Search

Tumors are composed of subpopulations of cancer cells with functionally distinct features. Intratumoral heterogeneity limits the therapeutic effectiveness of cancer drugs. To address this issue, it is important to understand the regulatory mechanisms driving a subclonal variety within a therapy-resistant tumor. We identified tumor subclones of HN9 head and neck cancer cells showing distinct responses to radiation with different levels of p62 expression. Genetically identical grounds but epigenetic heterogeneity of the p62 promoter regions revealed that radioresistant HN9-R clones displayed low p62 expression via the creation of repressive chromatin architecture, in which cooperation between DNMT1 (DNA methyltransferases 1) and HDAC1 (histone deacetylases 1) resulted in DNA methylation and repressive H3K9me3 and H3K27me3 marks in the p62 promoter. Combined inhibition of DNMT1 and HDAC1 by genetic depletion or inhibitors enhanced the suppressive effects on proliferative capacity and in vivo tumorigenesis following irradiation. Importantly, ectopically p62-overexpressed HN9-R clones increased the induction of senescence along with p62-dependent autophagy activation. These results demonstrate the heterogeneous expression of p62 as the key component of clonal variation within a tumor against irradiation. Understanding the epigenetic diversity of p62 heterogeneity among subclones allows for improved identification of the functional state of subclones and provides a novel treatment option to resolve resistance to current therapies.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has attracted attention as a potential candidate for cancer therapy. However, many primary cancers are resistant to TRAIL, even when combined with standard chemotherapy. The mechanism of TRAIL resistance in cancer cells has not been fully elucidated. The TRAIL death receptor (DR) 3&prime, untranslated region (3&prime, UTR) is reported to contain AU-rich elements (AREs) that are important for regulating DR mRNA stability. However, the mechanisms by which DR mRNA stability is determined by its 3&prime, UTR are unknown. We demonstrate that tristetraprolin (TTP), an ARE-binding protein, has a critical function of regulating DR mRNA stability. DR4 mRNA contains three AREs and DR5 mRNA contains four AREs in 3&prime, UTR. TTP bound to all three AREs in DR4 and ARE3 in DR5 and enhanced decay of DR4/5 mRNA. TTP overexpression in colon cancer cells changed the TRAIL-sensitive cancer cells to TRAIL-resistant cells, and down-regulation of TTP increased TRAIL sensitivity via DR4/5 expression. Therefore, this study provides a molecular mechanism for enhanced levels of TRAIL DRs in cancer cells and a biological basis for posttranscriptional modification of TRAIL DRs. In addition, TTP status might be a biomarker for predicting TRAIL response when a TRAIL-based treatment is used for cancer.

P53 is an important biomarker in response to genotoxic stress, but it is also known to plays a key role in the regulation of metabolic homeostasis. The loss of p53 is a well-established contributor to the malignant transformation and glycolytic phenotype acquisition of cells during cancer development. However, the role of p53 in genotoxic therapy-induced metabolic shift in cancers remains unclear. Here, we attempted to elucidate how p53 participates in the glycolytic shift of head and neck cancer cell lines following irradiation. We established a stable radioresistant head and neck cancer cells (HN30-R; p53 wild type and UMSCC1-R; p53 null type) through cumulative irradiation and then analyzed their glucose metabolic profiles and mitochondria respiration. As a result, the metabolic analysis revealed no changes glycolysis of HN30-R cells, but UMSCC1-R cells exhibited increased glycolysis through increased glucose uptake and lactate production and glycolytic intermediates as well as related glycolytic enzymes, compared to UMSCC1 cells. Also, we confirmed that the mitochondrial respiration was reduced by the maximal respiration parameters of oxygen consumption rate (OCR) and that abnormal mitochondria were accumulated by electron microscopy in UMSCC1-R cells. Thus, UMSCC1-R cells exhibited an increased sensitivity to glycolysis-targeting drugs such a hexokinases inhibitor (2-deoxy-D-glucoes; 2-DG) and a lactate dehydrogenase-A inhibitor (AT101), but HN30-R cells did not shown any changes. Moreover, we identified that mitophagy limits glycolytic shift through the p53-dependent clearance of abnormal mitochondria. Taken together, these results suggest that p53 null type cells increased aerobic glycolysis to overcome the accumulation of abnormal mitochondria in radioresistnat cells. Conversely, p53 wild type cells inhibited the glycolytic shift by regulating a integrity through p53-dependent mitophagy. Thus, glycolysis-targeted drugs could be an alternative strategy for overcoming recurrent cancers after radiotherapy, and p53 status could be a biomarker for selecting participants for clinical trials. Citation Format: Hyo Won Chang, Ji Won Kim, Mi Ra Kim, Hae Yun Nam, Myungjin Lee, Won Hyeok Lee, Song Hee Kim, Da Seul Seong, Myung Woul Han, Jong Cheol Lee, Jung Je Park, Ji-hyun Seo, Seong Who Kim, Sang Yoon Kim. P53-dependent mitophagy controls glycolytic shift in radioresistant head and neck cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2405.

