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Shammah is a smokeless tobacco product often mixed with lime, ash, black pepper and flavorings. Exposure to shammah has been linked with dental diseases and oral squamous cell carcinoma. There is limited literature on the prevalence of shammah and its role in pathobiology of oral cancer. In this study, we developed a cellular model to understand the effect of chronic shammah exposure on oral keratinocytes. Chronic exposure to shammah resulted in increased proliferation and invasiveness of non-transformed oral keratinocytes. Quantitative proteomics of shammah treated cells compared to untreated cells led to quantification of 4712 proteins of which 402 were found to be significantly altered. In addition, phosphoproteomics analysis of shammah treated cells compared to untreated revealed hyperphosphorylation of 36 proteins and hypophosphorylation of 83 proteins (twofold, p-value ≤ 0.05). Bioinformatics analysis of significantly altered proteins showed enrichment of proteins involved in extracellular matrix interactions, necroptosis and peroxisome mediated fatty acid oxidation. Kinase-Substrate Enrichment Analysis showed significant increase in activity of kinases such as ROCK1, RAF1, PRKCE and HIPK2 in shammah treated cells. These results provide better understanding of how shammah transforms non-neoplastic cells and warrants additional studies that may assist in improved early diagnosis and treatment of shammah induced oral cancer.

Epidermal growth factor receptor (EGFR) targeted therapies have shown limited efficacy in head and neck squamous cell carcinoma (HNSCC) patients despite its overexpression. Identifying molecular mechanisms associated with acquired resistance to EGFR-TKIs such as erlotinib remains an unmet need and a therapeutic challenge. In this study, we employed an integrated multi-omics approach to delineate mechanisms associated with acquired resistance to erlotinib by carrying out whole exome sequencing, quantitative proteomic and phosphoproteomic profiling. We observed amplification of several genes including AXL kinase and transcription factor YAP1 resulting in protein overexpression. We also observed expression of constitutively active mutant MAP2K1 (p.K57E) in erlotinib resistant SCC-R cells. An integrated analysis of genomic, proteomic and phosphoproteomic data revealed alterations in MAPK pathway and its downstream targets in SCC-R cells. We demonstrate that erlotinib-resistant cells are sensitive to MAPK pathway inhibition. This study revealed multiple genetic, proteomic and phosphoproteomic alterations associated with erlotinib resistant SCC-R cells. Our data indicates that therapeutic targeting of MAPK pathway is an effective strategy for treating erlotinib-resistant HNSCC tumors.

Carcinogenic effect of tobacco in oral cancer is through chewing and/or smoking. Significant differences exist in development of oral cancer between tobacco users and non-users. However, molecular alterations induced by different forms of tobacco are yet to be fully elucidated. We developed cellular models of chronic exposure to chewing tobacco and cigarette smoke using immortalized oral keratinocytes. Chronic exposure to tobacco resulted in increased cell scattering and invasiveness in immortalized oral keratinocytes. miRNA sequencing using Illumina HiSeq 2500 resulted in the identification of 10 significantly dysregulated miRNAs (4 fold; p ≤ 0.05) in chewing tobacco treated cells and 6 in cigarette smoke exposed cells. We integrated this data with global proteomic data and identified 36 protein targets that showed inverse expression pattern in chewing tobacco treated cells and 16 protein targets that showed inverse expression in smoke exposed cells. In addition, we identified 6 novel miRNAs in chewing tobacco treated cells and 18 novel miRNAs in smoke exposed cells. Integrative analysis of dysregulated miRNAs and their targets indicates that signaling mechanisms leading to oncogenic transformation are distinct between both forms of tobacco. Our study demonstrates alterations in miRNA expression in oral cells in response to two frequently used forms of tobacco.

This corrects the article DOI: 10.1038/srep36132.

