Enhanced CTLA-4 blockade anti-tumor immunity with APG-157 combination in a murine head and neck cancer.

BACKGROUND: A phase I clinical study for patients with locally advanced H&N cancer with a new class of botanical drug APG-157 provided hints of potential synergy with immunotherapy. We sought to evaluate the efficacy of the combination of APG-157 and immune checkpoint inhibitors. METHODS: CCL23, UM-SCC1 (human), and SCCVII (HPV-), MEER (HPV+) (murine) H&N cancer cell lines were utilized for in vitro and in vivo studies. We measured tumor growth by treating the mice with APG-157, anti-PD-1, and anti-CTLA-4 antibody combinations (8 groups). The tumor microenvironments were assessed by multi-color flow cytometry, immunohistochemistry, and RNA-seq analysis. Fecal microbiome was analyzed by 16S rRNA sequence. RESULTS: Among the eight treatment groups, APG-157 + anti-CTLA-4 demonstrated the best tumor growth suppression (p = 0.0065 compared to the control), followed by anti-PD-1 + anti-CTLA-4 treatment group (p = 0.48 compared to the control). Immunophenotype showed over 30% of CD8+ T cells in APG-157 + anti-CTLA-4 group compared to 4%-5% of CD8+ T cells for the control group. Differential gene expression analysis revealed that APG-157 + anti-CTLA-4 group showed an enriched set of genes for inflammatory response and apoptotic signaling pathways. The fecal microbiome analysis showed a substantial difference of lactobacillus genus among groups, highest for APG-157 + anti-CTLA-4 treatment group. We were unable to perform correlative studies for MEER model as there was tumor growth suppression with all treatment conditions, except for the untreated control group. CONCLUSIONS: The results indicate that APG-157 and immune checkpoint inhibitor combination treatment could potentially lead to improved tumor control.

Gigaxonin Suppresses Epithelial-to-Mesenchymal Transition of Human Cancer Through Downregulation of Snail.

Gigaxonin is an E3 ubiquitin ligase that plays a role in cytoskeletal stability. Its role in cancer is not yet clearly understood. Our previous studies of head and neck cancer had identified gigaxonin interacting with p16 for NFκB ubiquitination. To explore its role in cancer cell growth suppression, we analyzed normal and tumor DNA from cervical and head and neck cancers. There was a higher frequency of exon 8 SNP (c.1293 C>T, rs2608555) in the tumor (46% vs. 25% normal, P = 0.011) pointing to a relationship to cancer. Comparison of primary tumor with recurrence and metastasis did not reveal a statistical significance. Two cervical cancer cell lines, ME180 and HT3 harboring exon 8 SNP and showing T allele expression correlated with higher gigaxonin expression, reduced in vitro cell growth and enhanced cisplatin sensitivity in comparison with C allele expressing cancer cell lines. Loss of gigaxonin expression in ME180 cells through CRISPR-Cas9 or siRNA led to aggressive cancer cell growth including increased migration and Matrigel invasion. The in vitro cell growth phenotypes were reversed with re-expression of gigaxonin. Suppression of cell growth correlated with reduced Snail and increased e-cadherin expression. Mouse tail vein injection studies showed increased lung metastasis of cells with low gigaxonin expression and reduced metastasis with reexpression of gigaxonin. We have found an association between C allele expression and RNA instability and absence of multimeric protein formation. From our results, we conclude that gigaxonin expression is associated with suppression of epithelial-mesenchymal transition through inhibition of Snail. SIGNIFICANCE: Our results suggest that GAN gene exon 8 SNP T allele expression correlates with higher gigaxonin expression and suppression of aggressive cancer cell growth. There is downregulation of Snail and upregulation of e-cadherin through NFκB ubiquitination. We hypothesize that exon 8 T allele and gigaxonin expression could serve as diagnostic markers of suppression of aggressive growth of head and neck cancer.

EGFR pathway targeting drugs in head and neck cancer in the era of immunotherapy.

