David Reisman

Compromised HOXA5 function can limit p53 expression in human breast tumours

Expression of the p53 gene protects cells against malignant transformation. Whereas control of p53 degradation has been a subject of intense scrutiny, little is known about the factors that regulate p53 synthesis. Here we show that p53 messenger RNA levels are low in a large proportion of breast tumours. Seeking potential regulators of p53 transcription, we found consensus HOX binding sites in the p53 promoter. Transient transfection of Hox/HOXA5 activated the p53 promoter. Expression of HOXA5 in epithelial cancer cells expressing wild-type p53, but not in isogenic variants lacking the p53 gene, led to apoptotic cell death. Moreover, breast cancer cell lines and patient tumours display a coordinate loss of p53 and HOXA5 mRNA and protein expression. The HOXA5 promoter region was methylated in 16 out of 20 p53-negative breast tumour specimens. We conclude that loss of expression of p53 in human breast cancer may be primarily due to lack of expression of HOXA5.

Transactivation of the human p53 tumor suppressor gene by c-Myc/Max contributes to elevated mutant p53 expression in some tumors.

Elevated levels of mutant forms of the p53 tumor suppressor are a hallmark of many transformed cells. Multiple mechanisms such as increased stability of the protein and increased transcription of the gene can account for elevated p53 expression. Recent findings indicate that c-Myc/Max heterodimers can bind to an essential CA(C/T)GTG-containing site in the p53 promoter and elevate its expression. We have addressed the possibility that elevated mutant p53 expression is due to deregulated c-Myc expression. Here we demonstrate that the human p53 promoter is transactivated by high c-Myc expression and repressed by high Max expression. In examining the relative levels of c-Myc and p53 in human Burkitts lymphomas and other B-lymphoid lines, we found that there is a correlation between the levels of c-Myc protein and p53 mRNA expression. In particular, cells that express very low levels of c-Myc protein also express low levels of p53 mRNA, while cells that express high levels of c-Myc tend to express high levels of p53 mRNA. To determine whether the p53 gene can be a target for c-Myc in vivo, we assayed the effects of antisense c-myc RNA on the levels of endogenous p53 mRNA. The results indicate that the presence of antisense c-myc RNA leads to a reduction in the levels of c-Myc protein, p53 mRNA, and expression from the p53 promoter. Taken together, our findings support a direct role for c-Myc in elevating expression of the mutant p53 gene in some tumors.

Transcriptional repression of p53 by human T-cell leukemia virus type I Tax protein.

The human T-cell leukemia virus type I oncoprotein Tax transcriptionally deregulates a wide variety of viral and cellular genes. Tax deregulation of gene expression is mediated through interaction with a variety of structurally unrelated cellular transcription factors, as Tax does not bind DNA in a sequence-specific manner. Although most of these cellular transcription factors have been shown to mediate activation by Tax, we have recently demonstrated that members of the basic helix-loop-helix (bHLH) family of transcription factors, which play a critical role in progression through the cell cycle, mediate repression by Tax. In this report, we examined whether Tax might repress transcription of the tumor suppressor p53, as the p53 gene has recently been demonstrated to be regulated by the bHLH protein c-Myc. Furthermore, loss or inactivation of the p53 gene has been shown to be causally associated with oncogenic transformation. We show that Tax represses transcription of the p53 gene and that this repression is dependent upon the bHLH recognition element in the p53 promoter. Together, these results suggest that Tax may promote malignant transformation through repression of p53 transcription.

