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.

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.

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.

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.

Key Players of the Immunosuppressive Tumor Microenvironment and Emerging Therapeutic Strategies.

The tumor microenvironment (TME) is a complex, dynamic battlefield for both immune cells and tumor cells. The advent of the immune checkpoint inhibitors (ICI) since 2011, such as the anti-cytotoxic T-lymphocyte associated protein (CTLA)-4 and anti-programmed cell death receptor (PD)-(L)1 antibodies, provided powerful weapons in the arsenal of cancer treatments, demonstrating unprecedented durable responses for patients with many types of advanced cancers. However, the response rate is generally low across tumor types and a substantial number of patients develop acquired resistance. These primary or acquired resistance are attributed to various immunosuppressive elements (soluble and cellular factors) and alternative immune checkpoints in the TME. Therefore, a better understanding of the TME is absolutely essential to develop therapeutic strategies to overcome resistance. Numerous clinical studies are underway using ICIs and additional agents that are tailored to the characteristics of the tumor or the TME. Some of the combination treatments are already approved by the Food and Drug Administration (FDA), such as platinum-doublet chemotherapy, tyrosine kinase inhibitor (TKI) -targeting vascular endothelial growth factor (VEGF) combined with anti-PD-(L)1 antibodies or immuno-immuno combinations (anti-CTLA-4 and anti-PD-1). In this review, we will discuss the key immunosuppressive cells, metabolites, cytokines or chemokines, and hypoxic conditions in the TME that contribute to tumor immune escape and the prospect of relevant clinical trials by targeting these elements in combination with ICIs.

Curcumin: A review of anti-cancer properties and therapeutic activity in head and neck squamous cell carcinoma.

Curcumin (diferuloylmethane) is a polyphenol derived from the Curcuma longa plant, commonly known as turmeric. Curcumin has been used extensively in Ayurvedic medicine for centuries, as it is nontoxic and has a variety of therapeutic properties including anti-oxidant, analgesic, anti-inflammatory and antiseptic activity. More recently curcumin has been found to possess anti-cancer activities via its effect on a variety of biological pathways involved in mutagenesis, oncogene expression, cell cycle regulation, apoptosis, tumorigenesis and metastasis. Curcumin has shown anti-proliferative effect in multiple cancers, and is an inhibitor of the transcription factor NF-κB and downstream gene products (including c-myc, Bcl-2, COX-2, NOS, Cyclin D1, TNF-α, interleukins and MMP-9). In addition, curcumin affects a variety of growth factor receptors and cell adhesion molecules involved in tumor growth, angiogenesis and metastasis. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and treatment protocols include disfiguring surgery, platinum-based chemotherapy and radiation, all of which may result in tremendous patient morbidity. As a result, there is significant interest in developing adjuvant chemotherapies to augment currently available treatment protocols, which may allow decreased side effects and toxicity without compromising therapeutic efficacy. Curcumin is one such potential candidate, and this review presents an overview of the current in vitro and in vivo data supporting its therapeutic activity in head and neck cancer as well as some of the challenges concerning its development as an adjuvant chemotherapeutic agent.

CAR mediates efficient tumor engraftment of mesenchymal type lung cancer cells.

The coxsackie-adenovirus receptor (CAR) is a developmentally regulated intercellular adhesion molecule that was previously observed to be required for efficient tumor formation. To confirm that observation, we compared the tumorigenicity of clonally derived test and control cell subsets that were genetically modified for CAR. Silencing CAR in lung cancer cells with high constitutive expression reduced engraftment efficiency. Conversely, overexpressing CAR in lung cancer cells with low constitutive expression did not affect tumor formation or growth kinetics. A blocking antibody to the extracellular domain of CAR inhibited tumor engraftment, implicating that domain as being important to this process. However, differences in adhesion properties attributable to this domain (barrier function and aggregation) could not be distinguished in the test groups in vitro, and the mechanisms underlying CAR's contribution to tumor engraftment remain elusive. Because high CAR cells displayed a spindle-shaped morphology at baseline, we considered whether this expression was an accompaniment of other mesenchymal features in these lung cancer cells. Molecular correlates of CAR were compared in model epithelial and mesenchymal type lung cancer cells. CAR expression is associated with an absence of E-cadherin, diminished expression of alpha- and gamma-catenin, and increased Zeb1, Snail, and vimentin expression in lung cancer cells. In contrast, epithelial type (NCI-H292, Calu3) lung cancer cells show comparatively low CAR expression. These data suggest that if the mesenchymal cell phenotype is an accurate measure of an undifferentiated and invasive state, then CAR expression may be more closely aligned with this phenotype of lung cancer cells.

