Identification and cloning of two overexpressed genes, U21B31/PRAD1 and EMS1, within the amplified chromosome 11q13 region in human carcinomas.

Amplification of the chromosome 11q13 region is frequently found in human breast cancer and in squamous cell carcinomas of the head and neck, and has been associated with an unfavourable clinical course of disease. The known oncogenes within the amplified 11q13 region, INT2 and HSTF1, are rarely expressed in these tumours, indicating that another, hitherto unidentified, gene or genes confer(s) the biological (prognostic) significance to the amplification of the 11q13 region. To identify the gene or genes, we have constructed a cDNA library from a cell line with an 11q13 amplification and have performed differential cDNA cloning using [32P]dCTP-labelled cDNAs from human squamous cell carcinoma cell lines with and without an 11q13 amplification. We isolated two cDNA clones, U21B31 and U21C8, which recognize two genes amplified and overexpressed in cell lines harbouring an 11q13 amplification. In breast carcinomas and in squamous cell carcinomas amplification of both the U21B31 and the U21C8 gene was found in most tumours with an amplification of the 11q13 region, despite the large distance between both genes. Sequence analysis of the U21C8 cDNA clone revealed no homology to known genes; we call this gene EMS1. The U21B31 cDNA clone corresponded to the 3' end of the PRAD1 proto-oncogene, recently cloned from a parathyroid adenoma. Both gene products are of interest as potential markers to identify tumours with an 11q13 amplification.     

Overexpression of the eukaryotic translation initiation factor 4G (eIF4G-1) in squamous cell lung carcinoma

eIF4G-1 belongs to the family of translational initiation factors and is recognized as the central organizing protein in recruitment of mRNA during translational initiation. Previously published studies have provided some evidence that overexpression of translational factors is a general event in the process of carcinogenesis. We have characterized the expression of the eIF4G-1 protein in 33 squamous cell carcinoma (SCC) of the lung by Western blotting. Overexpression of the eIF4G-1 protein was detected in 61% of the tumors compared to the respective normal lung tissue. In addition, we analyzed the expression of this protein by immunohistochemistry in 138 SCC of the lung using a newly generated antibody that is specific for eIF4G-1 as determined by Western blotting. This anti-eIF4G-1 antibody was suitable for the immunohistochemistry of paraffin-embedded tissues. There is a strong cytoplasmic staining detected in the tumor areas that is consistent with the cytoplasmic localization of the translation factor eIF4G-1. In 72% of the examined tissue sections of SCCs of the lung, we detected an overexpression of the eIF4G-1 protein compared to the surrounding connective tissue. Two tumors that were analyzed by both methods showed an overexpression of eIF4G-1 both with Western blot analysis and immunohistochemical staining. Overexpression of eIF4G-1 may result in an increased amount of the translation initiation complex eIF4F, which in turn may activate the translation of the same target mRNAs as eIF4E.     

Amplification of protooncogenes in surgical specimens of human lung carcinomas

The possible existence of amplification or rearrangement of protooncogenes was examined in more than 100 surgical specimens of human lung carcinoma. Protooncogenes were amplified in 28% of the carcinomas. About 90% of the amplified genes were of the myc, ras, or erbB family. Of the myc family genes, myc was amplified in 14 of 137 tumors and L-myc in four of 108 tumors, but N-myc was not amplified. A high frequency of amplification of myc was observed in squamous cell carcinomas (seven of 37) and of L-myc in small cell carcinomas (two of six). Of the ras family genes, K-ras-2 was amplified in six of the 137 tumors and N-ras in two of the 137 tumors, but no amplification of H-ras-1 was detected. Seven of the eight cases of amplified ras genes were in advanced pathological stages. Of the erbB family genes, erbB-1 (epidermal growth factor receptor) was amplified in 10 of 114 tumors and erbB-2 (HER-2/neu) in one of 51 tumors. Amplifications of the myc, ras, and erbB family genes might be one of the crucial DNA abnormalities involved in the development of human lung carcinomas.     

Key contribution of eIF4H-mediated translational control in tumor promotion

Dysregulated expression of translation initiation factors has been associated with carcinogenesis, but underlying mechanisms remains to be fully understood. Here we show that eIF4H (eukaryotic translation initiation factor 4H), an activator of the RNA helicase eIF4A, is overexpressed in lung carcinomas and predictive of response to chemotherapy. In lung cancer cells, depletion of eIF4H enhances sensitization to chemotherapy, decreases cell migration and inhibits tumor growth in vivo, in association with reduced translation of mRNA encoding cell-proliferation (c-Myc, cyclin D1) angiogenic (FGF-2) and anti-apoptotic factors (CIAP-1, BCL-xL). Conversely, each isoform of eIF4H acts as an oncogene in NIH3T3 cells by stimulating transformation, invasion, tumor growth and resistance to drug-induced apoptosis together with increased translation of IRES-containing or structured 5′UTR mRNAs. These results demonstrate that eIF4H plays a crucial role in translational control and can promote cellular transformation by preferentially regulating the translation of potent growth and survival factor mRNAs, indicating that eIF4H is a promising new molecular target for cancer therapy.     