A subset of patients with oral squamous cell carcinoma (OSCC), the most common subtype of head and neck squamous cell carcinoma (HNSCC), harbor dysplastic lesions (often visually identified as leukoplakia) prior to cancer diagnosis. Although evidence suggest that leukoplakia represents an initial step in the progression to cancer, signaling networks driving this progression are poorly understood. Here, we applied in silico Pathway Activation Network Decomposition Analysis (iPANDA), a new bioinformatics software suite for qualitative analysis of intracellular signaling pathway activation using transcriptomic data, to assess a network of molecular signaling in OSCC and pre-neoplastic oral lesions. In tumor samples, our analysis detected major conserved mitogenic and survival signaling pathways strongly associated with HNSCC, suggesting that some of the pathways identified by our algorithm, but not yet validated as HNSCC related, may be attractive targets for future research. While pathways activation landscape in the majority of leukoplakias was different from that seen in OSCC, a subset of pre-neoplastic lesions has demonstrated some degree of similarity to the signaling profile seen in tumors, including dysregulation of the cancer-driving pathways related to survival and apoptosis. These results suggest that dysregulation of these signaling networks may be the driving force behind the early stages of OSCC tumorigenesis. While future studies with larger leukoplakia data sets are warranted to further estimate the values of this approach for capturing signaling features that characterize relevant lesions that actually progress to cancers, our platform proposes a promising new approach for detecting cancer-promoting pathways and tailoring the right therapy to prevent tumorigenesis., Competing Interests: EM, IO, KL, MT and AZ are affiliated with Insilico Medicine, Inc., a company engaged in aging research, which uses and provides both paid and free services using a variety of pathway activation scoring algorithms and hence may have competing financial interests. The remaining authors declare no conflict of interest.

Despite advances in clinical management, 5-year survival rate in patients with late-stage head and neck squamous cell carcinoma (HNSCC) has not improved significantly over the past decade. Targeted therapies have emerged as one of the most promising approaches to treat several malignancies. Though tyrosine phosphorylation accounts for a minority of total phosphorylation, it is critical for activation of signaling pathways and plays a significant role in driving cancers. To identify activated tyrosine kinase signaling pathways in HNSCC, we compared the phosphotyrosine profiles of a panel of HNSCC cell lines to a normal oral keratinocyte cell line. Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) was one of the kinases hyperphosphorylated at Tyr-321 in all HNSCC cell lines. Inhibition of DYRK1A resulted in an increased apoptosis and decrease in invasion and colony formation ability of HNSCC cell lines. Further, administration of the small molecular inhibitor against DYRK1A in mice bearing HNSCC xenograft tumors induced regression of tumor growth. Immunohistochemical labeling of DYRK1A in primary tumor tissues using tissue microarrays revealed strong to moderate staining of DYRK1A in 97.5% (39/40) of HNSCC tissues analyzed. Taken together our results suggest that DYRK1A could be a novel therapeutic target in HNSCC.

In spite of significant technical advances, genesis and progression of non-small cell lung cancer (NSCLC) remain poorly understood. We undertook an integrated genetic approach to discover novel microRNAs that were deregulated in NSCLCs. A total 119 primary NSCLCs with matched normal were analyzed for genome-wide copy number changes. We also tested a subset of matched samples by microRNA expression array, and integrated them to identify microRNAs positioned in allelic imbalance area. Our findings support that most of the identified deregulated microRNAs (miR-21, miR-23b, miR-31, miR-126, miR-150, and miR-205) were positioned in allelic imbalance areas. Among microRNAs tested in independent 114 NSCLCs, overexpression of miR-23b was revealed to be a significantly poor prognostic factor of recurrence free survival (HR = 2.40, P = 0.005, 95%CI: 1.32-4.29) and overall survival (HR = 2.35, P = 0.005, 95%CI: 1.30-4.19) in multivariable analysis. In addition, overexpression of miR-23b in H1838 cell line significantly increased cell proliferation, while inhibition of miR-23b in H1437 and H1944 cell lines significantly decreased cell doubling time. In summary, integration of genomic analysis and microRNA expression profiling could identify novel cancer-related microRNAs, and miR-23b could be a potential prognostic marker for early stage NSCLCs. Further biological studies of miR-23b are warranted for the potential development of targeted therapy.

Cetuximab, a monoclonal antibody against epidermal growth factor receptor (EGFR), was shown to be active in colorectal cancer. Although some patients who harbor K-ras wild-type tumors benefit from cetuximab treatment, 40 to 60% of patients with wild-type K-ras tumors do not respond to cetuximab. Currently, there is no universal marker or method of clinical utility that could guide the treatment of cetuximab in colorectal cancer. Here, we demonstrate a method to predict response to cetuximab in patients with colorectal cancer using OncoFinder pathway activation strength (PAS), based on the transcriptomic data of the tumors. We first evaluated our OncoFinder pathway activation strength model in a set of transcriptomic data obtained from patient-derived xenograft (PDx) models established from colorectal cancer biopsies. Then, the approach and models were validated using a clinical trial data set. PAS could efficiently predict patients' response to cetuximab, and thus holds promise as a selection criterion for cetuximab treatment in metastatic colorectal cancer.

