A phase 1 study of oral ASP5878, a selective small-molecule inhibitor of fibroblast growth factor receptors 1-4, as a single dose and multiple doses in patients with solid malignancies.

ASP5878 is a selective small-molecule inhibitor of fibroblast growth factor receptors (FGFRs). This study investigated safety, tolerability, and antitumor effect of single and multiple oral doses of ASP5878 in patients with solid tumors. This phase 1, open label, first-in-human study comprised dose-escalation and dose-expansion parts. Primary objectives of the dose-escalation part were to identify the dose-limiting toxicity (DLT), maximum tolerated dose, and recommended dose of ASP5878 for the dose-expansion part. Nine dose cohorts of ASP5878 were evaluated (0.5─2 mg once daily; 2─40 mg twice daily [BID]). A single dose of ASP5878 was followed by a 2-day pharmacokinetic collection, and then either 28-day cycles of daily dosing (ASP5878 ≤ 10 mg BID) or 5-day dosing/2-day interruption (ASP5878 ≥ 20 mg BID). The primary objective of the dose-expansion part was to determine the safety of ASP5878 (16 mg BID) administered in 28-day cycles of 5-day dosing/2-day interruption in patients with urothelial carcinoma, hepatocellular carcinoma, or squamous cell lung carcinoma with FGFR genetic alterations. Safety was assessed by monitoring adverse events (AEs). Thirty-five patients were enrolled and 31 discontinued in the dose-escalation part; 51 patients were enrolled and 51 discontinued in the dose-expansion part. In the dose-escalation part, 66.7% of patients in the 20 mg BID 5-day dosing/2-day interruption group reported DLTs of hyperphosphatemia. The recommended dose for the dose-expansion part was 16 mg BID. Common AEs included retinal detachment, diarrhea, and increased alanine aminotransferase. One death occurred that was not related to ASP5878. ASP5878 was well tolerated with manageable toxicities including hyperphosphatemia.

The relation between systemic inflammation and incident cancer in patients with stable cardiovascular disease: a cohort study.

AIMS: Low-grade inflammation, measured by elevated plasma concentrations of high-sensitive C-reactive protein (CRP), is a risk factor for cardiovascular disease (CVD). There is evidence that low-grade inflammation is also related to a higher risk of cancer. The present prospective cohort study evaluates the relation between low-grade systemic inflammation and risk of cancer in patients with stable CVD. METHODS AND RESULTS: In total, 7178 patients with stable CVD and plasma CRP levels ≤10 mg/L were included. Data were linked to the Dutch national cancer registry. Cox regression models were fitted to study the relation between CRP and incident CVD and cancer. After a median follow-up time of 8.3 years (interquartile range 4.6-12.3) 1072 incident cancer diagnoses were observed. C-reactive protein concentration was related to total cancer [hazard ratio (HR) 1.35; 95% confidence interval (CI) 1.10-1.65] comparing last quintile to first quintile of CRP. Especially lung cancer, independent of histopathological subtype, was related to CRP (HR 3.39; 95% CI 2.02-5.69 comparing last to first quintile of CRP). Incidence of epithelial neoplasms and especially squamous cell neoplasms were related to CRP concentration, irrespective of anatomical location. Sensitivity analyses after excluding patients with a cancer diagnosis within 1, 2, and 5 years of follow-up showed similar results. No effect modification was observed by smoking status or time since smoking cessation (P-values for interaction > 0.05). CONCLUSION: Chronic systemic low-grade inflammation, measured by CRP levels ≤10 mg/L, is a risk factor for incident cancer, markedly lung cancer, in patients with stable CVD. The relation between inflammation and incident cancer is seen in former and current smokers and is uncertain in never smokers.

Consequences of Vitamin A Deficiency: Immunoglobulin Dysregulation, Squamous Cell Metaplasia, Infectious Disease, and Death.