Receptor tyrosine kinases (RTKs) are cell surface receptors that bind growth factor ligands and initiate cellular signaling. Of the 20 classes of RTKs, 7 classes, I-V, VIII, and X, are linked to head and neck cancers (HNCs). We focus on the first class of RTK, epidermal growth factor receptor (EGFR), as it is the most thoroughly studied class. EGFR overexpression is observed in 20% of tumors, and expression of EGFR variant III is seen in 15% of aggressive chemoradiotherapy resistant HNCs. Currently, the EGFR monoclonal antibody (mAb) cetuximab is the only FDA approved RTK-targeting drug for the treatment of HNCs. Clinical trials have also included EGFR mAbs, with tyrosine kinase inhibitors, and small molecule inhibitors targeting the EGFR, MAPK, and mTOR pathways. Additionally, Immunotherapy has been found to be effective in 15 to 20% of patients with recurrent or metastatic HNC as a monotherapy. Thus, attempts are underway for the combinatorial treatment of immunotherapy and EGFR mAbs to determine if the recruitment of immune cells in the tumor microenvironment can overcome EGFR resistance.

In Vitro Effects of Permethrin on Sinonasal Epithelia.

Inhalant toxicants are postulated to contribute to the pathogenesis of chronic rhinosinusitis. Permethrin is a pesticide widely used in agricultural, industrial, and residential settings. The objective of this pilot study is to investigate the in vitro effects of permethrin on sinonasal epithelial cells (SNECs). Sinus mucosa was collected from 4 patients undergoing transsphenoidal pituitary surgery without a history of chronic rhinosinusitis. Cultured SNECs were exposed to varied concentrations of permethrin (0-156 µM) for 6 days. Cell viability and proliferation were determined via the MTT colorimetric assay and the Incucyte Live Cell Imaging System. Cellular reactive oxygen species (ROS) activity was measured by the DCFDA ROS detection assay. A statistically significant reduction in cell viability and proliferation was observed between the exposure and control groups at certain concentrations, and a dose-dependent increase in ROS activity was also observed. These findings indicate that permethrin may have deleterious effects on SNECs in a dose-dependent manner.

Cell-Free RNA as a Novel Biomarker for Response to Therapy in Head & Neck Cancer.

Liquid biopsies are gaining more traction as non-invasive tools for the diagnosis and monitoring of cancer. In a new paradigm of cancer treatment, a synergistic botanical drug combination (APG-157) consisting of multiple molecules, is emerging as a new class of cancer therapeutics, targeting multiple pathways and providing a durable clinical response, wide therapeutic window and high level of safety. Monitoring the efficacy of such drugs involves assessing multiple molecules and cellular events simultaneously. We report, for the first time, a methodology that uses circulating plasma cell-free RNA (cfRNA) as a sensitive indicator of patient response upon drug treatment. Plasma was collected from six patients with head and neck cancer (HNC) and four healthy controls receiving three doses of 100 or 200 mg APG-157 or placebo through an oral mucosal route, before treatment and on multiple points post-dosing. Circulating cfRNA was extracted from plasma at 0-, 3- and 24-hours post-treatment, followed by RNA sequencing. We performed comparative analyses of the circulating transcriptome and were able to detect significant perturbation following APG-157 treatment. Transcripts associated with inflammatory response, leukocyte activation and cytokine were upregulated upon treatment with APG-157 in cancer patients, but not in healthy or placebo-treated patients. A platelet-related transcriptional signature could be detected in cancer patients but not in healthy individuals, indicating a platelet-centric pathway involved in the development of HNC. These results from a Phase 1 study are a proof of principle of the utility of cfRNAs as non-invasive circulating biomarkers for monitoring the efficacy of APG-157 in HNC.

Dysregulation of hsa-miR-34a and hsa-miR-449a leads to overexpression of PACS-1 and loss of DNA damage response (DDR) in cervical cancer.

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, Lys382-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 G1-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.

A sensitive LC-MS assay using derivatization with boron trifluoride to quantify curcuminoids in biological samples.

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.

A randomized, phase 1, placebo-controlled trial of APG-157 in oral cancer demonstrates systemic absorption and an inhibitory effect on cytokines and tumor-associated microbes.