Liposome-encapsulated curcumin suppresses growth of head and neck squamous cell carcinoma in vitro and in xenografts through the inhibition of nuclear factor kappaB by an AKT-independent pathway.

PURPOSE: The purpose of this study was to determine whether a liposomal formulation of curcumin would suppress the growth of head and neck squamous cell carcinoma (HNSCC) cell lines CAL27 and UM-SCC1 in vitro and in vivo. EXPERIMENTAL DESIGN: HNSCC cell lines were treated with liposomal curcumin at different doses and assayed for in vitro growth suppression using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. A reporter gene assay was done on cell lines to study the effect of liposomal curcumin on nuclear factor kappaB (NFkappaB) activation. Western blot analysis was done to determine the effect of curcumin on the expression of NFkappaB, phospho-IkappaBalpha, phospho-AKT (pAKT), phospho-S6 kinase, cyclin D1, cyclooxygenase-2, matrix metalloproteinase-9, Bcl-2, Bcl-xL, Mcl-1L, and Mcl-1S. Xenograft mouse tumors were grown and treated with intravenous liposomal curcumin. After 5 weeks, tumors were harvested and weighed. Immunohistochemistry and Western blot analyses were used to study the effect of liposomal curcumin on the expression of NFkappaB and pAKT. RESULTS: The addition of liposomal curcumin resulted in a dose-dependent growth suppression of both cell lines. Liposomal curcumin treatment suppressed the activation of NFkappaB without affecting the expression of pAKT or its downstream target phospho-S6 kinase. Expression of cyclin D1, cyclooxygenase-2, matrix metalloproteinase-9, Bcl-2, Bcl-xL, Mcl-1L, and Mcl-1S were reduced, indicating the effect of curcumin on the NFkappaB pathway. Nude mice xenograft tumors were suppressed after 3.5 weeks of treatment with i.v. liposomal curcumin, and there was no demonstrable toxicity of liposomal curcumin upon autopsy. Immunohistochemistry and Western blot analysis on xenograft tumors showed the inhibition of NFkappaB without affecting the expression of pAKT. CONCLUSIONS: Liposomal curcumin suppresses HNSCC growth in vitro and in vivo. The results suggest that liposomal curcumin is a viable nontoxic therapeutic agent for HNSCC that may work via an AKT-independent pathway.

Inactivation of the cystatin E/M tumor suppressor gene in cervical cancer.

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 overexpressed 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. Reexpression 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. Reexpression of cystatin E/M resulted in decreased intracellular and extracellular expression of cathepsin L. Overexpression 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.

Adipose-Derived Mesenchymal Stromal Cells Persist in Tissue-Engineered Vocal Fold Replacement in Rabbits.

OBJECTIVES:: Cell therapies using mesenchymal stromal cells (MSCs) have been proposed as a promising new tool for the treatment of vocal fold scarring. However, the mechanisms by which MSCs promote healing as well as their duration of survival within the host vocal fold have yet to be defined. The aim of this work was to assess the persistence of embedded MSCs within a tissue-engineered vocal fold mucosal replacement in a rabbit model of vocal fold injury. METHODS:: Male rabbit adipose-derived MSCs were embedded within a 3-dimensional fibrin gel, forming the cell-based outer vocal fold replacement. Four female rabbits underwent unilateral resection of vocal fold epithelium and lamina propria and reconstruction with cell-based outer vocal fold replacement implantation. Polymerase chain reaction and fluorescent in situ hybridization for the sex-determining region of the Y chromosome (SRY-II) in the sex-mismatched donor-recipient pairs sought persistent cells after 4 weeks. RESULTS:: A subset of implanted male cells was detected in the implant site at 4 weeks. Many SRY-II-negative cells were also detected at the implant site, presumably representing native female cells that migrated to the area. No SRY-II signal was detected in contralateral control vocal folds. CONCLUSIONS:: The emergent tissue after implantation of a tissue-engineered outer vocal fold replacement is derived both from initially embedded adipose-derived stromal cells and infiltrating native cells. Our results suggest this tissue-engineering approach can provide a well-integrated tissue graft with prolonged cell activity for repair of severe vocal fold scars.

