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We have observed overexpression of PACS-1, a cytosolic sorting protein in primary cervical tumors. Absence of exonic mutations and overexpression at the RNA level suggested a transcriptional and/or posttranscriptional regulation. University of California Santa Cruz genome browser analysis of PACS-1 micro RNAs (miR), revealed two 8-base target sequences at the 3′ terminus for hsa-miR-34a and hsa-miR-449a. Quantitative RT-PCR and Northern blotting studies showed reduced or loss of expression of the two microRNAs in cervical cancer cell lines and primary tumors, indicating dysregulation of these two microRNAs in cervical cancer. Loss of PACS-1 with siRNA or exogenous expression of hsa-miR-34a or hsa-miR-449a in HeLa and SiHa cervical cancer cell lines resulted in DNA damage response, S-phase cell cycle arrest, and reduction in cell growth. Furthermore, the siRNA studies showed that loss of PACS-1 expression was accompanied by increased nuclear γH2AX expression, Lys(382)-p53 acetylation, and genomic instability. PACS-1 re-expression through LNA-hsa-anti-miR-34a or -449a or through PACS-1 cDNA transfection led to the reversal of DNA damage response and restoration of cell growth. Release of cells post 24-h serum starvation showed PACS-1 nuclear localization at G(1)-S phase of the cell cycle. Our results therefore indicate that the loss of hsa-miR-34a and hsa-miR-449a expression in cervical cancer leads to overexpression of PACS-1 and suppression of DNA damage response, resulting in the development of chemo-resistant tumors.

The oncogenic activation of the ETS-related gene (ERG) due to gene fusions is present in over half of prostate cancers in Western countries. Because of its high incidence and oncogenic role, ERG and components of ERG network have emerged as potential drug targets for prostate cancer. Utilizing gene expression datasets, from matched normal and prostate tumor epithelial cells, an association of NOTCH transcription factors with ERG expression status was identified, confirming that NOTCH factors are direct transcriptional targets of ERG. Inhibition of ERG in TMPRSS2-ERG–positive VCaP cells led to decreased levels of NOTCH1 and 2 proteins and downstream transcriptional targets and partially recapitulated the phenotypes associated with ERG inhibition. Regulation of NOTCH1 and 2 genes by ERG were also noted with ectopic ERG expression in LNCaP (ERG-negative prostate cancer) and RWPE-1 (benign prostate–derived immortalized) cells. Furthermore, inhibition of NOTCH by the small-molecule γ-secretase inhibitor 1, GSI-1, conferred an increased sensitivity to androgen receptor (AR) inhibitors (bicalutamide and enzalutamide) or the androgen biosynthesis inhibitor (abiraterone) in VCaP cells. Combined treatment with bicalutamide and GSI-1 showed strongest inhibition of AR, ERG, NOTCH1, NOTCH2, and PSA protein levels along with decreased cell growth, cell survival, and enhanced apoptosis. Intriguingly, this effect was not observed in ERG-negative prostate cancer cells or immortalized benign/normal prostate epithelial cells. These data underscore the synergy of AR and NOTCH inhibitors in reducing the growth of ERG-positive prostate cancer cells. Implications: Combinational targeting of NOTCH and AR signaling has therapeutic potential in advanced ERG-driven prostate cancers. Mol Cancer Res; 15(10); 1308–17. ©2017 AACR.

A procedure is described to measure curcumin (C), demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC), tetrahydrocurcumim (TC) and their glucuronidated metabolites (CG, DMCG, and BDMCG) in plasma, brain, liver and tumor samples. The procedure involves converting the analytes to their boron difluoride derivatives and analyzing them by combined liquid chromatography coupled to an ion trap mass spectrometer operating in the negative ion MSn scan mode. The method has superb limits of detection of 0.01 nM for all curcuminoids and 0.5 nM for TC and the glucuroniated metabolites, and several representative chromatograms of biological samples containing these analytes are provided. In addition, the pharmacokinetic profile of these compounds in one human who daily consumed an over-the-counter curcuminoid product shows the peak and changes in circulating concentrations achieved by this mode of administration.