Inhibition of the putative tumor suppressor gene TIMP-3 by tumor-derived p53 mutants and wild type p53

The p53 gene is a tumor suppressor that regulates the expression of genes required for cell cycle arrest or apoptosis. Mutations in p53 have been observed in over 60% of all human cancers. Certain classes of mutant p53 proteins maintain some of their activities or acquire novel activities and thus may contribute to the transformed phenotype. By carrying out an analysis of differential gene expression using cDNA expression arrays, we compared the expression patterns of cells expressing no p53 to isogenic lines expressing the codon 248 Arg to Trp mutant p53 allele (R248W). In this report, we show that the R248W and D281G p53 mutants, two of the more commonly occurring mutations, as well as wild type p53, repress transcription of the tissue inhibitor of metalloproteinases type 3 (TIMP-3) gene by greater than tenfold. TIMP-3 expression has been observed to be repressed in many tumors and its reduced expression is thought to contribute to tumor metastasis and invasiveness by allowing increased activity of metalloproteinases in the extracellular matrix. Since mutant forms of p53 tend to be expressed at greatly elevated levels in many human tumors, the retention of their ability to repress TIMP-3 illustrate one mechanism by which mutant forms of the p53 gene may contribute to tumorigenesis.

C/EBPβ Participates in Regulating Transcription of the p53 Gene in Response to Mitogen Stimulation

The tightly regulated expression of p53 contributes to genomic stability, and transcription of the p53 gene is induced prior to cells entering S phase, possibly as a mechanism to ensure a rapid p53 response in the event of DNA damage. We have previously described the cloning of an additional 1000 bp of upstream p53 sequences that we have demonstrated play a role in the regulated expression of p53. As described in an earlier report, we preliminarily identified that a member of the CAAT/enhancer-binding protein (C/EPB) family of transcription factors may play a role in regulating p53. Here we have demonstrated that a particular C/EBPβ isoform, C/EBPβ-2, efficiently binds to the p53 promoter and induces its expression in a fashion that reflects the pattern of p53 expression seen as cells are induced to enter S phase and is absent from cells that are defective in proper p53 regulation. We conclude from these findings that C/EBPβ-2 plays a central role in the regulating of p53 transcription during the transition into S phase.

AP-1 DNA binding activity induced by hyperosmolality in the rat hypothalamic supraoptic and paraventricular nuclei

Immediate early gene products (c-fos, c-jun and their cognates) act as transcription factors coupling physiologically relevant stimuli to long-term responses by binding to the AP-1 site in the promoter region of target genes. The induction of c-fos has been identified in the paraventricular (PVN) and supraoptic (SON) hypothalamic magnocellular nuclei after hyperosmotic stimulation by using in situ hybridization and immunocytochemistry. In this study, AP-1 DNA binding activity, an indicator of the functional form of the c-fos transcription factor, was examined in nuclear extracts prepared from these brain regions using an electrophoretic mobility shift assay and a labeled oligonucleotide containing the AP-1 consensus sequence. Two hours after hypertonic saline injection (i.p.), rats were killed and nuclear proteins were extracted from tissue punches of brain regions to assess AP-1 binding activity. Hyperosmolality induced an increase of AP-1 binding activity in nuclear protein from SON and PVN, but not striatum. This binding was competitively displaced by excess unlabeled AP-1 oligonucleotide whereas addition of increasing amounts of unlabeled SP-1 oligonucleotide (promoter site on housekeeping genes for the ubiquitous SP-1 transcription factor) did not decrease the binding. The binding protein was shown to contain c-Fos/Fra and c-Jun since addition of c-Fos/Fra antiserum formed a supershift of the DNA, protein and antibody complex, and c-Jun antibody blocked the protein DNA binding. These data suggest that hyperosmolality leads to a selective and specific increase in AP-1 DNA binding activity which may be responsible for regulating secondary target gene expression in the hypothalamic SON and PVN.

Lack of p53 expression in human myeloid leukemias is not due to mutations in transcriptional regulatory regions of the gene.

Lack of p53 expression in human myeloid leukemias is not due to mutations in transcriptional regulatory regions of the gene

HOXB7 promotes malignant progression by activating the TGFβ signaling pathway.