Vitamin A is an important regulator of immune protection, but it is often overlooked in studies of infectious disease. Vitamin A binds an array of nuclear receptors (e.g., retinoic acid receptor, peroxisome proliferator-activated receptor, retinoid X receptor) and influences the barrier and immune cells responsible for pathogen control. Children and adults in developed and developing countries are often vitamin A-deficient or insufficient, characteristics associated with poor health outcomes. To gain a better understanding of the protective mechanisms influenced by vitamin A, we examined immune factors and epithelial barriers in vitamin A deficient (VAD) mice, vitamin D deficient (VDD) mice, double deficient (VAD+VDD) mice, and mice on a vitamin-replete diet (controls). Some mice received insults, including intraperitoneal injections with complete and incomplete Freund's adjuvant (emulsified with PBS alone or with DNA + Fus-1 peptide) or intranasal inoculations with Sendai virus (SeV). Both before and after insults, the VAD and VAD+VDD mice exhibited abnormal serum immunoglobulin isotypes (e.g., elevated IgG2b levels, particularly in males) and cytokine/chemokine patterns (e.g., elevated eotaxin). Even without insult, when the VAD and VAD+VDD mice reached 3-6 months of age, they frequently exhibited opportunistic ascending bacterial urinary tract infections. There were high frequencies of nephropathy (squamous cell hyperplasia of the renal urothelium, renal scarring, and ascending pyelonephritis) and death in the VAD and VAD+VDD mice. When younger VAD mice were infected with SeV, the predominant lesion was squamous cell metaplasia of respiratory epithelium in lungs and bronchioles. Results highlight a critical role for vitamin A in the maintenance of healthy immune responses, epithelial cell integrity, and pathogen control.

Enrichment of genes associated with squamous differentiation in cancer initiating cells isolated from urothelial cells transformed by the environmental toxicant arsenite.

Arsenic is an environmental toxicant with long-term exposure associated with the development of urothelial carcinomas. Our lab has developed an in-vitro model of urothelial carcinoma by exposing the immortal, but non-tumorigenic bladder cell line, the UROtsa, to arsenite (As3+). These transformed cells form tumors in immune-compromised mice, which resemble urothelial carcinomas with components of the tumor exhibiting squamous differentiation. The goal of the present study was to determine the differences in global gene expression patterns between the As3+-transformed UROtsa cells and the urospheres (spheroids containing putative cancer initiating cells) isolated from these cell lines and to determine if the genes involved in the development of squamous differentiation were enriched in the urospheres. The results obtained in this study show an enrichment of genes such as KRT1, KRT5, KRT6A, KRT6B, KRT6C, KRT14 and KRT16 associated with squamous differentiation, a characteristic feature seen in aggressive basal subtypes of urothelial cell carcinoma (UCC) in the urospheres isolated from As3+-transformed UROtsa cells. In addition, there is increased expression of several of the small proline-rich proteins (SPRR) in the urospheres and overexpression of these genes occur in UCC's displaying squamous differentiation. In conclusion, the cancer initiating cells present in the urospheres are enriched with genes associated with squamous differentiation.

Clinical outcomes of muscle invasive bladder Cancer according to the BASQ classification.

BACKGROUND: We evaluated the clinical efficacy and prognosis of muscle-invasive bladder cancer according to the basal/squamous-like (BASQ) classification system based on immunohistochemical staining [CK5/6(+), CK14(+), GATA3(-), and FOXA1(-)]. METHODS: One hundred patients diagnosed with muscle-invasive bladder cancer (cT2-4 N0-3 M0) were included in the study. All patients underwent radical cystectomy after transurethral removal of bladder tumor. Immunostaining was performed for CK5/6, CK14, FOXA1, and GATA3 antibodies on tissue microarray slides, and expression patterns were quantitatively analyzed using a scanning program. RESULTS: The median follow-up time was 77.4 (interquartile range: 39-120.9) months. The mean age of the patients was 65.1 ± 11.2 years. FOXA1 or CK14 expression greater than 1% was respectively positively and negatively correlated with overall survival (OS; p = 0.011 and p = 0.042, respectively), cancer-specific survival (CSS; p = 0.050 for both), and recurrence-free survival (RFS; p = 0.018 and p = 0.040, respectively). For CK5/6+ and GATA3- or FOXA1- expression, 10% CK5/6+ cells were negatively correlated with OS (p = 0.032 and p = 0.039, respectively) and with RFS in combination with FOXA1- only (p = 0.050). CONCLUSIONS: In this study, CK14 expression was associated with a poor prognosis. The new classification system of bladder cancer based on molecular characteristics is expected to helpful tool for the establishment of personalized treatment strategies and associated prediction of therapeutic responses.