The molecular mechanism of p16-mediated senescence in cisplatin-treated cancer cells is not fully understood. Here we show that cisplatin treatment of head and neck cancer cells results in nuclear transport of p16 leading to a molecular modification of NFκB. Chromatin immunoprecipitation assays show that this modification is associated with the inhibition of NFκB interacting with its DNA binding sequences, leading to decreased expression of NFκB-transcribed proteins. LCMS proteomic analysis of LAP-TAP-purified proteins from HeLa cells containing a tetracycline-inducible GFP-S peptide-NFκB expression system identified gigaxonin, an ubiquitin E3 ligase adaptor, as an NFκB-interacting protein. Immunoblotting and siRNA studies confirmed the NFκB-gigaxonin interaction and the dependence of this binding on p16-NFκB binding. Using gel shift assays, we have confirmed p16-NFκB and gigaxonin-NFκB interactions. Furthermore, we have observed increased NFκB ubiquitination with cisplatin treatment that is abolished in the absence of p16 and gigaxonin expression. Analysis of 103 primary tumors has shown that increased nuclear p16 expression correlates with enhanced survival of head and neck cancer patients (p < 0.0000542), indicating the importance of nuclear p16 expression in prognosis. Finally, p16 expression is associated with reduced cytokine expression and the presence of human papilloma virus in chemoradiation-sensitive basaloid tumors. However, the absence of p16 expression is associated with enhanced cytokine expression and the absence of human papilloma virus in aggressive tumors. These results clearly demonstrate that nuclear p16 and gigaxonin play an important role in chemosensitivity of head and neck cancers through ubiquitination of NFκB.

Cloning and sequencing of 5.5kb deletion at chromosome 11q13.1 from the HeLa cells, tumorigenic hybrids and two fibroblast cell lines has revealed homologous recombination between AluSx and AluY resulting in the deletion of intervening sequences. Long-range PCR of the 5.5kb sequence in 494 normal lymphocyte samples showed heterozygous deletion in 28.3% of African- American ancestry samples but only in 4.8% of Caucasian samples (p

We and others have shown that the cystatin E/M gene is inactivated in primary human tumors, pointing to its role as a tumor suppressor gene. However, the molecular mechanism of tumor suppression is not yet understood. Using plasmid-directed cystatin E/M gene overexpression, a lentivirus-mediated tetracycline-inducible vector system, and human papillomavirus 16 (HPV 16) E6 and E7 gene-immortalized normal human epidermal keratinocytes, we demonstrated intracellular and non-cell-autonomous apoptotic growth inhibition of tumor cell lines and that growth inhibition is associated with cytoplasmic retention of NF-κB. We further demonstrated decreased phosphorylation of IκB kinase (IKKβ) and IκBα in the presence of tumor necrosis factor alpha (TNF-α), confirming the role of cystatin E/M in the regulation of the NF-κB signaling pathway. Growth suppression of nude mouse xenograft tumors carrying a tetracycline-inducible vector system was observed with the addition of doxycycline in drinking water, confirming that the cystatin E/M gene is a tumor suppressor gene. Finally, immunohistochemical analyses of cervical carcinoma in situ and primary tumors have shown a statistically significant inverse relationship between the expression of cystatin E/M and cathepsin L and a direct relationship between the loss of cystatin E/M expression and nuclear expression of NF-κB. We therefore propose that the cystatin E/M suppressor gene plays an important role in the regulation of NF-κB.

We have previously shown TP16 MTS1/CDK41 gene deletion in more than 50% of a cohort of squamous cell carcinoma of the head and neck (SCCHN) patients using polymerase chain reaction (PCR). We have performed fluorescence in situ hybridization (FISH) on paraffin-embedded SCCHN specimens from the same cohort to identify the deletion of TP16 MTS1/CDK41CDK41 gene. Twenty normal and 19 SCCHN specimens were studied. An α-satellite DNA probe specific for chromosome 9 and a cosmid probe for the TP16 MTS1/CDK41CDK41 gene were used. Of the 19 tumors examined by FISH, 6 had homozygous deletions, 7 were hemizygously deleted, and the remaining 6 showed no evidence of deletion of the TP16 MTS1/CDK41 gene. None of the normal specimens showed TP16 gene deletion. Data obtained from FISH highly correlated with the PCR results for the identification of TP16 MTS1/CDK41 gene deletions. Patients with deletion of the TP16 MTS1/CDK41 gene show a greater tendency toward the development of recurrence and metastasis.