BACKGROUND: Although curcumin's effect on head and neck cancer has been studied in vitro and in vivo, to the authors' knowledge its efficacy is limited by poor systemic absorption from oral administration. APG-157 is a botanical drug containing multiple polyphenols, including curcumin, developed under the US Food and Drug Administration's Botanical Drug Development, that delivers the active components to oromucosal tissues near the tumor target. METHODS: A double-blind, randomized, placebo-controlled, phase 1 clinical trial was conducted with APG-157 in 13 normal subjects and 12 patients with oral cancer. Two doses, 100 mg or 200 mg, were delivered transorally every hour for 3 hours. Blood and saliva were collected before and 1 hour, 2 hours, 3 hours, and 24 hours after treatment. Electrocardiograms and blood tests did not demonstrate any toxicity. RESULTS: Treatment with APG-157 resulted in circulating concentrations of curcumin and analogs peaking at 3 hours with reduced IL-1ß, IL-6, and IL-8 concentrations in the salivary supernatant fluid of patients with cancer. Salivary microbial flora analysis showed a reduction in Bacteroidetes species in cancer subjects. RNA and immunofluorescence analyses of tumor tissues of a subject demonstrated increased expression of genes associated with differentiation and T-cell recruitment to the tumor microenvironment. CONCLUSIONS: The results of the current study suggested that APG-157 could serve as a therapeutic drug in combination with immunotherapy. LAY SUMMARY: Curcumin has been shown to suppress tumor cells because of its antioxidant and anti-inflammatory properties. However, its effectiveness has been limited by poor absorption when delivered orally. Subjects with oral cancer were given oral APG-157, a botanical drug containing multiple polyphenols, including curcumin. Curcumin was found in the blood and in tumor tissues. Inflammatory markers and Bacteroides species were found to be decreased in the saliva, and immune T cells were increased in the tumor tissue. APG-157 is absorbed well, reduces inflammation, and attracts T cells to the tumor, suggesting its potential use in combination with immunotherapy drugs.

Synergistic Activity with NOTCH Inhibition and Androgen Ablation in ERG-Positive Prostate Cancer Cells.

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.

Human papillomavirus in oropharyngeal cancer: The changing face of a disease.

The last decade has brought about an unexpected rise in oropharyngeal squamous cell carcinoma (OPSCC) primarily in white males from the ages of 40-55years, with limited exposure to alcohol and tobacco. This subset of squamous cell carcinoma (SCC) has been found to be associated with human papillomavirus infection (HPV). Other Head and Neck Squamous Cell carcinoma (HNSCC) subtypes include oral cavity, hypopharyngeal, nasopharyngeal, and laryngeal SCC which tend to be HPV negative. HPV associated oropharyngeal cancer has proven to differ from alcohol and tobacco associated oropharyngeal carcinoma in regards to the molecular pathophysiology, presentation, epidemiology, prognosis, and improved response to chemoradiation therapy. Given the improved survival of patients with HPV associated SCC, efforts to de-intensify treatment to decrease treatment related morbidity are at the forefront of clinical research. This review will focus on the important differences between HPV and tobacco related oropharyngeal cancer. We will review the molecular pathogenesis of HPV related oropharyngeal cancer with an emphasis on new paradigms for screening and treating this disease.

Cystatin E/M Suppresses Tumor Cell Growth through Cytoplasmic Retention of NF-κB.

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 (IKKß) 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.

A review of gigaxonin mutations in giant axonal neuropathy (GAN) and cancer.

Gigaxonin, the product of GAN gene localized to chromosome 16, is associated with the early onset neuronal degeneration disease giant axonal neuropathy (GAN). Gigaxonin is an E3 ubiquitin ligase adaptor protein involved in intermediate filament processing in neural cells, and vimentin filaments in fibroblasts. Mutations of the gene cause pre-neural filaments to accumulate and form giant axons resulting in the inhibition of neural cell signaling. Analysis of the catalog of somatic mutations in cancer, driver DB and IDGC data portal databases containing 21,000 tumor genomic sequences has identified GAN patient mutations in cancer cell lines and primary tumors. The database search has also shown the presence of identical missense and nonsense gigaxonin mutations in GAN and colon cancer. These mutations frequently occur in the domains associated with protein homodimerization and substrate interaction such as Broad-Complex, Tramtrack and Bric a brac (BTB), BTB associated C-terminal KELCH (BACK), and KELCH repeats. Analysis of the International HapMap Project database containing 1200 normal genomic sequences has identified a single nucleotide polymorphism (SNP), rs2608555, in exon 8 of the gigaxonin sequence. While this SNP is present in >40 % of Caucasian population, it is present in less than 10 % of Japanese and Chinese populations. Although the role of gigaxonin polymorphism is not yet known, CFTR and MDR1 gene studies have shown that silent mutations play a role in the instability and aberrant splicing and folding of mRNAs. We believe that molecular and functional investigation of gigaxonin mutations including the exon 8 polymorphism could lead to an improved understanding of the relationship between GAN and cancer.