Frequent amplification of the bcl-1 locus in poorly differentiated squamous cell carcinoma of the lung. The Lung Cancer Study Group

The bcl-1 locus on chromosome 11 at band q13 has been shown to be rearranged in some chronic B lymphoid malignancies with the t(11;14)(q13;q32). Chromosome 11 abnormalities occur in solid tumors including squamous cell cancers and adenocarcinomas. We have recently reported the frequency amplification of the bcl-1 locus in squamous cell carcinomas of head and neck origin, and increased copy number also has been found in some breast adenocarcinomas. In this investigation, we analyzed the arrangement, copy number and expression of this locus in 111 primary non-small cell lung cancers. Bcl-1 was found to be amplified three- to 10-fold in seven fresh tumors. Whereas none of 51 adenocarcinomas showed bcl-1 amplification, six of 46 squamous cell carcinomas revealed an increase in bcl-1 copy number. This amplification was more frequently associated with larger and more poorly differentiated tumors. None of the cancers showed rearrangement of this locus.     

HST1 and INT2 Gene Coamplification in a Squamous Cell Carcinoma of the Gallbladder

The HST1 gene has previously been found to be amplified in over40% of squamous cell carcinomas of the esophagus. We performedSouthern blot analyses on squamous cell carcinomas of the lung,nasal cavity, uterine cervix and gallbladder, using HST1, INT2and five other on cogenes as probes. The HST1 and INT2 genes,both of which were mapped to chromosome 11 at band q13, werecoamplified in a squamous cell carcinoma of the gallbladder.The degree of amplification exceeded eight fold.     

Identification of a novel tumor transforming gene GAEC1 at 7q22 which encodes a nuclear protein and is frequently amplified and overexpressed in esophageal squamous cell carcinoma

By comparative DNA fingerprinting, we identified a 357-bp DNA fragment frequently amplified in esophageal squamous cell carcinomas (ESCC). This fragment overlaps with an expressed sequence tag mapped to 7q22. Further 5' and 3'-rapid amplification of cDNA ends revealed that it is part of a novel, single-exon gene with full-length mRNA of 2052 bp and encodes a nuclear protein of 109 amino acids ( approximately 15 kDa). This gene, designated as gene amplified in esophageal cancer 1 (GAEC1), was located within a 1-2 Mb amplicon at 7q22.1 identified by high-resolution 1 Mb array-comparative genomic hybridization in 6/10 ESCC cell lines. GAEC1 was ubiquitously expressed in normal tissues including esophageal and gastrointestinal organs; with amplification and overexpression in 6/10 (60%) ESCC cell lines and 34/99 (34%) primary tumors. Overexpression of GAEC1 in 3T3 mouse fibroblasts caused foci formation and colony formation in soft agar, comparable to H-ras and injection of GAEC1-transfected 3T3 cells into athymic nude mice formed undifferentiated sarcoma in vivo, indicating that GAEC1 is a transforming oncogene. Although no significant correlation was observed between GAEC1 amplification and clinicopathological parameters and prognosis, our study demonstrated that overexpressed GAEC1 has tumorigenic potential and suggest that overexpressed GAEC1 may play an important role in ESCC pathogenesis.     

High incidence of coamplification of hst-1 and int-2 genes in human esophageal carcinomas

We analyzed the alteration of the hst-1 and int-2 genes in 36 cases of esophageal squamous cell carcinoma, 42 cases of gastric adenocarcinoma, and 52 cases of colorectal adenocarcinoma. Coamplification of the hst-1 and int-2 genes was observed in 19 of 36 esophageal carcinomas (52%), 16 of 34 primary tumor tissues (47%), and 10 of 10 metastatic tumors (100%). The degree of amplification ranged from 4- to 8-fold. The incidence of hst-1 and int-2 gene coamplification was significantly higher in male patients than that in female patients (P less than 0.05). The coamplification of the hst-1 and int-2 genes had a tendency to correlate with clinical stage. The progesterone receptor gene, which is mapped to chromosome 11 at band q21-23, was not amplified in these esophageal carcinomas. Coamplification of the hst-1 and int-2 gene does not seem to imply increased numbers of chromosome 11, and the hst-1 and int-2 genes appear to be in same amplification unit on chromosome 11 at band q13. No coamplification of the hst-1 and int-2 genes was detected in gastric carcinomas and colorectal carcinomas. These results suggest that amplification of chromosomal locus of the hst-1 and int-2 genes might participate in carcinogenesis, in progression, and particularly in metastasis of esophageal carcinomas.     

Frequent amplification of chromosomal region 20q12-q13 in ovarian cancer.