Previous molecular genetic studies on HeLa cell (a cervical cancer cell line) derived non-tumorigenic and tumorigenic hybrids have localized a tumor suppressor gene to the long arm of chromosome 11. Analysis of cervical cancer cell lines using chromosome 11 specific probes showed deletion and translocation of 11q13 sequences in five out of eight cell lines. Fluorescence in situ hybridization (FISH), using 11q13 specific probes, has shown interstitial deletion of 11q13 sequences in the HeLa cells. In order to determine whether 11q13 deletions occur in primary cervical tumors, we analysed 36 tumors using 20 different microsatellite and RFLP markers. Semi automated fluorescein based allelotyping was performed to identify loss of heterozygosity (LOH) in tumors. The results showed allelic loss in 17 (47%) tumors. Three different regions of loss, one near MEN1, the second near D11S913, and the third near INT2 locus were observed. The smallest region of deletion overlap at the D11S913 locus was localized to a 300 Kb distance between D11S4908 and D11S5023. Fluorescence in situ hybridization (FISH), using 11q13 specific cosmid and BAC (bacterial artificial chromosome) probes, confirmed allelic deletion in the tumors. PCR analysis further identified homozygous deletion of 11q13 sequences in a primary tumor, in HeLa cells and in two HeLa cell derived tumorigenic hybrid cell lines. The homozygous deletion in the cell lines was mapped to a 5.7 kb sequence of 11q13. We hypothesize therefore that a putative cervical cancer tumor suppressor gene exists within the 300 kb of chromosome 11q13.

Nasopharyngeal carcinoma (NPC) is a malignancy that is particularly prevalent among populations from Southern China and Southeast Asian countries. Evidence for a genetic contribution to the disease has been documented, although the genetic basis for NPC development is not yet fully understood. Previous functional evidence of tumor-suppressive activity on chromosome band 11q13 in NPC was obtained using a microcell-mediated chromosome-transfer approach with HONE1 NPC cells. In the present study, this region was subjected to a detailed investigation of microcell hybrids and their tumor segregants using microsatellite analysis to narrow down the region of tumor-suppressive activity. Fluorescence in situ hybridization was also performed with BAC and cosmid probes to confirm the microsatellite data. The critical region responsible for tumor suppression was narrowed down to a 1.8-Mb interval, which does not tolerate an additional normal allele by chromosome transfer. One or two alleles from either endogenous or exogenous chromosomes at 11q13 were consistently eliminated during tumor growth. Results of this study suggest that a candidate tumor-suppressor gene, not the MEN1 gene, maps between D11S4907 and GSTP1 in NPC.

Gigaxonin, a product of the Giant Axonal Neuropathy (GAN) gene located on chromosome 16, is involved in intermediate filament processing of neural cells and vimentin fibers in fibroblasts. Previous studies have shown an interaction between p16 and gigaxonin in cisplatin mediated ubiquitination of NFkB. Genomic studies have indicated higher frequency (44.25%) of exon 8 single nucleotide polymorphism (SNP) (c.1293 C>T) of gigaxonin in the individuals of Caucasian population compared to normal population (22%). The polymorphism frequency is much lower in individuals of other ethnicities. To determine the relationship to tumors, we analyzed exon 8 polymorphism in HPV positive and negative cervical and head and tumors. Our studies showed a 47.25% polymorphic frequency in these tumors. There was no relationship between the presence of polypmorphism and HPV status. However, there was an inverse relationship between polymorphism and tumor recurrence. Our studies have further shown higher expression of gigaxonin protein in cancer cell lines containing the polymorphic T allele. Growth assays in vitro and in soft agar have shown a direct relationship between the presence of the T allele and slower cell line growth. Our results therefore indicate that in addition to p16 expression, exon 8 SNP could also serve as a diagnostic marker of chemo sensitivity in human tumors. Citation Format: Eri S. Srivatsan, Kimberly J. Hwang, Albert Ko, Jenna R. Chatoff, Saroj K. Basak, Natarajan Venkatesan, Fernando Palma-Diaz, Michael S. Lewis, Pascale Bomont, Marilene B. Wang. Inverse relationship between exon 8 single nucleotide polymorphism (c.1293 C>T) of gigaxonin and human tumor cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4457. doi:10.1158/1538-7445.AM2017-4457