Head and neck squamous cell carcinoma (HNSCC) cells exposed to cisplatin (CIS) displayed a dramatic ATM-dependent phosphorylation of ΔNp63α that leads to the transcriptional regulation of downstream mRNAs. Here, we report that phospho (p)-ΔNp63α transcriptionally deregulates miRNA expression after CIS treatment. Several p-ΔNp63α-dependent microRNA species (miRNAs) were deregulated in HNSCC cells upon CIS exposure, including miR-181a, miR-519a, and miR-374a (downregulated) and miR-630 (upregulated). Deregulation of miRNA expression led to subsequent modulation of mRNA expression of several targets (TP53-S46, HIPK2, ATM, CDKN1A and 1B, CASP3, PARP1 and 2, DDIT1 and 4, BCL2 and BCL2L2, TP73, YES1, and YAP1) that are involved in the apoptotic process. Our data support the notion that miRNAs are critical downstream targets of p-ΔNp63α and mediate key pathways implicated in the response of cancer cells to chemotherapeutic drugs.

A mutation within one allele of the p53 tumor suppressor gene can inactivate the remaining wild-type allele in a dominant-negative manner and in some cases can exert an additional oncogenic activity, known as mutant p53 'gain of function' (GOF). To study the role of p53 mutations in prostate cancer and to discriminate between the dominant-negative effect and the GOF activity of mutant p53, we measured, using microarrays, the expression profiles of three immortalized prostate epithelial cultures expressing wild-type, inactivated p53 or mutated p53. Analysis of these gene expression profiles showed that both inactivated p53 and p53(R175H) mutant expression resulted in the upregulation of cell cycle progression genes. A second group, which was upregulated exclusively by mutant p53(R175H), was predominantly enriched in developmental genes. This group of genes included the Twist1, a regulator of metastasis and epithelial-mesenchymal transition (EMT). Twist1 levels were also elevated in metastatic prostate cancer-derived cell line DU145, in immortalized lung fibroblasts and in a subset of lung cancer samples, all in a mutant p53-dependent manner. p53(R175H) mutant bearing immortalized epithelial cells showed typical features of EMT, such as higher expression of mesenchymal markers, lower expression of epithelial markers and enhanced invasive properties in vitro. The mechanism by which p53(R175H) mutant induces Twist1 expression involves alleviation of the epigenetic repression. Our data suggest that Twist1 expression might be upregulated following p53 mutation in cancer cells.

HOP homeobox (HOPX) is an unusual homeobox gene encoding three spliced transcript variants, among which the only HOPX-beta promoter harbors CpG islands. The characteristics of its promoter methylation was analyzed using bisulfite sequencing and quantitative-methylation-specific polymerase chain reaction (Q-MSP), and the effects of HOPX expression were also examined. HOPX-beta expression was silenced in all gastric cancer cell lines tested; its expression could be restored by treatment with demethylating agent. On Q-MSP, HOPX-beta hypermethylation (cut-off value of 3.55) was found in 84% (67 out of 80) of primary tumor tissues and 10% (8 out of 80) of the corresponding normal tissues and could discriminate normal from tumor tissues (P<0.0001). The prognosis of the advanced cases with HOPX-beta hypermethylation was as poor as those with stage IV disease when cut-off value was set at 11.28. This finding was validated in an independent cohort of 90 advanced gastric cancers. The HOPX-beta hypermethylation was also an independent prognostic factor (P=0.029) on multivariate analysis. Exogenous HOPX expression significantly inhibited cell proliferation, colony formation and invasion as well as enhanced apoptosis. Taken together, HOPX-beta promoter methylation is a frequent and cancer-specific event in gastric cancer. Quantitative assessment of HOPX-beta methylation has great clinical potential as a marker of tumor aggressiveness.

2-Methoxyestradiol (2-ME), an endogenous estrogen metabolite of 17beta-estradiol, is known to induce mitochondria-mediated apoptosis through several mechanisms. We sought to study the effect of mitochondrialy targeted hOGG1 (MTS-hOGG1) on HeLa cells exposed to 2-ME. MTS-hOGG1-expressing cells exposed to 2-ME showed increased cellular survival and had significantly less G(2)/M cell cycle arrest compared to vector-only-transfected cells. In addition, 2-ME exposure resulted in an increase in mitochondrial membrane potential, increased apoptosis, accompanied by higher activation of caspase-3, -9, cleavage of Bid to tBid and protein poly(ADP-ribose) polymerase (PARP) cleavage in HeLa cells lacking MTS-hOGG1. Fas inhibitors cerulenin or C75 inhibited 2-ME-induced caspase activation, PARP cleavage, apoptosis and reversed mitochondrial membrane hyperpolarization, thereby recapitulating the increased expression of MTS-hOGG1. Hence, MTS-hOGG1 plays an important protective role against 2-ME-mediated mitochondrial damage by blocking apoptosis induced through the Fas pathway.

