Translation of an atypical human cDNA requires fidelity of apurine-pyrimidine repeat region and recoding.

Gain or loss of Migration inducting gene-7 (Mig-7) protein expression functional studies suggest it causes aggressive tumor cell invasion and tumor cell vessel-like structure formation. In addition, Mig-7 expression is apparently carcinoma and trophoblast cell-specific. Mig-7 is an example of an atypical gene that is unique in its induction, translation and apparent carcinoma-specific expression. However, studies of this predominantly integral membrane protein are hampered because of the cloning and expression techniques required for detection of Mig-7 protein. Because the encoding region possesses stop codons, repeat sequences and secondary structure, we hypothesized that genetically engineered E. coli are required to maintain the number of purine-pyrimidine repeats and reading frame when producing expression plasmids containing the Mig-7 sequence. Cloning Mig-7 sequence using E. coli genetically engineered to lack recombination and rearrangement capabilities prevented extension of the repeat region. Because of multiple stop codons in the sequence, three different constructs starting from three different reading frame ATG sites were tested for protein production in a human carcinoma cell line. Mig-7 protein of ~23 kD is produced from Mig-7 cDNA that contains multiple stop codons downstream from the ATG in a Kozak consensus sequence. In silico analyses imply that multiple Mig-7 mRNA secondary structures may cause frameshifting, read-through, and/or recoding of the multiple stop codons. Experimental results support that one or more of these translational events take place. In this report, we detail requirements for cloning and expression of this novel, atypical, human gene. These techniques can be used to express this unique protein for further studies.

Hepatocyte growth factor-regulated genes in differentiated RAW 264.7 osteoclast and undifferentiated cells.

Hepatocyte Growth Factor (HGF) and its protooncogene receptor c-Met regulate osteoclast function by activating pp60(c-Src) kinase and alpha(v)beta3 integrin. HGF causes transcription yet in osteoclast cells, this gene regulation is currently unknown. To begin characterization of HGF-regulated gene expression in osteoclast cells, we used a well characterized model of osteoclast cells. Using microarray, relative RT-PCR, and Western blot analyses, we have identified and confirmed differentially expressed genes in RAW 264.7 osteoclast cells in response to HGF. HGF regulation of transcription of these genes was concordant with microarray results. We report that HGF downregulates transcription factors, Distal-less 5 (Dlx-5), Distal-less 6 (Dlx-6) and Aristaless 4 (Alx-4), in RAW 264.7 osteoclast cells but has an inverse effect in undifferentiated RAW 264.7 cells.

Carcinoma cell-specific Mig-7: a new potential marker for circulating and migrating cancer cells.

Identification of genes that are expressed in a cancer cell-specific manner can provide markers for detection, diagnosis, and disease progression. We have previously reported that receptor tyrosine kinase ligands in concert with ligation of alphavbeta5 integrin induce expression of Mig-7 restricted to carcinoma cells. Because of this highly specific expression, we hypothesized that Mig-7 could be used as a marker of occult tumor cells. The objective of this study was to begin to test this hypothesis by generating Mig-7 specific antisera and RT-PCR methods for detection of Mig-7 expression in tissues and blood from cancer patients as compared to those from normal subjects. By immunohistochemistry and by RT-PCR, we detected Mig-7 mRNA in lymph nodes from 7 out of 9 (77.8%) endometrial carcinoma xenograft mice but not from any of the 5 negative control animals. Mig-7 expression was more specific than Met expression, the RTK that binds Scatter factor and is used as a marker of poor progression, in endometrial carcinoma as compared to normal endometrial tissue samples. In 87.3% of tumors from various tissues including breast, lung, colon and ovary, we detected Mig-7 expression. Blood samples from untreated metastatic cancer patients also displayed Mig-7 mRNA in contrast to a lack of expression in chemotherapy treated or normal individuals. In conclusion, we report the first immunohistochemical and RT-PCR assays for Mig-7 and discuss its highly specific localization to cancer cells in contrast to an absence in normal cells. Our preliminary data indicate that Mig-7 may be a potential early marker of migrating and circulating carcinoma cells.