Background. The neural cell adhesion molecule (NCAM) has numerous isoforms resulting from alternative splicing of mRNA. The 3 major isoforms found in adult tissue are (1) a 120-kDa protein that is linked to the plasma membrane by glycosylphosphatidylinositol; (2) a 140-kDa form that has a transmembrane component and a cytoplasmic tail with unknown function; and (3) a 180-kDa isoform that has an intracellular protein that binds the cytoskeleton. NCAM is capable of homotypic binding and therefore plays a role in cell-cell adhesion for cells expressing the 180-kDa isoform by anchoring groups of cells into epithelial sheets. NCAM-180 is the isoform found in colonocytes, and loss of expression is associated with clinically aggressive colon cancers. Methods. Western blotting and reverse transcriptase-polymerase chain reaction were used to screen commercially available cell lines for NCAM-180 expression. For cell-line pairs with differential NCAM-180 expression, exon analysis was performed with reverse transcriptase-polymerase chain reaction to determine where the molecule was spliced, culminating in failed expression. These results were confirmed with exon analysis in colon cancers harvested at the time of laparotomy. Results. Analysis of a SW480 cell line (derived from a patient's primary colon cancer lesion) revealed NCAM-180 expression, whereas no expression was found in the SW620 cell line (derived from a metastatic lesion from the same patient). Exon analysis of NCAM mRNA transcripts from SW620 revealed that the transcripts were truncated after exon 12. This region correlates to an area between 2 fibronectin-III domains on the NCAM protein. Conclusions. The most common site for NCAM alternative splicing is between the 2 fibronectin-III domains corresponding to the border between exons 12 and 13 of the NCAM gene. Loss of NCAM-180 expression in aggressive colon carcinoma results from a splice defect in the same area, which may result in defective intracellular adhesion between colonocytes. (Surgery 2001;130:834-43.)

Problem: Cyclin D1 is a regulatory factor essential in the progression of the cell cycle from G1 through S phase. Amplification and overexpression of cyclin D1 have been observed in many human cancers including head and neck squamous cell carcinoma (HNSCC). We have previously transfected a HNSCC control cell line (CCL23) with an antisense cyclin D1 plasmid and demonstrated inhibition of cell proliferation in vitro. In this study, we examine whether antisense cyclin D1 could inhibit tumor growth in vivo. Methods/measures: The CCL23 and its antisense cyclin D1 transfected clone (CCL23 AS) were injected into the flanks of nude mice. Tumor growth was monitored weekly. After 5 weeks, tumors were removed and studied for tumor size, cyclin D1 expression, cyclin D1-dependent kinase activity, and retinoblastoma (Rb) phosphorylation. Results: Compared with the control tumors, 11 of 19 antisense tumors were smaller, 7 tumors were of equal size, and 1 tumor was larger. Immunohistochemical analysis with an anti-cyclin D1 antibody demonstrated decreased cyclin D1 expression in CCL23 AS and the smaller antisense tumors. Cyclin D1-dependent kinase activity was reduced in CCL23 AS and the smaller antisense tumors, and this was accompanied by a relative decrease in phosphorylated Rb in these samples. Conclusion: Antisense cyclin D1 inhibits growth of HNSCC tumors. Cyclin D1 expression, cyclin D1-dependent kinase activity, and Rb phosphorylation are decreased in these tumors. Clinical significance: These findings lend support for the potential use of antisense cyclin D1 as gene therapy for HNSCC. (Otolaryngol Head Neck Surg 2001;124:656-62.)

Previous cytogenetic and molecular genetic studies have shown that the HeLa (cervical carcinoma) cell line D98/AH-2 contains two apparently normal copies of chromosome 11 and additional 11q13-25 material translocated onto a chromosome 3 marker. To determine the 11q13 breakpoint, we performed fluorescence in situ hybridization (FISH) using 18 different 11q13 specific BAC (bacterial artificial chromosome) and cosmid probes spanning a 5.6 Mb interval. Markers localized to the multiple endocrine neoplasia type 1 (MEN1) gene (menin) were also included in the analysis. The FISH study identified an interstitial deletion between markers D11S449 and GSTP1, an interval of 2.3 Mb, in the marker chromosome. This deletion did not include the MEN1 gene. Because point mutations and methylations can inactivate the MEN1 gene, single stranded conformational polymorphism (SSCP) and Northern and Western blot analyses were performed with MEN1 specific probes and antibody. SSCP did not reveal mutations of the MEN1 gene in HeLa or in seven other cervical cancer cell lines. Northern and Western blot studies revealed normal levels of expression of this gene in the cervical cancer cell lines as well as in HeLa cell derived tumorigenic hybrids. Because deletions of tumor suppressor genes often occur in cancer progression, we hypothesize that the inactivation of a tumor suppressor gene other than MEN1, localized to the 2.3 Mb interval on 11q13, might play a role in the abnormal growth behavior of HeLa cells in vitro or in vivo.