To identify novel methylated gene promoters, we compared differential RNA expression profiles of colorectal cancer (CRC) cell lines with or without treatment of 5-aza-2'-deoxycytidine (5-aza-dC). Out of 1776 genes that were initially 'absent (that is, silenced)' by gene expression array analysis, we selected 163 genes that were increased after 5-aza-dC treatment in at least two of three CRC cell lines. The microarray results were confirmed by Reverse Transcription-PCR, and CpG island of the gene promoters were amplified and sequenced for examination of cancer-specific methylation. Among the genes identified, the deafness, autosomal dominant 5 gene, DFNA5, promoter was found to be methylated in primary tumor tissues with high frequency (65%, 65/100). Quantitative methylation-specific PCR of DFNA5 clearly discriminated primary CRC tissues from normal colon tissues (3%, 3/100). The mRNA expression of DFNA5 in four of five colon cancer tissues was significantly downregulated as compared to normal tissues. Moreover, forced expression of full-length DFNA5 in CRC cell lines markedly decreased the cell growth and colony-forming ability whereas knockdown of DFNA5 increased cell growth in culture. Our data implicate DFNA5 as a novel tumor suppressor gene in CRC and a valuable molecular marker for human cancer.

N-methyl-D-aspartate receptors (NMDARs) are the predominant excitatory neurotransmitter receptors in the mammalian brain. We found that among the three NMDARs examined (NMDAR1, NMDAR2A, NMDAR2B), only NMDAR2A was silenced in colorectal carcinoma (CRC) cell lines at basal line and reactivated by the demethylating agent, 5-aza-2'-deoxycytidine. NMDAR2A was expressed in normal colon epithelium, while expression was hardly detectable in colon cancer tissues. Promoter methylation of NMDAR2A was confirmed by bisulfite sequencing and combined bisulfite restriction analysis in the CRC cell lines and primary tumors. Quantitative methylation-specific PCR demonstrated NMDAR2A promoter hypermethylation in 82 of 100 primary human CRC, 15 of 100 normal corresponding epithelial tissues and 1 of 11 (9%) normal colon mucosa samples obtained from patients without cancer. Moreover, forced expression of full-length NMDAR2A in CRC cell lines induced apoptosis and almost abolished the ability of the cells to form colonies in culture, while NMDAR2A knockdown increased cell growth. Thus, NMDAR2A is commonly hypermethylated in primary human CRC and possesses tumor-suppressive activity.

Identification of tumor suppressor genes (TSG) silenced by methylation uncovers mechanisms of tumorigenesis and identifies new epigenetic tumor markers for early cancer detection. Both nasopharyngeal carcinoma (NPC) and esophageal carcinoma are major tumors in Southern China and Southeast Asia. Through expression subtraction of NPC, we identified Deleted in Liver Cancer 1 (DLC1)/ARHGAP7 (NM&#95;006094)--an 8p22 TSG as a major downregulated gene. Although expressed in all normal tissues, DLC1 was silenced or downregulated in 11/12 (91%) NPC, 6/15 (40%) esophageal, 5/8 (63%) cervical and 3/9 (33%) breast carcinoma cell lines. No genetic deletion of DLC1 was detected in NPC although a hemizygous deletion at 8p22-11 was found by 1-Mb array-CGH in some cell lines. We then located the functional DLC1 promoter by 5'-RACE and promoter activity assays. This promoter was frequently methylated in all downregulated cell lines and in a large collection of primary tumors including 89% (64/72) NPC (endemic and sporadic types), 51% (48/94) esophageal, 87% (7/8) cervical and 36% (5/14) breast carcinomas, but seldom in paired surgical marginal tissues and not in any normal epithelial tissue. The transcriptional silencing of DLC1 could be reversed by 5-aza-2'-deoxycytidine or genetic double knock-out of DNMT1 and DNMT3B. Furthermore, ectopic expression of DLC1 in NPC and esophageal carcinoma cells strongly inhibited their colony formation. We thus found frequent epigenetic silencing of DLC1 in NPC, esophageal and cervical carcinomas, and a high correlation of methylation with its downregulation, suggesting a predominant role of epigenetic inactivation. DLC1 appears to be a major TSG implicated in the pathogenesis of these tumors, and should be further tested as a molecular biomarker in patients with these cancers.