Curcumin suppresses growth of head and neck squamous cell carcinoma.

PURPOSE: The purpose of this study was to determine whether curcumin would trigger cell death in the head and neck squamous cell carcinoma (HNSCC) cell lines CCL 23, CAL 27, and UM-SCC1 in a dose-dependent fashion. EXPERIMENTAL DESIGN: HNSCC cells were treated with curcumin and assayed for in vitro growth suppression using 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide and fluorescence-activated cell sorting analyses. Expression of p16, cyclin D1, phospho-Ikappabeta, and nuclear factor-kappabeta (NF-kappabeta) were measured by Western blotting, gel shift, and immunofluorescence. RESULTS: Addition of curcumin resulted in a dose-dependent growth inhibition of all three cell lines. Curcumin treatment resulted in reduced nuclear expression of NF-kappabeta. This effect on NF-kappabeta was further reflected in the decreased expression of phospho-Ikappabeta-alpha. Whereas the expression of cyclin D1, an NF-kappabeta-activated protein, was also reduced, there was no difference in the expression of p16 at the initial times after curcumin treatment. In vivo growth studies were done using nude mice xenograft tumors. Curcumin was applied as a noninvasive topical paste to the tumors and inhibition of tumor growth was observed in xenografts from the CAL27 cell line. CONCLUSIONS: Curcumin treatment resulted in suppression of HNSCC growth both in vitro and in vivo. Our data support further investigation into the potential use for curcumin as an adjuvant or chemopreventive agent in head and neck cancer.

Cisplatin-induced growth arrest of head and neck cancer cells correlates with increased expression of p16 and p53.

OBJECTIVES: To determine expression of cell cycle and apoptotic genes, biochemical analysis of CCL23 and antisense cyclin D1-transfected CCL23 (CCL23AS) cells in the presence of cisplatin was performed. In addition, biochemical analysis of CAL27 cells before and after treatment with cisplatin was performed to determine expression of cell cycle genes. DESIGN: CCL23, CCL23AS, and CAL27 cell lines were treated with cisplatin. Western blot analysis, fluorescence-activated cell sorting, and apoptosis assays were performed. SETTING: In vitro study of head and neck cancer cell lines CCL23, CCL23AS, and CAL27. INTERVENTION: CCL23, CCL23AS, and CAL27 cells were treated with cisplatin. MAIN OUTCOME MEASURES: Expression of p16, p21, p53, Bcl-xL, Bcl-xS, p27, DP1, MDM2, Bcl-2, c-Jun, and Jun-D were assessed using Western blot analysis. RESULTS: There was increased expression of p16, p21, p53, BCLxL, and BCLxS genes with cisplatin treatment in the CCL23 and CCL23AS cells. Expression of p27, DP1, MDM2, BCL2, c-iun, and jun-D remained unaltered after treatment. There was decreased phosphorylation of Rb protein with complete absence of hyperphosphorylated Rb in the maximally sensitized antisense cyclin D1-transfected (CCL23AS) cells. Fluorescence-activated cell sorter analysis revealed a decreased G2 phase of the cell cycle and an increased proportion of apoptotic cells in the CCL23AS cell line compared with parental CCL23 cells. Cell killing also occurred in the presence of caspase-3 inhibitor. While CCL23 cells contain wild-type p53, the CAL27 cells have a point mutation in codon 193 (A-->T transversion) of exon 6. However, CAL27 cells still exhibited increased expression of p21 after treatment with cisplatin. CONCLUSIONS: These results, in combination with increased expression of the p53 downstream effecter p21, indicate that the cisplatin-induced cell cycle arrest operates through the p16/p53-dependent pathway, and a caspase-independent pathway may be involved. Combination treatment of head and neck squamous cell carcinoma via cell cycle inhibition and cisplatin holds promise as a potential therapy in the clinical setting.

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.

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.

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.

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.].

Curcumin treatment suppresses IKKß kinase activity of salivary cells of patients with head and neck cancer: a pilot study.