Multiple endocrine neoplasia type 1 syndrome (MEN1, MIM 131100), an autosomal dominant disease, is characterized by parathyroid hyperplasia, pancreatic endocrine tumors, and pituitary adenomas. These tumors also occur sporadically. Both the familial (MEN1) and the sporadic tumors reveal loss of heterozygosity (LOH) for chromosome band 11q13 sequences. Based on prior linkage and LOH analyses, the MEN1 gene was localized between PYGM and D11S460. Recently, the MEN1 gene (menin) has been cloned from sequences 30-kb distal to PYGM. We performed deletion mapping on 25 endocrine tumors (5 MEN1 and 20 sporadic) by using 21 polymorphic markers on chromosome band 11q13. Of these, two (137C7A, 137C7B) were derived from PYGM-containing BAC (bacterial artificial chromosome-137C7) sequences, one from INT2-containing cosmid sequences and the marker D11S4748, a (CA)20 repeat marker that was developed by us. The LOH analysis shows that the markers close to the MEN1 (menin) gene were not deleted in three of the tumors. These tumors, however, showed LOH for distal markers. Thus, the data suggest the existence of a second tumor suppressor gene on chromosome band 11q13. Genes Chromosomes Cancer 22:130–137, 1998. © 1998 Wiley-Liss, Inc.

Previous experiments have shown that curcumin or cisplatin treatment suppresses growth of head and neck squamous cell carcinoma (HNSCC). To study the potential cooperative effect of both agents, two HNSCC cell lines were treated with curcumin or cisplatin alone or in combination. In vivo studies consisted of intravenous tail vein injection of liposomal curcumin, with intraperitoneal cisplatin, into nude mice growing xenograft HNSCC tumors. Introduction of curcumin and suboptimal concentrations of cisplatin showed a significant suppressive effect compared with treatment with either agent alone. Reduced expression of cyclin D1, IκBα, phospho-IκBα, and IKKβ occurred in cisplatin- and curcumin-treated cell lines. Confocal microscopy showed expression of IKKβ in the nucleus of the cell lines. Chromatin immunoprecipitation assay on DNA isolated from IKKβ immunoprecipitated samples showed PCR amplification of interleukin-8 promoter sequences, a binding site of NFκB, indicating an interaction between IKKβ and NFκB. Curcumin inhibited IKKβ in the cytoplasm and nucleus, leading to reduced NFκB activity, with no effect on phospho-AKT. In vivo studies showed significant growth inhibition of xenograft tumors treated with a combination of liposomal curcumin and cisplatin. The suppressive effect of curcumin was mediated through inhibition of cytoplasmic and nuclear IKKβ, resulting in inhibition of NFκB activity. Cisplatin treatment led to cellular senescence, indicating an effect mediated by p53 activation. The mechanisms of the two agents through different growth signaling pathways suggest potential for the clinical use of subtherapeutic doses of cisplatin in combination with curcumin, which will allow effective suppression of tumor growth while minimizing the toxic side effects of cisplatin. Mol Cancer Ther; 9(10); 2665–75. ©2010 AACR.

We have previously localized a cervical cancer tumor suppressor gene to a 300 kb interval of 11q13. Analysis of candidate genes revealed loss of expression of cystatin E/M, a lysosomal cysteine protease inhibitor, in 6 cervical cancer cell lines and 9 of 11 primary cervical tumors. Examination of the three exons in four cervical cancer cell lines, 19 primary tumors, and 21 normal controls revealed homozygous deletion of exon 1 sequences in one tumor. Point mutations were observed in six other tumors. Two tumors contained mutations at the consensus binding sites for cathepsin L, a lysosomal protease over-expressed in cervical cancer. Introduction of these two point mutations using site directed mutagenesis resulted in reduced binding of mutated cystatin E/M to cathepsin L. Although mutations were not observed in any cell lines, four cell lines and 12 of 18 tumors contained promoter hypermethylation. Re-expression of cystatin E/M was observed after 5′aza 2-deoxycytidiene and/or Trichostatin A treatment of cervical cancer cell lines, HeLa and SiHa, confirming promoter hypermethylation. Ectopic expression of cystatin E/M in these two cell lines resulted in growth suppression. There was also suppression of soft agar colony formation by HeLa cells expressing the cystatin E/M gene. Re-expression of cystatin E/M resulted in decreased intracellular and extracellular expression of cathepsin L. Over-expression of cathepsin L resulted in increased cell growth which was inhibited by the reintroduction of cystatin E/M. We conclude, therefore, that cystatin E/M is a cervical cancer suppressor gene and that the gene is inactivated by somatic mutations and promoter hypermethylation.