Somatic mitochondrial DNA (mtDNA) mutations have been increasingly observed in primary human cancers. As each cell contains many mitochondria with multiple copies of mtDNA, it is possible that wild-type and mutant mtDNA can co-exist in a state called heteroplasmy. During cell division, mitochondria are randomly distributed to daughter cells. Over time, the proportion of the mutant mtDNA within the cell can vary and may drift toward predominantly mutant or wild type to achieve homoplasmy. Thus, the biological impact of a given mutation may vary, depending on the proportion of mutant mtDNAs carried by the cell. This effect contributes to the various phenotypes observed among family members carrying the same pathogenic mtDNA mutation. Most mutations occur in the coding sequences but few result in substantial amino acid changes raising questions as to their biological consequence. Studies reveal that mtDNA play a crucial role in the development of cancer but further work is required to establish the functional significance of specific mitochondrial mutations in cancer and disease progression. The origin of somatic mtDNA mutations in human cancer and their potential diagnostic and therapeutic implications in cancer are discussed. This review article provides a detailed summary of mtDNA mutations that have been reported in various types of cancer. Furthermore, this review offers some perspective as to the origin of these of mutations, their functional consequences in cancer development, and possible therapeutic implications.

In humans, the oxidatively induced DNA lesion 8-hydroxyguanine (8-oxoG) is removed from DNA by hOgg1, a DNA glycosylase/AP lyase that specifically incises 8-oxoG opposite cytosine. We analysed the expression of hOGG1 mRNA in 18 lung cancer and three normal cell lines. Although hOGG1 was overexpressed in most cell lines, 2/18 (11.1%) showed a lower hOGG1 mRNA and protein expression (approximately 80% decrease) relative to normal cell lines. Liquid chromatography/mass spectrometry analysis showed increased levels of 8-oxoG in the two cell lines with the lowest hOGG1 mRNA expression. We examined the ability of nuclear and mitochondrial extracts to incise 8-oxoG lesion in cell lines H1650 and H226 expressing lower hOGG1 mRNA and H1915 and H1975 with higher than normal hOGG1 mRNA expression. Both nuclear and mitochondrial extracts from H1915 and H1975 cells were proficient in 8-oxoG removal. However, both cell lines with the lowest hOGG1 mRNA expression exhibited a severe reduction in 8-oxoG incision in both nuclear and mitochondrial extracts. Under-expression of hOGG1 mRNA and hOgg1 protein was associated with a decrease in mitochondrial DNA repair in response to oxidative damaging agents. These results provide evidence for defective incision of 8-oxoG in both nuclear and mitochondria of H1650 and H226 lung cancer cell lines. These results may implicate 8-oxoG repair defects in certain lung cancers.

The distal short arm of chromosome 1 is commonly deleted in a variety of human neoplasms including gastrointestinal cancer. Genetic alterations of the retinoblastoma protein-interacting zing-finger gene (RIZ)1 including loss of heterozygosity (LOH) at 1p36, frameshift mutations, and promoter hypermethylation were reported previously in several cancers. In this study, we evaluated RIZ1 in 30 primary gastric cancers and found frameshift mutations in two cases (6.7%). Moreover, using real-time quantitative methylation-specific PCR, methylation of the RIZ1 promoter was detected in 11 (37%) cases. In all 11 cases with methylation, inactivation of the second allele occurred through frameshift mutation, LOH or promoter methylation. Our results suggest that RIZ1 is a specific target of inactivation in human gastric cancer.

Aquaporins (AQPs) are important in controlling water permeability. As AQP1 is known as a serum-responsive gene, we hypothesized that AQP expression may be involved in the development of human cancer. By reverse transcriptase-polymerase chain reaction analysis, expression of AQPs 1, 3, and 5 was found in seven colon and colorectal cancer cell lines. Western blot analysis confirmed their expression in four of these cell lines. In situ hybridization demonstrated that during colorectal carcinogenesis, the expression of AQPs 1 and 5 was induced in early-stage disease (early dysplasia) and maintained through the late stages of colon cancer development. Expression of AQPs 1 and 5 was maintained even in metastatic lesions in the liver. These findings demonstrate that the expression of several AQPs is found in tumor cells and is associated with an early stage of colorectal cancer development. These novel observations suggest that multiple AQP expression may be advantageous to tumorigenesis, which may lead to a better understanding of colorectal carcinogenesis.