Development of a Multiplexed Liquid Chromatography Multiple-Reaction-Monitoring Mass Spectrometry (LC-MRM/MS) Method for Evaluation of Salivary Proteins as Oral Cancer Biomarkers.

Multiple (selected) reaction monitoring (MRM/SRM) of peptides is a growing technology for target protein quantification because it is more robust, precise, accurate, high-throughput, and multiplex-capable than antibody-based techniques. The technique has been applied clinically to the large-scale quantification of multiple target proteins in different types of fluids. However, previous MRM-based studies have placed less focus on sample-preparation workflow and analytical performance in the precise quantification of proteins in saliva, a noninvasively sampled body fluid. In this study, we evaluated the analytical performance of a simple and robust multiple reaction monitoring (MRM)-based targeted proteomics approach incorporating liquid chromatography with mass spectrometry detection (LC-MRM/MS). This platform was used to quantitatively assess the biomarker potential of a group of 56 salivary proteins that have previously been associated with human cancers. To further enhance the development of this technology for assay of salivary samples, we optimized the workflow for salivary protein digestion and evaluated quantification performance, robustness and technical limitations in analyzing clinical samples. Using a clinically well-characterized cohort of two independent clinical sample sets (total n = 119), we quantitatively characterized these protein biomarker candidates in saliva specimens from controls and oral squamous cell carcinoma (OSCC) patients. The results clearly showed a significant elevation of most targeted proteins in saliva samples from OSCC patients compared with controls. Overall, this platform was capable of assaying the most highly multiplexed panel of salivary protein biomarkers, highlighting the clinical utility of MRM in oral cancer biomarker research.

Motor Neuron and Pancreas Homeobox 1 (MNX1) Is Involved in Promoting Squamous Cervical Cancer Proliferation via Regulating Cyclin E.

BACKGROUND Cervical cancer is one of the most lethal gynecologic malignancies worldwide. The objective of this study was to assess the role of MNX1 in cervical cancer and its underlying mechanisms. MATERIAL AND METHODS The expression of motor neuron and pancreas homeobox 1 (MNX1) in immortal epithelial cervical cell line ECT, cervical cancer cell HeLa, and SiHa and cervical cancer, as well as in adjacent noncancer tissues, was detected and analyzed. CCK-8 and colony formation assays were performed to evaluate the effects of MNX1 overexpression on cervical cancer cell proliferation. Transwell assay was used to detect migration and invasion after MNX1 knockdown or overexpression. Real-time PCR and Western blotting were used to examine MNX1 and cell cycle regulator expression. RESULTS Data from our study indicated that MNX1 was upregulated both in cervical cancer cell lines and cervical cancer tissues. The high levels of MNX1 are related to advanced stages and lymph nodes metastasis. The overexpression of MNX1 promoted cervical cancer cells proliferation, migration, and invasion. Moreover, MNX1 upregulated 2 critical cell cycle regulators, CCNE1 and CCNE2. CONCLUSIONS These findings reveal MNX1 as a novel oncogene of cervical cancer and indicate MNX1 is a promising therapeutic and prognostic biomarker.

New targets bring hope in squamous cell lung cancer: neurotrophic tyrosine kinase gene fusions.

Neurotrophic tyrosine kinase genes encode for the Trk-family proteins TrkA, TrkB, and TrkC, which have an important role in the development of the nervous system; however, they have been identified as oncogenic fusions in solid tumors (NTK-1, NTRK-2, and NTRK-3) and are associated with poor survival in lung cancer. These three new fusions can be detected by fluorescent in situ hybridization or next-generation sequencing in less than 5% of the lung tumors. There are several ongoing clinical trials of NTRK oncogenes in lung cancer and other tumors. The agents entrectinib (RXDX-101), a multi-kinase small molecule inhibitor that selectively inhibits NTRK1, NTRK2, and NTRK3, ROS1 and ALK, and LOXO-101, an ATP-competitive pan-NTRK inhibitor, have shown responses in patients with lung cancer with an acceptable toxicity profile. Although these oncogenic fusions are not very prevalent, the high prevalence of lung cancer makes these findings very relevant and suggests the feasibility of these oncogenes as targets in lung cancer. New data from Ozono and collaborators presented in this issue suggest that BDNF/TrkB signal promotes proliferating migratory and invasive phenotypes and cellular plasticity in squamous cell carcinoma (SCC) of the lung but that it also represents a druggable target that may bring hope to squamous lung cancer patients.

