Over the past decade, therapies targeting the VEGF/VEGFR and mTOR pathways have served as the standard of care for the clinical management of renal cell carcinoma (RCC) patients. Albeit promising, these targeted drugs have attained only modest clinical benefits with limited prolonged progression-free survival. Therefore, alternative reasonable and applicable therapeutic approaches should be introduced to improve the clinical outcome of RCC patients.

The adaptive cellular response to low oxygen tensions is mediated by the hypoxia-inducible factors (HIFs), a family of heterodimeric transcription factors composed of HIF-α and HIF-β subunits. Prolonged HIF expression is a key contributor to cellular transformation, tumorigenesis and metastasis. As such, HIF degradation under hypoxic conditions is an essential homeostatic and tumour-suppressive mechanism. LIMD1 complexes with PHD2 and VHL in physiological oxygen levels (normoxia) to facilitate proteasomal degradation of the HIF-α subunit. Here, we identify

Head and neck squamous cell carcinoma (HNSCC) is a global disease and mortality burden, necessitating the elucidation of its molecular progression for effective disease management. The study aims to understand the molecular profile of three candidate cell cycle regulatory genes, RBSP3, LIMD1 and CDC25A in the basal/ parabasal versus spinous layer of normal oral epithelium and during head and neck tumorigenesis. Immunohistochemical expression and promoter methylation was used to determine the molecular signature in normal oral epithelium. The mechanism of alteration transmission of this profile during tumorigenesis was then explored through additional deletion and mutation in HPV/ tobacco etiological groups, followed byclinico-pathological correlation. In basal/parabasal layer, the molecular signature of the genes was low protein expression/ high promoter methylation of RBSP3, high expression/ low methylation of LIMD1 and high expression of CDC25A. Dysplastic epithelium maintained the signature of RBSP3 through high methylation/ additional deletion with loss of the signatures of LIMD1 and CDC25A via deletion/ additional methylation. Similarly, maintenance and / or loss of signature in invasive tumors was by recurrent deletion/ methylation. Thus, differential patterns of alteration of the genes might be pre-requisite for the development of dysplastic and invasive lesions. Etiological factors played a key role in promoting genetic alterations and determining prognosis. Tobacco negative HNSCC patients had significantly lower alterations of LIMD1 and CDC25A, along with better survival among tobacco negative/ HPV positive patients. Our data suggests the necessity for perturbation of normal molecular profile of RBSP3, LIMD1 and CDC25A in conjunction with etiological factors for head and neck tumorigenesis, implying their diagnostic and prognostic significance.

To understand the mechanism of cellular stress in basal-parabasal layers of normal cervical epithelium and during different stages of cervical carcinoma, we analyzed the alterations (expression/methylation/copy number variation/mutation) of HIF-1α and its associated genes LIMD1, VHL and VEGF in disease-free normal cervix (

Head and neck squamous cell carcinoma is one of the leading cancers in terms of incidence and mortality. However, no reliable marker till date accurately predicts its progression when altered in healthy tissues. The study aims to identify alleles of microsatellites adjacent to important cell cycle regulatory, tumor suppressor genes altered in early head and neck lesions, viz. RBSP3, LIMD1 and CDC25A, which undergo frequent deletion and can be used for population screening and early detection. DNA for tumors and normal tissues was isolated from 143 patients in different stages of head and neck squamous cell carcinoma. The size of microsatellite present in normal tissues and their deletion in the corresponding tumor was identified, along with the correlation of expression in normal epithelium with respect to allele size. The results revealed a range of alleles (CA