Overexpression of HOXB7 in breast cancer cells induces an epithelial-mesenchymal transition and promotes tumor progression and lung metastasis. However, the underlying mechanisms for HOXB7-induced aggressive phenotypes in breast cancer remain largely unknown. Here, we report that phosphorylation of SMAD3 was detected in a higher percentage in primary mammary tumor tissues from double-transgenic MMTV-Hoxb7/Her2 mice than tumors from single-transgenic Her2/neu mice, suggesting activation of TGFβ/SMAD3 signaling by HOXB7 in breast tumor tissues. As predicted, TGFβ2 was high in four MMTV-Hoxb7/Her2 transgenic mouse tumor cell lines and two breast cancer cell lines transfected with HOXB7, whereas TGFβ2 was low in HOXB7-depleted cells. HOXB7 directly bound to and activated the TGFβ2 promoter in luciferase and chromatin immunoprecipitation assays. Increased migration and invasion as a result of HOXB7 overexpression in breast cancer cells were reversed by knockdown of TGFβ2 or pharmacologic inhibition of TGFβ signaling. Furthermore, knockdown of TGFβ2 in HOXB7-overexpressing MDA-MB-231 breast cancer cells dramatically inhibited metastasis to the lung. Interestingly, HOXB7 overexpression also induced tumor-associated macrophage (TAM) recruitment and acquisition of an M2 tumor-promoting phenotype. TGFβ2 mediated HOXB7-induced activation of macrophages, suggesting that TAMs may contribute to HOXB7-promoted tumor metastasis. Providing clinical relevance to these findings, by real-time PCR analysis, there was a strong correlation between HOXB7 and TGFβ2 expression in primary breast carcinomas. Taken together, our results suggest that HOXB7 promotes tumor progression in a cell-autonomous and non-cell-autonomous manner through activation of the TGFβ signaling pathway.

Transcriptional Regulation of the p53 Tumor Suppressor Gene in S-Phase of the Cell-Cycle and the Cellular Response to DNA Damage

The p53 tumor suppressor induces the transcription of genes that negatively regulate progression of the cell cycle in response to DNA damage or other cellular stressors and thus participates in maintaining genome stability. Numerous studies have demonstrated that p53 transcription is activated before or during early S-phase in cells progressing from G0/G1 into S-phase through the combined action of two DNA-binding factors RBP-Jκ and C/EBPβ-2. Here, we review evidence that this induction occurs to provide available p53 mRNA in order to prepare the cell for DNA damage in S-phase, this ensuring a rapid response to DNA damage before exiting this stage of the cell cycle.

A novel double-negative feedback loop between miR-489 and the HER2-SHP2-MAPK signaling axis regulates breast cancer cell proliferation and tumor growth

Human epidermal growth factor receptor 2 (HER2 or ErBb2) is a receptor tyrosine kinase overexpressed in 20-30% of breast cancers and associated with poor prognosis and outcome. Dysregulation of several microRNAs (miRNAs) plays a key role in breast cancer progression and metastasis. In this study, we screened and identified miRNAs dysregualted in HER2-positive breast cancer cells. Our molecular study demonstrated that miR-489 was specifically downregulated by the HER2-downstream signaling, especially through the MAPK pathway. Restoration or overexpression of miR-489 in HER2-positive breast cancer cells significantly inhibited cell growth in vitro and decreased the tumorigenecity and tumor growth in xenograft mice. Mechanistically, we found that overexpression of miR-489 led to the decreased levels of HER2 and SHP2 and thus attenuated HER2-downstream signaling. Furthermore, we for the first time demonstrated that HER2 is a direct target of miR-489 and therefore HER2-SHP2-MAPK and miR-489 signaling pathways form a mutually inhibitory loop. Using quantitative real-time PCR analysis and Fluorescent in situ hybridization technique (FISH), we found that miR-489 was expressed at significantly lower level in tumor tissues compared to the adjacent normal tissues. Downregulation of miR-489 in breast cancers was associated with aggressive tumor phenotypes. Overall, our results define a double-negative feedback loop involving miR-489 and the HER2-SHP2-MAPK signaling axis that can regulate breast cancer cell proliferation and tumor progression and might have therapeutic relevance for HER2-positive breast cancer.