Smoking is directly responsible for approximately 90% of lung cancers and is also strongly associated with cancers of the head and neck, esophagus and urinary bladder. Our growing understanding of the molecular changes that underlie cancer progression has contributed to the development of novel molecular approaches for the detection of cancer. In this study, we review a number of recent studies that have used molecular techniques to detect neoplastic DNA from lung, head and neck, esophagus and bladder cancer. The majority of these approaches are based on polymerase chain reaction (PCR) based assays. These PCR-based techniques can detect a few clonal cancer cells containing a specific DNA mutation, microsatellite alteration, or CpG island methylation among an excess background of normal cells. The ability to accurately detect a small number of malignant cells in a wide range of clinical specimens including sputum, saliva, bronchoalveolar lavage fluid, urine, serum, plasma, or tissue has significant implications for screening high-risk individuals (such as cigarette smokers) for cancer.

PGP9.5 (UCH-L1) is a member of the ubiquitin C-terminal hydrolase (UCH) family of proteins that is expressed in neuronal tissues. Our previous studies have shown that PGP9.5 was highly expressed in primary lung cancers and lung cancer cell lines. Additionally, the frequency of PGP9.5 over expression increases with tumor stage, indicating that PGP9.5 may play a role in lung cancer tumorigenesis. We used the yeast two-hybrid system to identify proteins that interact with PGP9.5. We show that PGP9.5 interacts with at least three proteins, one of which is JAB1, a Jun activation domain binding protein that can bind to p27(Kip1) and is involved in the cytoplasmic transportation of p27(Kip1) for its degradation. We also show that PGP9.5 is associated with JAB1 in vitro and in vivo; and that both proteins can be a part of a heteromeric complex containing p27(Kip1) in the nucleus in lung cancer cells. Furthermore, under serum-restimulation, nuclear translocation of both PGP9.5 and JAB1 coincides with a reduced level of p27(Kip1) in the nucleus. In contrast, when cells are contact inhibited, both PGP9.5 and JAB1 became more perinuclear and cytoplasmic in localization while p27(Kip1) was present only in the nucleus. Therefore, PGP9.5 may contribute to p27(Kip1) degradation via its interaction and nuclear translocation with JAB1.

We recently demonstrated the existence of specific patterns of somatic mitochondrial DNA (mtDNA) mutations in several cancers. Here we sought to identify the presence of mtDNA mutations in prostate cancer and their paired PIN lesions. The D-loop region, 16S rRNA, and the NADH subunits of complex I were sequenced to identify mtDNA mutations in 16 matched PIN lesions and primary prostate cancers. Twenty mtDNA mutations were detected in the tumor tissue of three patients. Identical mutations were also identified in the PIN lesion from one patient. This patient with multiple point mutations also harbored a high frequency of microsatellite instability (MSI-H) in nuclear mononucleotide repeat markers. Remarkably, identical mutations were also detected in all (3/3) matched urine and plasma samples obtained from these patients. Although mitochondrial mutations are less common in prostate adenocarcinoma, they occur early in cancer progression and they can be detected in bodily fluids of early stage disease patients. The identification of MtDNA mutations may complement other early detection approaches for prostate cancer.

Methylation of 5' CpG islands in promoter and upstream coding regions has been identified as a mechanism for transcriptional inactivation of tumor suppressor genes. The purpose of this study was to determine whether hypermethylation of the adenomatous polyposis coli (APC) gene promoter occurs in primary non-small cell lung cancer (NSCLC), and whether hypermethylated APC has any relationship with survival. APC promoter 1A methylation was determined in normal and corresponding tumor tissue from 91 NSCLC patients and in a control group of 10 patients without cancer, using a quantitative fluorogenic real-time PCR (Taqman) system. APC promoter methylation was detectable in 86 (95%) of 91 tumor samples, but also in 80 (88%) of 91 normal samples of NSCLC patients, and in only two (20%) of 10 normal lung tissues of the control group. The median level of APC promoter methylation was 4.75 in tumor compared to 1.57 in normal lung tissue (P<0.001). Patients with low methylation status showed significantly longer survival than did patients with high methylation status (P=0.041). In a multivariate analysis of prognostic factors, APC methylation was a significant independent prognostic factor (P=0.044), as were pT (P=0.050) and pN (P<0.001) classifications. This investigation shows that APC gene promoter methylation occurs in the majority of primary NSCLCs. High APC promoter methylation is significantly associated with inferior survival, showing promise as a biomarker of biologically aggressive disease in NSCLC.

The protein product of the tumor suppressor gene p53 is phosphorylated on multiple residues by several protein kinases. Using a battery of 10 antibodies developed against different phosphorylated and acetylated residues of p53, we compared the pattern of p53 phosphorylation and acetylation in tumor-derived cell lines, tumor samples, and non-neoplastic cells. Irrespective of tumor types or the presence of p53 mutation, phosphorylation and acetylation of p53 was substantially higher in samples obtained from tumor tissues than those found in non-transformed samples. Among the 10 sites analysed, phosphorylation of residues 15, 81, 392, and acetylation were among the more frequent modifications. Analysis of two of the more abundant phosphorylation or acetylation sites on p53 is sufficient to detect 72% of tumor-derived p53 proteins. The distinct pattern of p53 phosphorylation and acetylation in human tumors may offer a new means to monitor the status and activity of p53 in the course of tumor development and progression.