The division of diffuse large B-cell lymphoma (DLBCL) into germinal center B-cell-like (GCB) and activated B-cell-like (ABC) subtypes based on gene expression profiling has proved to be a landmark in understanding the pathogenesis of the disease. This study aims to identify a novel biomarker to facilitate the translation of research into clinical practice. Using a training set of 350 patients, we identified a two-gene expression signature, "LIMD1-MYBL1 Index", which is significantly associated with cell-of-origin subtypes and clinical outcome. This two-gene index was further validated in two additional dataset. Tested against the gold standard method, the LIMD1-MYBL1 Index achieved 81% sensitivity, 89% specificity for ABC group and 81% sensitivity, 87% specificity for GCB group. The ABC group had significantly worse overall survival than the GCB group (hazard ratio = 3.5, P = 0.01). Furthermore, the performance of LIMD1-MYBL1 Index was satisfactory compared with common immunohistochemical algorithms. Thus, the LIMD1-MYBL1 Index had considerable clinical value for DLBCL subtype classification and prognosis. Our results might prompt the further development of this two-gene index to a simple assay amenable to routine clinical practice.

Since the application of molecular biology in cancer biology, lung cancer research has classically focused on molecular drivers of disease. One such pathway, the hypoxic response pathway, is activated by reduced local oxygen concentrations at the tumor site. Hypoxia-driven gene and protein changes enhance epithelial-to-mesenchymal transition, remodel the extracellular matrix, drive drug resistance, support cancer stem cells and aid evasion from immune cells. However, it is not the tumor cells alone which drive this response to hypoxia, but rather their interaction with a complex milieu of supporting cells. This review will focus on recent advances in our understanding of how these cells contribute to the tumor response to hypoxia in non-small-cell lung cancer.

The lymphocyte-specific transcription factor Interferon (IFN) Regulatory Factor 4 (IRF4) is implicated in certain types of lymphoid and myeloid malignancies. However, the molecular mechanisms underlying its interactions with these malignancies are largely unknown. In this study, we have first profiled molecular signatures associated with IRF4 expression in associated cancers, by analyzing existing gene expression profiling datasets. Our results show that IRF4 is overexpressed in melanoma, in addition to previously reported contexts including leukemia, myeloma, and lymphoma, and that IRF4 is associated with a unique gene expression pattern in each context. A pool of important genes involved in B-cell development, oncogenesis, cell cycle regulation, and cell death including BATF, LIMD1, CFLAR, PIM2, and CCND2 are common signatures associated with IRF4 in non-Hodgkin B cell lymphomas. We confirmed the correlation of IRF4 with LIMD1 and CFLAR in a panel of cell lines derived from lymphomas. Moreover, we profiled the IRF4 transcriptome in the context of EBV latent infection, and confirmed several genes including IFI27, IFI44, GBP1, and ARHGAP18, as well as CFLAR as novel targets for IRF4. These results provide valuable information for understanding the IRF4 regulatory network, and improve our knowledge of the unique roles of IRF4 in different hematological malignancies.

INTRODUCTION: To understand the role of two interacting proteins LIMD1 and pRB in development of head and neck squamous cell carcinoma (HNSCC), alterations of these genes were analyzed in 25 dysplastic head and neck lesions, 58 primary HNSCC samples and two HNSCC cell lines.
METHODS: Deletions of LIMD1 and RB1 were analyzed along with mutation and promoter methylation analysis of LIMD1. The genotyping of LIMD1 linked microsatellite marker, hmlimD1, was done to find out any risk allele. The mRNA expression of LIMD1 and RB1 were analyzed by Q-PCR. Immunohistochemical analysis of RB1 was performed. Alterations of these genes were correlated with different clinicopathological parameters.
RESULTS: High frequency [94% (78/83)] of LIMD1 alterations was observed in the samples studied. Compare to frequent deletion and methylation, mutation of LIMD1 was increased during tumor progression (P = 0.007). Six novel mutations in exon1 and one novel intron4/exon5 splice-junction mutation were detected in LIMD1 along with a susceptible hmlimD1 (CA)20 allele. Some of these mutations [42% (14/33)] produced non-functional proteins. RB1 deletion was infrequent (27%). Highly reduced mRNA expression of LIMD1 (25.1 +/- 19.04) was seen than RB1 (3.8 +/- 8.09), concordant to their molecular alterations. The pRB expression supported this data. Tumors with LIMD1 alterations in tobacco addicted patients without HPV infection showed poor prognosis. Co-alterations of these genes led the worse patients' outcome.
CONCLUSIONS: Our study suggests LIMD1 inactivation as primary event than inactivation of RB1 in HNSCC development.