Tristetraprolin (TTP) is an AU-rich element-binding protein that regulates mRNA stability. Here, we report that TTP suppress the growth of human colon cancer cells both in vivo and in vitro by regulating of the expression of vascular endothelial growth factor (VEGF). TTP protein expression in human colonic tissues was markedly decreased in colonic adenocarcinoma compared with in normal mucosa and adenoma. VEGF expression was higher in colonic adenocarcinoma than in normal mucosa and adenoma. Specific inhibition of TTP expression by RNA-interference increased the expression of VEGF in cultured human colon cancer cells, and TTP overexpression markedly decreased it. In addition, elevated expression of TTP decreased the expression level of luciferase linked to a 3' terminal AU-rich element (ARE) of VEGF mRNA. Colo320/TTP cells overexpressing TTP grew slowly in vitro and became tumors small in size when xenografted s.c into nude mice. These findings demonstrate that TTP acts as a negative regulator of VEGF gene expression in colon cancer cells, suggesting that it can be used as novel therapeutic agent to treat colon cancer.

There are limited therapeutic options for patients with recurrent head and neck cancer after radiation therapy failure. To assess the use of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as a salvage chemotherapeutic agent for recurrent cancer after radiation failure, we investigated the effect of clinically relevant cumulative irradiation on TRAIL-induced apoptosis.Using a previously established HN3 cell line from a laryngeal carcinoma patient, we generated a chronically irradiated HN3R isogenic cell line. Viability and apoptosis in HN3 and HN3R cells treated with TRAIL were analyzed with MTS and PI/annexin V-FITC assays. Western blotting and flow cytometry were used to determine the underlying mechanism of TRAIL resistance. DR4 expression was semiquantitatively scored in a tissue microarray with 107 laryngeal cancer specimens. Methylation-specific polymerase chain reaction and bisulfite sequencing for DR4 were performed for genomic DNA isolated from each cell line.HN3R cells were more resistant than HN3 cells to TRAIL-induced apoptosis because of significantly reduced levels of the DR4 receptor. The DR4 staining score in 37 salvage surgical specimens after radiation failure was lower in 70 surgical specimens without radiation treatment (3.03 ± 2.75 vs 5.46 ± 3.30, respectively; P.001). HN3R cells had a methylated DR4 CpG island that was partially demethylated by the DNA demethylating agent 5-aza-2'-deoxycytidine.Epigenetic silencing of the TRAIL receptor by hypermethylation of a DR4 CpG island might be an underlying mechanism for TRAIL resistance in recurrent laryngeal carcinoma treated with radiation.