Nonrandom deletion of chromosome 11q13 sequences is a significant event in a number of human tumors. We have recently identified a 300-kb minimal area of deletion in primary cervical tumors that overlaps with deletions observed in endocrine and nasopharyngeal tumors. We have also observed a 5.7-kb homozygous deletion within this interval in HeLa cells (a cervical cancer cell line), HeLa cell-derived tumorigenic hybrids, and a primary cervical tumor, suggesting the presence of a tumor suppressor gene in this region. In the present investigation, we have constructed a 700-kb contig map encompassing the 300-kb deletion using the human genome sequence database and confirmed the map using various STS markers from the region. Our map also shows the overlap of a previously published rare, heritable fragile site, FRA11A, with the cervical cancer deletion locus. The mapped region contains highly repetitive GC-poor sequences. We have identified and characterized eight different polymorphic microsatellite markers from the sequences within and surrounding the deletion. Further, expression studies performed with 18 different ESTs localized adjacent to the homozygous deletion showed the presence of a transcript for only one of the ESTs, AA282789. This EST mapping within the homozygous deletion is also expressed in HeLa cells, thereby excluding the EST as the putative tumor suppressor gene. Additionally, analysis of four candidate genes (SF3B2, BRMS1, RIN1, and RAB1B) from the region showed expression of the expected size message in both the nontumorigenic and the tumorigenic HeLa cell hybrids, thereby excluding them as the putative tumor suppressor gene(s). However, Northern blot analysis with a fifth candidate gene, PACS1 (phosphofurin acidic cluster sorting protein), mapped to the deletion/FRA11A overlap region showed the expression of an 8-kb transcript in HeLa and five other tumor cell lines in addition to the expected 4.5-kb transcript. Since the gene shows abundant expression in normal tissues and an altered transcript is observed in tumor cell lines, we hypothesize that this gene could represent sequences of the putative tumor suppressor gene. Finally, we have observed a perfect 48-bp CAG/CCG repeat 99 kb proximal to D11S913, the marker linked to the neurodegenerative disorder spinocerebellar ataxia 5. The physical and transcription maps and the microsatellite markers of the 700-kb region of chromosome 11q13 should be helpful in the cloning of the cervical cancer tumor suppressor gene.

Studies on nontumorigenic and tumorigenic human cell hybrids derived from the fusion of HeLa (a cervical cancer cell line) with GM00077 (a normal skin fibroblast cell line) have demonstrated "functional" tumor-suppressor activity on chromosome 11. It has been shown that several of the neoplastically transformed radiation-induced hybrid cells called GIMs (gamma ray induced mutants), isolated from the nontumorigenic CGL1 cells, have lost one copy of the fibroblast chromosome 11. We hypothesized, therefore, that the remaining copy of the gene might be mutated in the cytogenetically intact copy of fibroblast chromosome 11. Because a cervical cancer tumor suppressor locus has been localized to chromosome band 11q13, we performed deletion-mapping analysis of eight different GIMs using a total of 32 different polymorphic and microsatellite markers on the long arm (q arm) of chromosome 11. Four irradiated, nontumorigenic hybrid cell lines, called CONs, were also analyzed. Allelic deletion was ascertained by the loss of a fibroblast allele in the hybrid cell lines. The analysis confirmed the loss of a fibroblast chromosome 11 in five of the GIMs. Further, homozygous deletion (complete loss) of chromosome band 11q13 band sequences, including that of D11S913, was observed in two of the GIMs. Detailed mapping with genomic sequences localized the homozygous deletion to a 5.7-kb interval between EST AW167735 and EST F05086. Southern blot hybridization using genomic DNA probes from the D11S913 locus confirmed the existence of homozygous deletion in the two GIM cell lines. Additionally, PCR analysis showed a reduction in signal intensity for a marker mapped 31 kb centromeric of D11S913 in four other GIMs. Finally, Northern blot hybridization with the genomic probes revealed the presence of a novel >15-kb transcript in six of the GIMs. These transcripts were not observed in the nontumorigenic hybrid cell lines. Because the chromosome 11q13 band deletions in the tumorigenic hybrid cell lines overlapped with the minimal deletion in cervical cancer, the data suggest that the same gene may be involved in the development of cervical cancer and in radiation-induced carcinogenesis. We propose that a gene localized in proximity to the homozygous deletion is the candidate tumor-suppressor gene.