Loss of heterozygosity (LOH) and homozygous deletions at chromosome 3p21.3 are common in both small and nonsmall cell lung cancers, indicating the likely presence of tumor suppressor genes (TSGs). Although genetic and epigenetic changes within this region have been identified, the functional significance of these changes has not been explored. Concurrent protein expression and genetic analyses of human lung tumors coupled with functional studies have not been done. Here, we show that expression of the 3p21.3 gene, LIMD1, is frequently down-regulated in human lung tumors. Loss of LIMD1 expression occurs through a combination of gene deletion, LOH, and epigenetic silencing of transcription without evidence for coding region mutations. Experimentally, LIMD1 is a bona fide TSG. Limd1(-/-) mice are predisposed to chemical-induced lung adenocarcinoma and genetic inactivation of Limd1 in mice heterozygous for oncogenic K-Ras(G12D) markedly increased tumor initiation, promotion, and mortality. Thus, we conclude that LIMD1 is a validated chromosome 3p21.3 tumor-suppressor gene involved in human lung cancer development. LIMD1 is a LIM domain containing adapter protein that localizes to E-cadherin cell-cell adhesive junctions, yet also translocates to the nucleus where it has been shown to function as an RB corepressor. As such, LIMD1 has the potential to communicate cell extrinsic or environmental cues with nuclear responses.

The aim of our study was to analyze the alterations of some candidate tumor suppressor genes (TSGs) viz. LIMD1, LTF, CDC25A, SCOTIN, RASSF1A and CACNA2D2 located in the chromosomal region 3p21.31 associated with the development of early dysplastic lesions of head and neck. In analysis of 72 dysplastic lesions and 116 squamous cell carcinoma of head and neck, both deletion and promoter methylation have been seen in these genes except for CDC25A and SCOTIN where no methylation has been detected. The alteration of LIMD1 was highest (50%) in the mild dysplastic lesions and did not change significantly during progression of tumor indicating its association with this stage of the disease. It was evident that alterations of LTF, CDC25A and CACNA2D2 were associated with development of moderate dysplastic lesions, while alterations in RASSF1A and CACNA2D2 were needed for progression. Novel somatic mutations were seen in exon 1 of LIMD1 (7%), intron 3/exon4 splice junction of LTF (2%) and exon 7 of cdc25A (10%). Quantitative RT-PCR analysis revealed mean reduced expression of the genes in the following order: LTF (67.6 +/- 16.8) > LIMD1 (53.2 +/- 20.1) > CACNA2D2 (23.7 +/- 7.1) > RASSF1A (15.1 +/- 5.6) > CDC25A (5.3 +/- 2.3) > SCOTIN (0.58 +/- 0.54). Immunohistochemical analysis of CDC25A showed its localization both in cytoplasm and nucleus in primary lesions and oral cancer cell lines. In absence of HPV infection, LTF and RASSF1A alterations jointly have adverse impact on survival of tobacco addicted patients. Thus, our data suggested that multiple candidate TSGs in the chromosomal 3p21.31 region were differentially associated with the early dysplastic lesions of head and neck.