Targeting migration inducting gene-7 inhibits carcinoma cell invasion, early primary tumor growth, and stimulates monocyte oncolytic activity.

Expression of Migration inducting gene-7 (Mig-7) is limited to tumor cells and to date not found in normal tissues. Multiple tumor microenvironment factors, such as epidermal and hepatocyte growth factors, in concert with alphavbeta5 integrin ligation, induce Mig-7 mRNA expression. Gain or loss of Mig-7 protein studies shows that Mig-7 promotes invasion of colon and endometrial carcinoma cells. These data led us to hypothesize that targeting Mig-7 through various methods could decrease invasion, enhance monocyte cell killing of tumor cells, and inhibit disease progression. To begin testing this hypothesis, an in vitro chemoinvasion assay of endometrial carcinoma cells treated with Mig-7-specific or control antibodies was used. Mig-7 antibody significantly reduced invasion by >60% compared with controls. In another approach to test this hypothesis, an in vitro analysis of peptide-stimulated human peripheral blood monocyte cells and their killing of MCF-7 breast carcinoma cells was used. Mig-7 peptide treatment increased monocyte cell tumor necrosis factor expression and killing of MCF-7 cells 30-fold over no peptide stimulation and 3-fold over MUC-1 or control peptide treatments. Furthermore, stably expressing Mig-7-specific short hairpin RNA resulted in significantly reduced Mig-7 protein levels and early primary tumor growth in a xenograft nude mouse model. Reduced phosphorylation of ERK1/2, Akt, and S6 kinase as well as decreased membrane-type 1 matrix metalloproteinase activity were mechanisms through which Mig-7 protein caused these effects. Based on these collective data, Mig-7 expression could be a potential candidate for future targeted cancer therapies.

Matrix-coated transwell-cultured TM4 sertoli cell testosterone-regulated gene expression mimics in vivo expression.

In vitro culture systems are needed to mimic in vivo epithelial cell environments for identifying cell signaling, gene expressions, and molecular mechanisms. One such system is matrix-coated transwell cultures. However, no data exist on culturing Sertoli cells in this manner with respect to testosterone-regulated gene expression. Because the TM4 mouse Sertoli-like cell line expresses androgen receptor, our objective was to determine if testosterone treatment added to the bottom chamber of a matrix-coated transwell system induces some gene expressions found in Sertoli cells in vivo. After serum starvation, transwell-cultured TM4 cells were treated with testosterone or left untreated for 24 h. Microarray analyses initially identified differentially expressed genes either induced or repressed by testosterone treatment. By Northern blot analyses, Pem mRNA, a frequently used marker of Sertoli cell testosterone responsiveness, was induced. Proteins of the transcripts induced by testosterone in the in vitro system were immunolocalized to Sertoli cells in testosterone-dependent stages of spermatogenesis in mouse testes. By immunohistochemistry analyses of sectioned mouse testes, gene expression induced by testosterone in transwell-cultured TM4 cells, profilin as well as secreted protein acidic and rich in cysteines (SPARC) are localized to Sertoli cells in testosterone-dependent stages of spermatogenesis. Findings include localizations of SPARC and profilin, as well as an apparent germ cell communication required for translation of Pem mRNA in Sertoli cells. Taken together, results of these studies suggest that this TM4 transwell-culture system could be used to study these testosterone-regulated Sertoli gene expressions in vitro.

Overexpression of carcinoma and embryonic cytotrophoblast cell-specific Mig-7 induces invasion and vessel-like structure formation.