Cyclin D1 and cyclin G are essential regulatory factors in the progression of the cell cycle from G0 through G1 and S phase. Aberrations in expression of these cyclins may lead to dysregulated cellular proliferation that could result in neoplasia. Amplification and overexpression of cyclin D1 have been observed in many human cancers, whereas cyclin G is a new cyclin recently described in osteosarcoma cells. This study was performed to determine whether these cyclins were amplified in head and neck squamous cell carcinoma (HNSCC) tumors. Polymerase chain reaction of DNA extracted from 22 HNSCC primary tumors and three HNSCC cell lines did not reveal amplification of cyclin D1 in any of the tumor samples. Southern blot analysis identified amplification of cyclin D1 in a single tumor. Amplification of cyclin G was not observed in any of the tumors by Southern blot hybridization with a cyclin G probe. HNSCC cell lines transfected with antisense cyclin D1 were tested for cell proliferation by the incorporation of 3 H-thymidine into cells grown in serum-free media. By 72 hours of incubation, there was a greater than 30% reduction in proliferation of cells transfected with antisense cyclin D1 as compared with nontransfected control cells. The results indicate that cyclin D1 may play an important role in the growth and proliferation of HNSCC cells. (Otolaryngol Head Neck Surg 1998;119:593-9.)

Tumor suppressor genes APC, RB1, and DCC, as well as genes localized to 3p and 11q, have been implicated in the development of a number of human tumors. To determine whether allelic deletions occur at these loci in squamous cell carcinomas (SSCs) of the head and neck, 25 primary, 1 metastatic, and 3 recurrent tumors, along with the corresponding constitutional tissues, were analyzed by using a battery of polymorphic DNA markers. For two primary tumors, we also analyzed subsequent metastatic tumors of the lung. Polymerase chain reaction–based restriction fragment length polymorphism studies demonstrated loss of heterozygosity for the APC gene in 2 of 12 (17%), the RB1 gene in 5 of 22 (23%), and the DCC gene in 5 of 13 (38%) informative cases. Alleles on chromosomes 3p, 11q13, and 18q21.1 were lost in 7 of 20 (35%), 9 of 23 (39%), and 4 of 17 (24%) informative cases, respectively. A breakpoint was identified within the chromosomal region 3p13–21.2 in a SCC of the tongue. Breakpoints within 11q13 were identified in 2 additional tumors. Thus, allelic deletions of DCC, 3p, and 11q13 appear to be common in head and neck cancers, suggesting that these genes play a critical and complex role in the development of these tumors. Furthermore, the present study provides definitive evidence for a tumor suppressor gene at chromosome band 11q13 and localizes this gene to the INT2–D11S533 interval for future cloning and sequencing.