PURPOSE: To determine whether curcumin would inhibit IκB kinase ß (IKKß) kinase activity and suppress expression of proinflammatory cytokines in head and neck squamous cell carcinoma cancer (HNSCC) patients. EXPERIMENTAL DESIGN: Saliva was collected before and after subjects chewed curcumin tablets. Protein was extracted and IKKß kinase activity measured. Interleukin (IL)-6 and IL-8 levels in the salivary supernatants were measured by ELISA. IL-6, IL-8, and other interleukin were also measured independently with ELISA to confirm the inhibitory effect of curcumin on expression and secretion of salivary cytokines. RESULTS: Curcumin treatment led to a reduction in IKKß kinase activity in the salivary cells of HNSCC patients (P < 0.05). Treatment of UM-SCC1 cells with curcumin as well as with post-curcumin salivary supernatant showed a reduction of IKKß kinase activity. Significant reduction of IL-8 levels (P < 0.05) was seen in post-curcumin samples from patients with dental caries. Although there was reduced IL-8 expression in 8 of 21 post-curcumin samples of HNSCC patients, the data did not reach statistical significance. Saliva samples from HNSCC patients were also analyzed in a blinded fashion for expression of cytokines. IL-10, IFN-γ, IL-12p70, and IL-2 clustered together, and granulocyte macrophage colony stimulating factor and TNF-α clustered together. Log10 ratio analysis showed decrease in expression of all nine cytokines in both the salivary supernatant and salivary cells of curcumin-treated samples. CONCLUSIONS: Curcumin inhibited IKKß kinase activity in the saliva of HNSCC patients, and this inhibition correlated with reduced expression of a number of cytokines. IKKß kinase could be a useful biomarker for detecting the effect of curcumin in head and neck cancer.

Curcumin enhances the effect of cisplatin in suppression of head and neck squamous cell carcinoma via inhibition of IKKß protein of the NFκB pathway.

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 IKKß occurred in cisplatin- and curcumin-treated cell lines. Confocal microscopy showed expression of IKKß in the nucleus of the cell lines. Chromatin immunoprecipitation assay on DNA isolated from IKKß immunoprecipitated samples showed PCR amplification of interleukin-8 promoter sequences, a binding site of NFκB, indicating an interaction between IKKß and NFκB. Curcumin inhibited IKKß 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 IKKß, 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.

Suppression of interleukin 6 and 8 production in head and neck cancer cells with curcumin via inhibition of Ikappa beta kinase.

OBJECTIVES: To evaluate the effect of curcumin on production of interleukin 6 (IL-6) and 8 (IL-8) in head and neck squamous cell carcinoma (HNSCC) cell lines and to determine the mechanism by which these effects are modulated. Curcumin suppression of HNSCC is believed to be partly due to inhibition of the transcription factor nuclear factor-kappa beta (NF-kappa beta). Interleukin 6 and IL-8 are cytokines induced by NF-kappa beta activation with elevated levels in the serum of patients with HNSCC. DESIGN: We treated HNSCC cell lines CCL23, CAL27, UM-SCC1, and UM-SCC14A with increasing doses of curcumin and measured IL-6 and IL-8 levels using an enzyme-linked immunosorbent assay. SETTING: Levels of NF-kappa beta, Ikappa beta kinase (IKK), and phosphorylated Ikappa beta were analyzed by means of Western blot. The IKK activity was measured in UM-SCC14A cells using an IKK-specific Ikappa beta alpha substrate after treatment with curcumin. MAIN OUTCOME MEASURES: Reverse transcription-polymerase chain reaction was performed to determine the effect of curcumin on the expression of IL-6 and IL-8. RESULTS: Curcumin treatment resulted in dose-dependent inhibition of IL-6 and IL-8 in all cell lines. All cell lines had similar NF-kappa beta levels; however, UM-SCC1 and UM-SCC14A had significantly higher Ikappa beta kinase levels and required considerably higher doses of curcumin before inhibition of IL-6 and IL-8 occurred. Curcumin treatment resulted in inhibition of IKK activity and inhibition of IL-6 and IL-8 expression. CONCLUSIONS: Curcumin significantly reduces IL-6 and IL-8 levels in HNSCC cell lines. This mechanism appears to be mediated via inhibition of Ikappa beta-kinase activity in the NF-kappa beta pathway. Interleukins 6 and 8 have potential use as biomarkers to measure the efficacy of treatment with curcumin.