In an effort to identify tumor suppressor gene(s) associated with the frequent loss of heterozygosity observed on chromosome 6q25–q27, we constructed a contig derived from the sequences of bacterial artificial chromosome/P1 bacteriophage artificial chromosome clones defined by the genetic interval D6S1581–D6S1579–D6S305–D6S1599–D6S1008. Sequence analysis of this contig found it to contain eight known genes, including the complete genomic structure of the Parkin gene. Loss of heterozygosity (LOH) analysis of 40 malignant breast and ovarian tumors identified a common minimal region of loss, including the markers D6S305 (50%) and D6S1599 (32%). Both loci exhibited the highest frequencies of LOH in this study and are each located within the Parkin genomic structure. Whereas mutation analysis revealed no missense substitutions, expression of the Parkin gene appeared to be down-regulated or absent in the tumor biopsies and tumor cell lines examined. In addition, the identification of two truncating deletions in 3 of 20 ovarian tumor samples, as well as homozygous deletion of exon 2 in the lung adenocarcinoma cell lines Calu-3 and H-1573, supports the hypothesis that hemizygous or homozygous deletions are responsible for the abnormal expression of Parkin in these samples. These data suggest that the LOH observed at chromosome 6q25–q26 may contribute to the initiation and/or progression of cancer by inactivating or reducing the expression of the Parkin gene. Because Parkin maps to FRA6E , one of the most active common fragile sites in the human genome, it represents another example of a large tumor suppressor gene, like FHIT and WWOX , located at a common fragile site.

RB1 dual role in proliferation and apoptosis: cell fate control and implications for cancer therapy RB1 dual role in proliferation and apoptosis: Cell fate control and implications for cancer therapy

Overexpression of HMGA1 proteins is a constant feature of human carcinomas. Moreover, rearrangements of this gene have been detected in several human benign tumors of mesenchymal origin. To define the role of these proteins in cell transformation in vivo, we have generated transgenic mice overexpressing ubiquitously the HMGA1 gene. These mice developed mixed growth hormone/prolactin cell pituitary adenomas and natural killer (NK)-T/NK cell lymphomas. The HMGA1-induced expression of IL-2 and IL-15 proteins and their receptors may account for the onset of these lymphomas. At odds with mice overexpressing a wild-type or a truncated HMGA2 protein, adrenal medullar hyperplasia and pancreatic islet cell hyperplasia frequently occurred and no increase in body size and weight was observed in HMGA1 mice. Taken together, these data indicate an oncogenic role of the HMGA1 gene also in vivo.

Tumor growth requires neoangiogenesis. VEGF is the most potent proangiogenic factor. Dysregulation of hypoxia-inducible factor (HIF) or cytokine stimuli such as those involving the chemokine receptor 4/stromal-derived cell factor 1 (CXCR4/SDF-1) axis are the major cause of ectopic overexpression of VEGF in tumors. Although the CXCR4/SDF-1 pathway is well characterized, the transcription factors executing the effector function of this signaling are poorly understood. The multifunctional Yin Yang 1 (YY1) protein is highly expressed in different types of cancers and may regulate some cancer-related genes. The network involving CXCR4/YY1 and neoangiogenesis could play a major role in cancer progression. In this study we have shown that YY1 forms an active complex with HIF-1α at VEGF gene promoters and increases VEGF transcription and expression observed by RT-PCR, ELISA, and Western blot using two different antibodies against VEGFB. Long-term treatment with T22 peptide (a CXCR4/SDF-1 inhibitor) and YY1 silencing can reduce in vivo systemic neoangiogenesis ( P < 0.01 and P < 0.05 vs. control, respectively) during metastasis. Moreover, using an in vitro angiogenesis assay, we observed that YY1 silencing led to a 60% reduction in branches ( P < 0.01) and tube length ( P < 0.02) and a 75% reduction in tube area ( P < 0.001) compared with control cells. A similar reduction was observed using T22 peptide. We demonstrated that T22 peptide determines YY1 cytoplasmic accumulation by reducing its phosphorylation via down-regulation of AKT, identifying a crosstalk mechanism involving CXCR4/YY1. Thus, YY1 may represent a crucial molecular target for antiangiogenic therapy during cancer progression.

Purpose: Parkin, a gene mutated in autosomal recessive juvenile Parkinsonism and mapped to the common fragile site FRA6E on human chromosome 6q25-q27, is associated with a frequent loss of heterozygosity and altered expression in breast and ovarian carcinomas. In addition, homozygous deletions of exon 2 creating deleterious truncations of the Parkin transcript were observed in the lung adenocarcinoma cell lines Calu-3 and H-1573, suggesting that the loss of this locus and the resulting changes in its expression are involved in the development of these tumors. Experimental Design: We examined 20 paired normal and non-small cell lung cancer samples for the presence of Parkin alterations in the coding sequence and changes in gene expression. We also restored gene expression in the Parkin-deficient lung carcinoma cell line H460 by use of a recombinant lentivirus containing the wild-type Parkin cDNA. Results: Loss of heterozygosity analysis identified a common region of loss in the Parkin/FRA6E locus with the highest frequency for the intragenic marker D6S1599 (45%), and semi-quantitative reverse transcription-PCR revealed reduced expression in 3 of 9 (33%) lung tumors. Although we did not observe any in vitro changes in cell proliferation or cell cycle, ectopic Parkin expression had the ability to reduce in vivo tumorigenicity in nude mice. Conclusion: These data suggest that Parkin is a tumor suppressor gene whose inactivation may play an important role in non-small cell lung cancer tumorigenesis.

A drastic reduction in BRCA1 gene expression is a characteristic feature of aggressive sporadic breast carcinoma. However, the mechanisms underlying BRCA1 downregulation in breast cancer are not well understood. Here we report that both in vitro and in vivo HMGA1b protein binds to and inhibits the activity of both human and mouse BRCA1 promoters. Consistently, murine embryonic stem (ES) cells with the Hmga1 gene deleted display higher Brca1 mRNA and protein levels than do wild-type ES cells. Stable transfection of MCF-7 cells with the HMGA1b cDNA results in a decrease of BRCA1 gene expression and in a lack of BRCA1 induction after estrogen treatment. Finally, we found an inverse correlation between HMGA1 and BRCA1 mRNA and protein expression in human mammary carcinoma cell lines and tissues. These data indicate that HMGA1 proteins are involved in transcriptional regulation of the BRCA1 gene, and their overexpression may have a role in BRCA1 downregulation observed in aggressive mammary carcinomas.