Tristetraprolin (TTP) and let-7 microRNA exhibit suppressive effects on cell growth through down-regulation of oncogenes. Both TTP and let-7 are often repressed in human cancers, thereby promoting oncogenesis by derepressing their target genes. However, the precise mechanism of this repression is unknown. We here demonstrate that p53 stimulated by the DNA-damaging agent doxorubicin (DOX) induced the expression of TTP in cancer cells. TTP in turn increased let-7 levels through down-regulation of Lin28a. Correspondingly, cancer cells with mutations or inhibition of p53 failed to induce the expression of both TTP and let-7 on treatment with DOX. Down-regulation of TTP by small interfering RNAs attenuated the inhibitory effect of DOX on let-7 expression and cell growth. Therefore, TTP provides an important link between p53 activation induced by DNA damage and let-7 biogenesis. These novel findings provide a mechanism for the widespread decrease in TTP and let-7 and chemoresistance observed in human cancers.

Epidemiologic evidence supports a role for vitamin D in colorectal cancer (CRC) risk. Variants in vitamin D–related genes might modify the association between vitamin D levels and CRC risk. In this analysis, we did a comprehensive evaluation of common variants in the vitamin D receptor (VDR) and the vitamin D–binding protein (GC; group-specific component) genes using a population-based case–unaffected sibling control design that included 1,750 sibships recruited into the Colon Cancer Family Registry. We also evaluated whether any associations differed by calcium supplement use, family history of CRC, or tumor characteristics. Heterogeneity by calcium and vitamin D intake was evaluated for a subset of 585 cases and 837 sibling controls who completed a detailed food frequency questionnaire. Age- and sex-adjusted associations were estimated using conditional logistic regression. Overall, we did not find evidence for an association between any single-nucleotide polymorphism (SNP) in VDR or GC and risk for CRC (range of unadjusted P values 0.01-0.98 for VDR and 0.07-0.95 for GC). None of these associations was significant after adjustment for multiple comparisons. We also found no evidence that calcium or vitamin D intake (food and supplement) from the food frequency questionnaire modified the association estimates between VDR and GC SNPs and CRC. We did observe associations between SNPs in GC and microsatellite unstable CRC, although these results should be confirmed in additional studies. Overall, our results do not provide evidence for a role of common genetic variants in VDR or GC in susceptibility to CRC. Cancer Epidemiol Biomarkers Prev; 19(2); 525–36

The radiotherapy has been known as one of standard treatment methods in larynx cancer, and the many targeted therapies have been developed for improving radiotherapy. In this regard, the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be one promising candidate, because TRAIL has a high selectivity for tumor cells and radiation efficiently sensitizes tumor cells towards apoptosis induction by TRAIL. It has been showing that irradiation strongly sensitizes malignant cells to TRAIL-induced apoptosis without any synergistic effects in normal tissues. Up to now, however, most studies have focused on immediate TRAIL-effect after small amount of irradiation. In this study, we aim to investigate the effect of chronic irradiation on TRAIL response to see if TRAIL could be an effective targeted therapy in recurrent laryngeal squamous cell carcinoma. Using previously established AMC-HN3 cell line from laryngeal SCCa patient, we have generated isogenic model of chronic irradiated HN3R cell line after every 2 or 3 day irradiation of 2 Gy/day of X-rays (70 Gy cumulative doses). Interestingly, through MTS assay, HN3R cells show much more resistance to TRAIL induced apoptosis than HN3 cells. Among suggested mechanisms, we found that a significantly reduced DR4 receptor is the cause of this resistance. Reverse transcription-PCR, western blotting and FACS analysis for DR4 receptors show that the expression of mRNA and the total amount, and surface expression of DR4 protein was much less in HN3R cells. Inhibition of DR4 by antagonistic antibody or si-DR4 significantly decreased TRAIL sensitivity in HN3 cells, and overexpression of DR4 enhanced the TRAIL sensitivity in HN3R cells. To evaluate this result in laryngeal cancer specimens, tissue microarray with 81 advanced laryngeal cancer specimens was constructed. Immunohistochemical assessment score of DR4 staining in 23 salvage surgical specimens after radiation failure shows statistically significant lower score than 58 surgical specimens without radiation treatment (3.09±2.23 vs. 4.43±3.36, p=0.040). Postulating that DR4 gene might be silenced by hypermethylation after long-term irradiation, methylation-specific PCR for DR4 was undertaken. Compared to HN3 cells, HN3R cells had a methylated 5’ regulatory CpG island that was partially demethylated by the DNA demethylating agent 5-aza-2’-deoxycytidine (5-AZAdC). In conclusion, chronic irradiation induces the TRAIL resistance by hypermethylation of CpG island of DR4 receptor in recurrent laryngeal squamous cell carcinoma. Thus, the status of receptor methylation might be one of biomarker to predict TRAIL response especially after radiation failure. Citation Format: Jong Cheol Lee, Won Hyeok Lee, Young Joo Min, Yoon-Se Lee, Jung Je Park, Seong Who Kim, Sang Yoon Kim. TRAIL resistance by radiation induced hypermethylation of DR4 CpG island in recurrent laryngeal squamous cell carcinoma . [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 4431. doi:10.1158/1538-7445.AM2013-4431