Inactivation of the P16 (INK4A)/retinoblastoma (RB) or TP53 biochemical pathway is frequent event in most human cancers. Recent evidence has shown that P14ARF binds to MDM2 leading to an increased availability of wild type TP53 protein. Functional studies also support a putative tumor suppressor gene function for p14ARF suggesting that p14ARF or p53 inactivation may be functionally equivalent in tumorigenesis. To study the relative contribution of each pathway in tumorigenesis, we analysed and compared alterations of the p16, p14ARF and p53 genes in 38 primary non-small cell lung cancers (NSCLCs) (19 adenocarcinomas and 19 squamous carcinoma). The p16 tumor suppressor gene was inactivated in 22 of 38 (58%) tumors. Twelve of these samples (31%) had homozygous deletions by microsatellite analysis; eight of them (21%) had p16 promoter hypermethylation detected by Methylation Specific PCR (MSP) and the remaining two (5%) harbored a point mutation in exon 2 by sequence analysis. The absence of P16 protein in every case was confirmed by immunohistochemistry. Fourteen of the 22 tumors with p16 inactivation also inactivated the p14ARF gene (12 with homozygous deletions extending into INK4a/ARF and two with exon 2 mutations). Mutations of p53 were found in 18 (47%) of the tumors and nine of them (50%) harbored p14ARF inactivation. Thus, an inverse correlation was not found between p14ARF and p53 genetic alterations (P=0.18; Fisher Exact Test). Our data confirm that the p16 gene is frequently inactivated in NSCLC. Assuming that 9p deletion occurs first, the common occurrence of p53 and p14ARF alterations suggests that p14ARF inactivation is not functionally equivalent to abrogation of the TP53 pathway by p53 mutation.

Progression through the G1 phase of the cell cycle is mediated by phosphorylation of the retinoblastoma protein (pRb) resulting in the release of essential transcription factors such as E2F-1. The phosphorylation of pRb is regulated positively by cyclin D1/CDK4 and negatively by CDK inhibitors, such as p16 (CDKN2/MTS-1/INK4A). The p16/cyclin D1/Rb pathway plays a critical role in tumorigenesis and many tumor types display a high frequency of inactivation of at least one component of this pathway. In order to determine the overall contribution of these three components to progression of head and neck squamous cell carcinoma (HNSCC), we examined p16 inactivation, cyclin D1 amplification, and pRb expression in 23 primary HNSCC tumors and five cell lines. p16 inactivation was detected in 19/23 (83%) primary tumors by detailed genetic analysis and was confirmed by immunohistochemistry (IHC). Absence of Rb protein expression indicative of pRb inactivation was identified in 2/23 (9%) tumors. In this set of tumors, there was a perfect inverse correlation between p16 and pRb inactivation. Using fluorescence in situ hybridization (FISH) cyclin D1 amplification was identified in 4/5 (80%) cell lines and 4/11 (36%) primary tumors. However, 2/4 cell lines and all four primary tumors with cyclin D1 amplification contained a concomitant alteration of p16. Therefore 21/ 23 (91%) of primary HNSCC contained at least one alteration in the p16/cyclin D1/Rb pathway. Although p16 and Rb alteration are apparently exclusive, cyclin D1 amplification occurs concomitantly with the loss of p16 suggesting an additional role for this amplification in HNSCC.

A new tumor suppressor gene PTEN/MMAC1 was recently isolated at chromosome 10q23 and found to be inactivated by point mutation or homozygous deletion in glioma, prostate and breast cancer. PTEN/MMAC1 was also identified as the gene predisposing to Cowden disease, an autosomal dominant cancer predisposition syndrome associated with an increased risk of breast, skin and thyroid tumors and occasional cases of other cancers including bladder and renal cell carcinoma. We screened 345 urinary tract cancers by microsatellite analysis and found chromosome 10q to be deleted in 65 of 285 (23%) bladder and 15 of 60 (25%) renal cell cancers. We then screened the entire PTEN/MMAC1 coding region for mutation in 25 bladder and 15 renal cell primary tumors with deletion of chromosome 10q. Two somatic point mutations, a frameshift and a splicing variant, were found in the panel of bladder tumors while no mutation was observed in the renal cell carcinomas. To screen for homozygous deletion, we isolated two polymorphic microsatellite repeats from genomic BAC clones containing the PTEN/MMAC1 gene. Using these new informative markers, we identified apparent retention at the gene locus indicative of homozygous deletion of PTEN/MMAC1 in four of 65 bladder and 0 of 15 renal cell tumors with LOH through chromosome 10q. Identification of the second inactivation event in six bladder tumors with LOH of 10q implies that the PTEN/MMAC1 gene is occasionally involved in bladder tumorigenesis. However, the low frequency of biallelic inactivation suggests that either PTEN/MMAC1 is inactivated by other mechanisms or it is not the only target of chromosome 10q deletion in primary bladder and renal cell cancer.