Molecular requirements for carcinoma cell interactions with the microenvironment are critical for disease progression but are poorly understood. Integrin alpha v beta 5, which senses the extracellular matrix, is important for carcinoma cell dissemination in vivo. alpha v beta 5 signaling induces Mig-7, a novel human gene product that is apparently carcinoma-specific. We hypothesized that Mig-7 expression facilitates tumor cell dissemination by increasing invasion and vasculogenic mimicry. Results show that embryonic cytotrophoblasts up-regulated Mig-7 expression before they acquired an invasive phenotype capable of pseudovasculogenesis. Mig-7 protein primarily co-localized with vasculogenic mimicry markers factor VIII-associated antigen, vascular endothelial-cadherin, and laminin 5 gamma 2 chain domain III fragment in lymph node metastases. Overexpression of Mig-7 increased gamma 2 chain domain III fragments known to contain epidermal growth factor (EGF)-like repeats that can activate EGF receptor. Interestingly, EGF also induced Mig-7 expression. Carcinoma cell adhesion to laminins was significantly reduced by Mig-7 expression. Remarkably, in two-dimensional and three-dimensional Matrigel cultures, Mig-7 expression caused invasion and vessel-like structures. Melanoma cells, which were previously characterized to invade aggressively and to undergo vasculogenic mimicry, expressed Mig-7. Taken together, these data suggest that Mig-7 expression allows cells to sense their environment, to invade, and to form vessel-like structures through a novel relationship with laminin 5 gamma 2 chain domain III fragments.

Novel hepatocyte growth factor/scatter factor isoform transcripts in the macaque endometrium and placenta.

Hepatocyte growth factor/scatter factor (HGF/SF) induces proliferation, motility and morphogenesis of cells that express the proto-oncogene for the tyrosine kinase receptor, c-Met. Because these cellular events occur in the endometrium during the menstrual cycle and in placenta during development, we have initiated studies of this growth factor in these tissues from macaques. Several HGF/SF alternatively spliced transcripts have been previously reported in other tissues. However, expression of HGF/SF isoforms in the endometrium has not been studied. Here we describe the relative transcript amounts of HGF/SF isoforms in the endometrium and placenta using RNase protection analyses. During these analyses, we discovered two unexpected protected bands that were found through sequence analyses to represent isoforms similar to the previously reported NK1 and NK2 except that they encode a five amino acid deletion in the first kringle domain. We designated these two isoforms as dNK1 and dNK2. Endometrium expressed all of the isoforms; however, dNK2 was consistently expressed at higher levels than NK2 transcripts. In contrast, placenta expressed NK2 and dNK2 mRNA at equal levels, and both NK1 and dNK1 were undetectable in placenta. HGF/SF function in endometrium and placenta may involve complex interactions between the isoforms of HGF/SF and those of c-Met.

HGF and ligation of alphavbeta5 integrin induce a novel, cancer cell-specific gene expression required for cell scattering.

Hepatocyte growth factor (HGF), a cytokine involved in tumorigenesis and most metastases, initiates cell migration by binding to the protooncogene c-Met receptor. In epithelial carcinoma cells, c-Met activation causes the breakdown of E-cadherin cell-cell contacts leading to cell spreading. While the breakdown of E-cadherin contacts is immediate, HGF-induced migration requires transcription. To test the hypothesis that this de novo mRNA synthesis includes cancer cell-specific transcripts, we performed subtraction hybridization to isolate HGF-induced transcripts from an endometrial epithelial carcinoma cell line, RL95-2 (RL95), known to migrate but not to proliferate with HGF treatment. One novel cDNA we call Mig-7 is induced by HGF in endometrial epithelial carcinoma cell lines RL95 and HEC-1A before migration ensues. Ovarian, oral squamous cell, and colon metastatic tumors but not normal tissues express Mig-7. HGF did not induce Mig-7 in normal primary endometrial epithelial cells. In addition, blocking antibodies to alphavbeta5 integrin inhibited HGF induction of Mig-7 in RL95 cells. Most importantly, Mig-7-specific antisense oligonucleotides inhibited scattering of RL95 cells in vitro. These results are the first to demonstrate that Mig-7 expression may be used as a cancer cell-specific target to inhibit cell scattering.