DNA amplification at chromosomal region 20q12-q13, which is common in breast cancer, has recently been described also in ovarian tumors. We studied the amplification of the recently identified candidate oncogenes in this region in 24 sporadic, 3 familial and 4 hereditary ovarian carcinomas, and in 8 ovarian cancer cell lines. High-level amplification of at least one of the five nonsyntenic regions at 20q12-q13.2 was found in 13 sporadic (54%) and in all four hereditary tumors. Typically, two or more distinct amplicons (separated by nonamplified DNA) were found coamplified in various combinations. The regions defined by the AIB1 and PTPN1 genes (at 20q12 and 20q13.1, respectively) were amplified in 25% and 29% of the sporadic tumors, also without simultaneous coamplification of other regions. Amplification of AIB1 (a steroid receptor coactivator gene) was associated with estrogen receptor positivity in sporadic ovarian carcinomas (P = 0.01) and showed a tendency to correlate with poor survival of patients. Of the genes amplified in breast cancer, the BTAK gene was amplified in 21%, the MYBL2 gene in 17%, and the ZNF217 gene in 12.5% of the sporadic tumors. The high frequency of gene amplification at 20q12-q13.2 suggests that the genes amplified therein may play a central role in the pathogenesis of sporadic and hereditary ovarian carcinoma.     

Squamous cell carcinoma related oncogene/DCUN1D1 is highly conserved and activated by amplification in squamous cell carcinomas

Chromosomal amplification at 3q is common to multiple human cancers, but has a specific predilection for squamous cell carcinomas (SCC) of mucosal origin. We identified and characterized a novel oncogene, SCC-related oncogene (SCCRO), which is amplified along the 3q26.3 region in human SCC. Amplification and overexpression of SCCRO in these tumors correlate with poor clinical outcome. The importance of SCCRO amplification in malignant transformation is established by the apoptotic response to short hairpin RNA against SCCRO, exclusively in cancer cell lines carrying SCCRO amplification. The oncogenic potential of SCCRO is underscored by its ability to transform fibroblasts (NIH-3T3 cells) in vitro and in vivo. We show that SCCRO regulates Gli1--a key regulator of the hedgehog (HH) pathway. Collectively, these data suggest that SCCRO is a novel component of the HH signaling pathway involved in the malignant transformation of squamous cell lineage.     

High incidence of amplification of the epidermal growth factor receptor gene in human squamous carcinoma cell lines

Southern blot-hybridization analysis of DNAs from human tumors demonstrated amplification of the epidermal growth factor (EGF) receptor gene in 10 of 12 squamous cell carcinoma cell lines tested and in none of 18 tumor cell lines of nonsquamous cell carcinomas. The degree of amplification in the squamous cells varied from 2- to 50-fold relative to the epidermal keratinocyte. Hybridization analysis of the RNA showed that the amplification of the EGF receptor gene is accompanied with an increase of the 5.6 kilobases of EGF receptor mRNA. Scatchard plot analysis and sodium dodecyl sulfate-polyacrylamide gel analysis of the EGF receptor revealed that the synthesis of the EGF receptor is also greater in the cells with amplified EGF receptor gene. In contrast, Southern blot analysis of DNAs of primary tumors showed that incidence of amplification of the EGF receptor gene in squamous cells (1 of 6) was almost as frequent as in nonsquamous cells (1 of 4). These results show that amplification of the EGF receptor gene is commonly found in various tumors. In addition, our data suggest that primary squamous cell carcinomas with amplified EGF receptor gene may readily adapt to growth in tissue culture.     

Amplification of the hst-1 gene in human esophageal carcinomas

The hst-1 gene, previously designated as the hst gene, and seven other oncogenes were examined for possible structural changes in esophageal, gastric and colorectal carcinomas by Southern blot hybridization. The hst-1 gene was amplified in eight (42.1%) of the nineteen esophageal squamous cell carcinomas and in all four metastatic tumors of lymph nodes. The degree of amplification ranged from two to eight times. Coamplification of the hst-1 and c-erbB-1 gene was found in one case of esophageal carcinoma. However, no amplification of the hst-1 gene was detected in gastric and colorectal carcinomas.     

Overexpression of LRP12, a gene contained within an 8q22 amplicon identified by high-resolution array CGH analysis of oral squamous cell carcinomas

Chromosome 8q amplification is a common event observed in cancer. In this study, we used high-resolution array comparative genomic hybridization to resolve two neighboring regions on 8q that are both amplified in oral cancer. One region (at 8q24) contains the MYC oncogene, which is frequently overexpressed in many cancers, while the other region (at 8q22) represents a novel amplicon. The alignment of array comparative genomic hybridization profiles of 20 microdissected oral squamous cell carcinomas (OSCCs) revealed a approximately 5 Mbp region of frequent copy number alteration. This region harbors 16 known genes. Gene expression analysis comparing 15 microdissected OSCC with 16 normal epithelium samples revealed overexpression specific to LRP12 but not the neighboring genes, dihydropyrimidinase and FOG2, suggesting that LRP12 may function as an oncogene in oral tumors.