The tumour suppressor gene (TSG) LIM domain containing protein 1 (LIMD1) has been associated with transformation of epithelial cells of the lung and its expression is downregulated in all lung tumour samples tested compared to normal lung matched controls. In the first study of its kind we used an anti-LIMD1 specific monoclonal antibody to investigate expression/localisation of the LIMD1 protein in a well-characterised tissue microarray of breast cancers and normal adjacent epithelia. Comparison of tumour with adjacent normal and distant normal tissue demonstrated that LIMD1 expression is moderate to high compared to tumour. There was also a significant correlation with histological grade (p = 0.0001), tumour size (p = 0.013) and tumour type (p = 0.004) indicating an association with aggressive disease. Cytoplasmic LIMD1 expression was seen in 99.3% of cases, with 43.1% showing both nuclear and cytoplasmic localisation. Absence/loss of nuclear staining showed a strong correlation with patient survival and was indicative of poor prognosis (p = 0.033). There was no association with lymph node status and other clinicopathological parameters. Nuclear staining was more pronounced in better prognosis tumours and normal tissue. This study demonstrates that LIMD1 represents a novel prognostic marker for breast cancer. Combined with the fact that LIMD1 expression is downregulated in lung cancers this clearly indicates that LIMD1 may represent a critical TSG, the function of which is deregulated via overall loss of expression and/or relocalisation within the cell during tumour development. The possible functions of LIMD1 localisation within the nucleus and cytoplasm and its relationship to tumour prognosis are discussed.

The hLIMD1 gene is located at chromosome 3p21 and was identified as a putative tumor suppressor gene using an elimination test assay. Chromosome 3p21 loci are frequently deleted in a number of cancers, including breast. The 3p21.3 locus harbors a number of tumor suppressor candidates, including LIMD1, a member of the ZYXIN family of genes. LIMD1 directly interacts with RB and is thought to play a role in suppressing tumor growth. To investigate whether mutations in the LIMD1 gene could potentially be involved in breast cancer, we used single-stranded conformation polymorphism analysis on DNA from 235 breast cancers and 95 controls. We identified four novel coding region alterations, including two amino acid substitutions at positions 255 and 302. The two remaining novel variants were found at amino acid positions 246 and 647 and encoded silent alterations. The rare Ser255Arg variant was identified in only sporadic breast tumors (2/165 tumors). Some ZYXIN proteins are phosphorylated by serine/threonine kinases, and the Ser255Arg change is located in a region phosphorylated on serine residues. Together, the data suggest that this variant may warrant further characterization.

The TEL (ETV6)-AML1 (RUNX1) chimeric gene fusion is the most common genetic abnormality in childhood acute lymphoblastic leukemias. Evidence suggests that this chimeric gene fusion constitutes an initiating mutation that is necessary but insufficient for the development of leukemia. In a search for additional genetic events that could be linked to the development of leukemia, we applied a genome-wide array-comparative genomic hybridization technique to 24 TEL-AML1 leukemia samples and two cell lines. It was found that at least two chromosomal imbalances were involved in all samples. Recurrent regions of chromosomal imbalance (>10% of cases) and representative involved genes were gain of chromosomes 10 (17%) and 21q (25%; RUNX1) and loss of 12p13.2 (87%; TEL), 9p21.3 (29%; p16INK4a/ARF), 9p13.2 (25%; PAX5), 12q21.3 (25%; BTG1), 3p21 (21%; LIMD1), 6q21 (17%; AIM1 and BLIMP1), 4q31.23 (17%; NR3C2), 11q22-q23 (13%; ATM) and 19q13.11-q13.12 (13%; PDCD5). Enforced expression of TEL and to a lesser extent BTG1, both single genes known to be located in their respective minimum common region of loss, inhibited proliferation of the TEL-AML1 cell line Reh. Together, these findings suggest that some of the genes identified as lost by array-comparative genomic hybridization may partly account for the development of leukemia.