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: It is well established that the female sex steroids estrogen and progesterone are important in breast cancer etiology. It therefore seems plausible that variation in genes involved in biosynthesis and metabolism of these hormones may affect breast cancer risk. However, the results from genetic epidemiological studies investigating the role of variation in such genes so far have been mixed. We selected 27 genes in the estrogen and progesterone synthesis and metabolism pathway and investigated whether variation in these genes was associated with breast cancer risk. Methods: We analyzed data from a breast cancer case-control study nested in the California Teachers Study. We genotyped 365 tagging single nucleotide polymorphisms (SNPs) in 27 hormone pathway genes in 2746 non-Hispanic white women: 1351 cases and 1395 controls. Unconditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (CI), adjusting for age and geographic area. We calculated P values adjusted for multiple correlated tests (PACT) within a gene. Results: The most significant associations were observed for several SNPs in SLCO1B1, a solute carrier organic anion transporter gene. SLCO1B1 is important in estrogen metabolism as it transports estradiol-17β-glucuronide and estrone-3-sulfate from the blood into the hepatocytes. Among the 38 tagging SNPs of SLCO1B1, 9 SNPs were associated with breast cancer risk with the smallest p-value (P=0.005) for rs11045773. Several SNPs in HSD17B4 ([rs382719][1] and rs17388769) and HSD17B3 ([rs394243][2]), both involved in hormone metabolism, also showed associations with breast cancer risk, but these were not statistically significant after correcting for multiple testing. When we examined the associations separately by menopausal status, 10 SNPs in SLCO1B1 showed significant associations with postmenopausal breast cancer risk, and 5 of them (rs11045777, rs11045773, rs16923519, rs4149057, rs11045884) remained statistically significant after adjusting for multiple testing within this gene. The smallest P value among postmenopausal women was obtained for rs11045777, with OR=0.77 (95% CI=0.65-0.90, P=0.0009, PACT=0.019). Among premenopausal women, rs17388769 in HSD17B4 was associated with breast cancer risk (OR=1.56, 95% CI=1.15-2.13, P=0.004, PACT=0.051). Polymorphisms in other hormone pathway genes tested in this study were not associated with breast cancer risk in pre- or postmenopausal women. Furthermore, after additional Bonferroni adjustment for the number of genes tested, none of the genetic associations presented here were statistically significant. Conclusions: We found some evidence that genetic variation in SLCO1B1 is associated with breast cancer risk. 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 2856. [1]: /lookup/external-ref?link_type=GEN&access_num=rs382719&atom=%2Fcanres%2F70%2F8_Supplement%2F2856.atom [2]: /lookup/external-ref?link_type=GEN&access_num=rs394243&atom=%2Fcanres%2F70%2F8_Supplement%2F2856.atom