Liposome encapsulated curcumin-difluorinated (CDF) inhibits the growth of cisplatin resistant head and neck cancer stem cells.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer, with 600,000 new cases every year worldwide. Although chemotherapeutics exist, five-year survival is only 50%. New strategies to overcome drug resistance are required to improve HNSCC treatment. Curcumin-difluorinated (CDF), a synthetic analog of curcumin, was packaged in liposomes and used to evaluate growth inhibition of cisplatin resistant HNSCC cell lines CCL-23R and UM-SCC-1R generated from the parental cell lines CCL-23 and UM-SCC-1 respectively. Growth inhibition in vitro and expression levels of the CD44 (cancer stem cell marker), cytokines, and growth factors were investigated after liposomal CDF treatment. The in vivo growth inhibitory effect of liposomal CDF was evaluated in the nude mice xenograft tumor model of UM-SCC-1R and the inhibition of CD44 was measured. Treatment of the resistant cell lines in vitro with liposomal CDF resulted in a statistically significant growth inhibition (p < 0.05). The nude mice xenograft study showed a statistically significant tumor growth inhibition of UM-SCC-1R cells and a reduction in the expression of CD44 (p < 0.05), indicating an inhibitory effect of liposomal CDF on CSCs. Our results demonstrate that delivery of CDF through liposomes may be an effective method for the treatment of cisplatin resistant HNSCC.

p16 Protein and gigaxonin are associated with the ubiquitination of NFκB in cisplatin-induced senescence of cancer cells.

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.

One in four individuals of African-American ancestry harbors a 5.5kb deletion at chromosome 11q13.1.

Cloning and sequencing of 5.5 kb deletion at chromosome 11q13.1 from the HeLa cells, tumorigenic hybrids and two fibroblast cell lines have revealed homologous recombination between AluSx and AluY resulting in the deletion of intervening sequences. Long-range PCR of the 5.5 kb 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<0.0001). This observation is strengthened by the copy number variation (CNV) data of the HapMap samples which showed that this deletion occurs in 27% of YRI (Yoruba--West African) population but none in non-African populations. The HapMap analysis further identified strong linkage disequilibrium between 5 single nucleotide polymorphisms and the 5.5 kb deletion in people of African ancestry. Computational analysis of 175 kb sequence surrounding the deletion site revealed enhanced flexibility, low thermodynamic stability, high repetitiveness, and stable stem-loop/hairpin secondary structures that are hallmarks of common fragile sites.

Correction: The CD44high Tumorigenic Subsets in Lung Cancer Biospecimens Are Enriched for Low miR-34a Expression.

[This corrects the article on p. e73195 in vol. 8.].

The CD44(high) tumorigenic subsets in lung cancer biospecimens are enriched for low miR-34a expression.

Cellular heterogeneity is an integral part of cancer development and progression. Progression can be associated with emergence of cells that exhibit high phenotypic plasticity (including "de-differentiation" to primitive developmental states), and aggressive behavioral properties (including high tumorigenic potentials). We observed that many biomarkers that are used to identify Cancer Stem Cells (CSC) can label cell subsets in an advanced clinical stage of lung cancer (malignant pleural effusions, or MPE). Thus, CSC-biomarkers may be useful for live sorting functionally distinct cell subsets from individual tumors, which may enable investigators to hone in on the molecular basis for functional heterogeneity. We demonstrate that the CD44(hi) (CD44-high) cancer cell subsets display higher clonal, colony forming potential than CD44(lo) cells (n=3) and are also tumorigenic (n=2/2) when transplanted in mouse xenograft model. The CD44(hi) subsets express different levels of embryonal (de-differentiation) markers or chromatin regulators. In archived lung cancer tissues, ALDH markers co-localize more with CD44 in squamous cell carcinoma (n=5/7) than Adeno Carcinoma (n=1/12). MPE cancer cells and a lung cancer cell line (NCI-H-2122) exhibit chromosomal abnormalities and 1p36 deletion (n=3/3). Since miR-34a maps to the 1p36 deletion site, low miR-34a expression levels were detected in these cells. The colony forming efficiency of CD44(hi) cells, characteristic property of CSC, can be inhibited by mir-34a replacement in these samples. In addition the highly tumorigenic CD44(hi) cells are enriched for cells in the G2 phase of cell cycle.