Chromosome deletions do abound in cancer and are detected in certain regions in a non-random manner. Although their relevance remains elusive, it is a general agreement that segmental losses provide the cell with selective growth advantage. Consequently these may contain genes and/or regulatory sequences that control normal growth and inhibit malignancy. We have developed a monochromosomal hybrid based experimental model for the generation and functional analysis of deletions, that is called "elimination test" (Et). Focused on human chromosome 3 - that was known to carry multiple 3p deletions - the Et was expected to restrict a 3p tumor suppressor region to a sufficiently small segment that permits the selection of a critically important candidate gene. Surprisingly, we detected three regions that were lost in all or majority of tumors: CER1 (3p21.3, Mb: 43.32-45.74), CER2 (3p22, Mb: 37.83-39.06) and FER (3p14.3-p21.2, Mb: 50.12-58.03). In contrast a 3q26-qter region (CRR) was regularly retained. CER1 - our main focus - contains multiple genes that may inhibit tumor growth, but 3 genes, RIS1, LF (LTF) and LIMD1 have already the necessary experimental support to be considered bona fide tumor suppressors. Tumor suppressor region borders display instability features including: (1) they break in evolution and in tumors, (2) they evolve horizontally, and (3) they are enriched with pseudogene insertions. The most remarkable features at the breakpoint cluster regions were segmental duplications that drive horizontal evolution and contribute to cancer associated instability.

LIM domains-containing protein 1 (LIMD1) is encoded at chromosome 3p21.3, a region commonly deleted in many solid malignancies. However, the function of LIMD1 is unknown. Here we show that LIMD1 specifically interacts with retinoblastoma protein (pRB), inhibits E2F-mediated transcription, and suppresses the expression of the majority of genes with E2F1-responsive elements. LIMD1 blocks tumor growth in vitro and in vivo and is down-regulated in the majority of human lung cancer samples tested. Our data indicate that LIMD1 is a tumor-suppressor gene, the protein product of which functionally interacts with pRB and the loss of which promotes lung carcinogenesis.

We have previously shown that inoculation of human chromosome 3 (chr3)/A9 mouse fibrosarcoma microcell hybrids (MCHs) into severe combined immunodeficient (SCID) mice was followed by the regular elimination of some 3p regions whereas a 3q region was retained even after prolonged mouse passage. Using this approach, referred to as the elimination test (Et), we have defined a common eliminated region (CER) of approximately 7 cM at 3p21.3 that was absent in all of the 27 tumors generated from five MCHs. Later, CER was reduced to a 1-Mb region, designated as CER1. Another eliminated region (ER2) at 3p21.1-p14.2 was absent in 21 of the 27 tumors. ER2 borders at but does not include the fragile histidine triad (FHIT) gene, considered as a putative tumor suppressor gene. In the present work, two new and two previously studied MCHs, and 13 derived SCID mouse tumors were analyzed by fluorescence in situ hybridization (FISH) chromosome painting and by PCR, using 72 chr3p-specific and 11 chr3q-specific markers. Nine tumors generated from three MCHs that carried cytogenetically normal chr3, remained PCR-positive for all of the chr3 markers tested. Designated as "PCR+" tumors, they were examined by reverse transcription (RT)-PCR, together with four of six previously studied tumors derived from MCH910.7, which carried a del(3)(pter-p21.1), for the expression of 14 human genes: 5 genes within CER1 (LIMD1, CCR1, CCR2, CCR3, CCR5), 5 genes located within regions that were homozygously deleted in a variety of carcinomas (ITGA4L, LUCA1, PTPRG, FHIT, DUTT1), and 4 other genes in chr3p (VHL, MLH1, TGM4, UBE1L). We found that VHL, MLH1, ITGA4L, LIMD1, UBE1L, LUCA1, PTPRG, and DUTT1 were expressed in the MCH lines in vitro and also in the derived SCID tumors. No transcripts that originated from the four CCR genes or from TGM4 could be detected in any of the MCH lines. Alone among the 14 genes examined, FHIT showed a tumor growth-associated change. It was expressed in vitro in five of seven MCH lines. Nine of 13 derived tumors had no FHIT transcript. The remaining 4 expressed a truncated mRNA and a reduced amount of the full-length mRNA. We have previously found that FHIT was deleted at the DNA level in 17 of 21 tumors derived from four MCHs. The remaining 4 of 21 had no FHIT transcript. Our compiled data show that FHIT was either physically or functionally impaired in all 34 of the 34 analyzed tumors. Variants with deleted or down-regulated FHIT have a selective growth advantage.