The human TSG101 gene was cloned and mapped to chromosome 11p15, a site suspected to contain a tumor suppressor gene involved in a variety of human cancers. Subsequent investigation described the presence of abnormally spliced transcripts and point mutations of TSG101 in breast cancer. Thus, we performed RT-PCR amplification of the entire open reading frame of TSG101 to test for aberrant transcripts in various human tumor cell lines derived from breast, bladder, head and neck, and lung cancer. In addition, we performed RT-PCR on cDNA from primary human breast and Wilms' tumor tissue. We found a single band of the expected size in 10 of 11 breast cancers and 6 of 6 Wilms' tumor samples after the first round of PCR. The remaining breast cancer sample displayed a barely visible smaller band. However, aberrant bands appeared in most cases after performing nested PCR casting doubt on the physiologic relevance of these spliced variants. We then searched for small intragenic mutations by complete sequence analysis of TSG101 in breast cancer cell lines and tumors, as well as in Wilms' tumors and normal fetal and adult kidney. No point mutations were found in any of the samples, including four breast tumors with chromosomal loss at 11p15. We found no consistent evidence of aberrant splicing or point mutations in breast cancer or Wilms' tumor suggesting that TSG101 is not a primary target of inactivation in human cancer.

The tumor suppressor gene CDKN2A (p16/MTS1/INK4A), which encodes the cyclin-dependent kinase inhibitor p16(INK4a), is a target of 9p21 deletions during the malignant progression of human gliomas. This gene also encodes a second protein product (human p16beta, murine p19ARF), which originates from an unrelated exon of CDKN2A (exon 1beta) spliced onto exon 2 in an alternate reading frame. Cell cycle arrest by p16beta is caused by an as yet unidentified pathway. In order to test the candidacy of p16beta as a glioma suppressor, we replaced p16(INK4a), p15(INK4b) and p16beta wild-type as well as a series of seven glioma-derived p16beta alleles (R87H, A112V, R120H, A121V, G125R, A128A and A128V), into glioma cell lines that had either CDKN2A-/RB+ (U-87MG and U-251MG) or CDKN2A+/RB- (LN-319) endogenous backgrounds and demonstrated that p16beta can act as a functional glioma cell growth suppressor. Moreover, p16beta, but not p16(INK4a) or p15(INK4b) inhibited the growth of RB-negative LN-319 cells, indicating that p16beta likely exerts its effects through an RB-independent pathway. In vitro and in vivo assays of pRB phosphorylation were consistent with this interpretation. Since none of the glioma-derived p16beta mutations inactivated their growth suppressive activities, it appears that mutations in CDKN2A exon 2 (which is shared in the coding sequences of p16(INK4a) and p16beta) likely exclusively target p16(INK4a).

Tumour progression is a fundamental feature of the biology of cancer. Cancers do not arise de novo in their final form, but begin as small, indolent growths, which gradually acquire characteristics associated with malignancy. In the brain, for example, low-grade tumours (astrocytomas) evolve into faster growing, more dysplastic and invasive high-grade tumours (glioblastomas). To define the genetic events underlying brain tumour progression, we analysed the p53 gene in ten primary brain tumour pairs. Seven pairs consisted of tumours that were high grade both at presentation and recurrence (group A) and three pairs consisted of low-grade tumours that had progressed to higher grade tumours (group B). In group A pairs, four of the recurrent tumours contained a p53 gene mutation; in three of them, the same mutation was found in the primary tumour. In group B pairs, progression to high grade was associated with a p53 gene mutation. A subpopulation of cells were present in the low-grade tumours that contained the same p53 gene mutation predominant in the cells of the recurrent tumours that had progressed to glioblastoma. Thus, the histological progression of brain tumours was associated with a clonal expansion of cells that had previously acquired a mutation in the p53 gene, endowing them with a selective growth advantage. These experimental observations strongly support Nowell's clonal evolution model of tumour progression.