Hyperactivated JNK is a therapeutic target in pVHL-deficient renal cell carcinoma.

Clear cell renal cell carcinomas (RCC), the major histologic subtype of RCC accounting for more than 80% of cases, are typified by biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. Although accumulation of hypoxia-inducible factor alpha (HIF-α) is the most well-studied effect of VHL inactivation, direct inhibition of HIFα or restoration of wild-type pVHL protein expression has not proved readily feasible, given the limitations associated with pharmacologic targeting of transcription factors (i.e., HIF-α) and gene replacement therapy of tumor suppressor genes (i.e., VHL). Here, we have established that phosphorylated c-Jun, a substrate of the c-Jun-NH(2)-kinase (JNK), is selectively activated in clear cell RCC patient specimens. Using multiple isogenic cell lines, we show that HIF-α-independent JNK hyperactivation is unique to the pVHL-deficient state. Importantly, pVHL-deficient RCCs are dependent upon JNK activity for in vitro and in vivo growth. A multistep signaling pathway that links pVHL loss to JNK activation involves the formation of a CARD9/BCL10/TRAF6 complex as a proximal signal to sequentially stimulate TAK1 (MAPKKK), MKK4 (MAPKK), and JNK (MAPK). JNK stimulates c-Jun phosphorylation, activation, and dimerization with c-Fos to form a transcriptionally competent AP1 complex that drives transcription of the Twist gene and induces epithelial-mesenchymal transition. Thus, JNK represents a novel molecular target that is selectively activated in and drives the growth of pVHL-deficient clear cell RCCs. These findings can serve as the preclinical foundation for directed efforts to characterize potent pharmacologic inhibitors of the JNK pathway for clinical translation.

Cancer stem cells, microRNAs, and therapeutic strategies including natural products.

Embryonic stem cells divide continuously and differentiate into organs through the expression of specific transcription factors at specific time periods. Differentiated adult stem cells on the other hand remain in quiescent state and divide by receiving cues from the environment (extracellular matrix or niche), as in the case of wound healing from tissue injury or inflammation. Similarly, it is believed that cancer stem cells (CSCs), forming a smaller fraction of the tumor bulk, also remain in a quiescent state. These cells are capable of initiating and propagating neoplastic growth upon receiving environmental cues, such as overexpression of growth factors, cytokines, and chemokines. Candidate CSCs express distinct biomarkers that can be utilized for their identification and isolation. This review focuses on the known and candidate cancer stem cell markers identified in various solid tumors and the promising future of disease management and therapy targeted at these markers. The review also provides details on the differential expression of microRNAs (miRNAs), and the miRNA- and natural product-based therapies that could be applied for the treatment of cancer stem cells.

Cellular senescence and tumor suppressor gene p16.

Cellular senescence is an irreversible arrest of cell growth. Biochemical and morphological changes occur during cellular senescence, including the formation of a unique cellular morphology such as flattened cytoplasm. Function of mitochondria, endoplasmic reticulum and lysosomes are affected resulting in the inhibition of lysosomal and proteosomal pathways. Cellular senescence can be triggered by a number of factors including, aging, DNA damage, oncogene activation and oxidative stress. While the molecular mechanism of senescence involves p16 and p53 tumor suppressor genes and telomere shortening, this review is focused on the mechanism of p16 control. The p16-mediated senescence acts through the retinoblastoma (Rb) pathway inhibiting the action of the cyclin dependant kinases leading to G1 cell cycle arrest. Rb is maintained in a hypophosphorylated state resulting in the inhibition of transcription factor E2F1. Regulation of p16 expression is complex and involves epigenetic control and multiple transcription factors. PRC1 (Pombe repressor complex (1) and PRC2 (Pombe repressor complex (2) proteins and histone deacetylases play an important role in the promoter hypermethylation for suppressing p16 expression. While transcription factors YY1 and Id1 suppress p16 expression, transcription factors CTCF, Sp1 and Ets family members activate p16 transcription. Senescence occurs with the inactivation of suppressor elements leading to the enhanced expression of p16.