens). We placed a mutant truncated MAD1L1, found in a lymphoma sample, into HOS, Ht161 and SJSA cell lines and found that it was less inhibitory than wild type MAD1L1 at decreasing cell proliferation. Co-expression experiments showed that the mutant form had a dominant-negative effect. Furthermore, this mutant impaired the mitotic checkpoint as shown by decreased mitotic indices in HOS cells expressing mutant MAD1L1 after culture with the microtubule-disrupting agent, nocodazole. Our results suggest a pathogenic role of MAD1L1 mutations in various types of human cancer.

Integration of SV40 in human osteosarcoma DNA

Simian virus 40 (SV40) has been demonstrated in several types of tumors, including osteosarcoma, by polymerase chain reaction (PCR). We detected SV40 sequences by PCR, followed by hybridization, in nine of 35 osteosarcoma tumors and one of 11 osteosarcoma explants. PCR can detect fewer than one virus per cell but gives little detail of the gross structure and abundance of the virus. Analysis by Southern blotting of total DNA from ten osteosarcomas, positive for SV40 by PCR, found viral integration in half of these. Analysis showed integration of one to four copies per cell of rearranged SV40. No SV40 was detectable on blots of the remaining five SV40+ osteosarcomas, perhaps because of the lesser sensitivity of direct hybridization. Inactivation of the p53 and Rb tumor suppressors is a key activity of SV40 T-antigen. Unexpectedly, correlation of these findings with our prior studies indicated that five of ten osteosarcomas positive for SV40 DNA had mutations of p53, and two had deleted Rb. Apparently clonal integration with pre-existing alteration of a tumor suppressor gene, suggests that SV40 may play a role in the final conversion to malignant osteosarcoma.