We previously mapped a 5.5 kb homozygous deletion in cervical cancer cell lines and primary tumors to chromosome 11q13 within the 1st intron of PACS-1 gene, which encodes a membrane traffic regulator. This deletion correlates with elevated expression of 120 kDa PACS-1 in primary cervical tumors compared to normal tissues as well as the expression of a 140 kDa immunoreactive isoform in the nucleus specifically in cervical cancer cell lines. Inspection of the PACS-1 mRNA sequence revealed a target site for miR449a, which mediates cell fate. Northern blot analysis showed miRNA 449a expression was reduced in cervical cancer cell lines and primary tumors suggesting that dysregulation of miRNA449a or the intronic deletion contributed to aberrant PACS-1 expression and that the increased expression of PACS-1 may mediate cell viability and cell cycle progression. Consistent with this possibility, siRNA knockdown of PACS-1 or increased expression of miR449a caused cells to accumulate in S phase and induced expression of γH2AX. By contrast, overexpression of PACS-1 or expression of antimiR 449a reduced γH2AX expression. Whereas PACS-1 mediates secretory pathway trafficking, the effect of PACS-1 expression on genome stability suggested PACS-1 might shuttle between the cytoplasm and the nucleus to mediate genome stability. Consistent with this possibility, the PACS-1 sequence contains canonical NLS and NES motifs. Indeed, confocal microscopy studies revealed GFP-tagged PACS-1 accumulated in the nucleus in a leptomycin B-dependent manner. We hypothesize that aberrant expression of PACS-1 in cervical cancer cells results in the stabilization and replication of rearranged DNA sequences leading to tumor development. Citation Format: Mysore S. Veena, Saroj K. Basak, Natarajan Venkatesan, Alborz Zinabadi, Laurel Thomas, Gary Thomas, Eri S. Srivatsan. The membrane traffic regulator PACS-1 mediates genome stability at S-phase of the cell cycle. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5187. doi:10.1158/1538-7445.AM2013-5187

Restriction fragment length polymorphism (RFLP) analysis carried out on 45 primary neuroblastomas showed deletion of chromosome 11 sequences in 12 of 37 (32%) informative cases. Both 11p and 11q probes were informative in seven tumors; loss of all of chromosome 11, of only 11p sequences, and of only 11q sequences was observed in 4, 1, and 2 tumors, respectively. A cytogenetic abnormality involving translocation of chromosome arm 11q to chromosome arm 1p was observed in a primary tumor. Deletion of 14q was observed in 6 of 27 (22%) informative cases. Deletion of chromosome 11 but not 14q may correlate with regional and metastatic disease. These results suggest a possible role for sequences localized to chromosome 11 and to 14q in the development and/or progression of neuroblastoma.

Cervical cancer is one of the leading causes of female death world wide with approximately 50,000 deaths per year. We identified a 300 kb deletion of chromosome 11q13 in cervical cancer and mapped eleven genes to this region. Of these, phosphofurin acidic cluster sorting −1 (PACS-1) gene encodes a multi-functional sorting protein involved in secretory pathway membrane traffic. Expression studies showed the presence of an abnormal PACS-1 transcript in tumorigenic cell lines. Protein expression studies in paired normal and cervical tumors showed over-expression of PACS-1 protein in tumor specimens. RT-PCR indicated that protein over-expression was related to over-expression at the RNA level. These findings together with the absence of detectable mutations in PACS-1 exons in HeLa or SiHa cervical carcinoma cell lines raised the possibility that dysregulation of a microRNA (miRNA) may alter PACS-1 expression in cervical cancer. Consistent with this possibility, a UCSC genome database search showed the presence of binding sites for miRNA449a in the 3′ UTR (untranslated region) of the PACS-1 RNA transcript. Co-transfection of PACS-1 3′UTR luciferase reporter with miRNA449a resulted in reduced expression of luciferase. Expression of miR449a in HeLa and SiHa cells led to reduced expression of PACS-1 and increased expression of p53 and p21 proteins. FACS analysis of HeLa cells transfected with miR449a showed a reduction in S phase indicating the growth suppressive effect of miR449a. These results indicate that miR449a regulates PACS-1 expression and loss of miR449a expression leads to over-expression of PACS-1. This in turn seems to result in the destabilization of p53 in cervical cancer. Our findings suggest PACS-1 plays a role in cervical cancer development and its expression is regulated post-transcriptionally by miRNA449a. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4048.

The biochemical mechanisms that underlie hypoxia-induced NF-kappaB activity have remained largely undefined. Here, we find that prolonged hypoxia-induced NF-kappaB activation is restricted to cancer cell lines infected with high-risk human papillomavirus (HPV) serotypes. The HPV-encoded E6 protein is necessary and sufficient for prolonged hypoxia-induced NF-kappaB activation in these systems. The molecular target of E6 in the NF-kappaB pathway is the CYLD lysine 63 (K63) deubiquitinase, a negative regulator of the NF-kappaB pathway. Specifically, hypoxia stimulates E6-mediated ubiquitination and proteasomal degradation of CYLD. Given the established role of NF-kappaB in human carcinogenesis, these findings provide a potential molecular/viral link between hypoxia and the adverse clinical outcomes observed in HPV-associated malignancies.