High Expression of S100A6 Predicts Unfavorable Prognosis of Lung Squamous Cell Cancer.

BACKGROUND S100 family of proteins is mainly involved in regulation of intracellular calcium homeostasis. Aberrant expression of S100 family members has been reported in many types of cancers. However, as a member of S100 family, the prognostic value of S100A6 for lung squamous cell carcinoma (SCC) has not been well-studied. MATERIAL AND METHODS Using immunohistochemistry, we investigated the expression of S100A6 in 177 patients with SCC and further divided the cohort into a high S100A6 expression group and a low S100A6 expression group. The chi-square test was applied to analyze the correlation between S100A6 expression and clinicopathological factors. Univariate analysis using the Kaplan-Meier method was performed to compare the difference in survival rates between the high S100A6 expression group and the low S100A6 expression group; multivariate analysis with Cox regression model was used to identify independent prognostic risk factors. RESULTS In our experiment, we demonstrated that the expression of S100A6 was significantly associated with patient age and tumor differentiation. High-expression of S100A6 was shown to be substantially related to the unfavorable prognosis of SCC. Moreover, our results confirmed that S100A6 was an independent risk factor for SCC prognosis, and could predict unfavorable prognosis. CONCLUSIONS High-expression of S100A6 was identified as an independent unfavorable prognostic factor for SCC, suggesting that targeting S100A6 may result in the development of potential targeted drug for SCC.

Elevated GAPDH expression is associated with the proliferation and invasion of lung and esophageal squamous cell carcinomas.

Glyceraldehyde-3-phosphate dehydrogenase, is one of the most investigated housekeeping genes and widely used as an internal control in analysis of gene expression levels. The present study was designed to assess whether GAPDH is associated with cancer cell growth and progression and, therefore may not be a good internal control in cancer research. Our results from clinical tissue studies showed that the levels of GAPDH protein were significantly up-regulated in lung squamous cell carcinoma tissues, compared with the adjacent normal lung tissues, and this was confirmed by western blotting and immunohistochemistry. GAPDH knockdown by siRNA resulted in significant reductions in proliferation, migration, and invasion of lung squamous carcinoma cells in vitro. In a nude mouse cancer xenograft model, GAPDH knockdown significantly inhibited the cell proliferation and migration/invasion in vivo. In summary, GAPDH may not be an appropriate internal control for gene expression studies, especially in cancer research. The role of GAPDH in cancer development and progression should be further examined in pre-clinical and clinical studies.

Decorin in human oral cancer: a promising predictive biomarker of S-1 neoadjuvant chemosensitivity.

We reported previously that decorin (DCN) is significantly up-regulated in chemoresistant cancer cell lines. DCN is a small leucine-rich proteoglycan that exists and functions in stromal and epithelial cells. Accumulating evidence suggests that DCN affects the biology of several types of cancer by directly/indirectly targeting the signaling molecules involved in cell growth, survival, metastasis, and angiogenesis, however, the molecular mechanisms of DCN in chemoresistance and its clinical relevance are still unknown. Here we assumed that DCN silencing cells increase chemosusceptibility to S-1, consisted of tegafur, prodrug of 5-fluorouracil. We first established DCN knockdown transfectants derived from oral cancer cells for following experiments including chemosusceptibility assay to S-1. In addition to the in vitro data, DCN knockdown zenografting tumors in nude mice demonstrate decreasing cell proliferation and increasing apoptosis with dephosphorylation of AKT after S-1 chemotherapy. We also investigated whether DCN expression predicts the clinical responses of neoadjuvant chemotherapy (NAC) using S-1 (S-1 NAC) for oral cancer patients. Immunohistochemistry data in the preoperative biopsy samples was analyzed to determine the cut-off point for status of DCN expression by receiver operating curve analysis. Interestingly, low DCN expression was observed in five (83%) of six cases with complete responses to S-1 NAC, and in one (10%) case of 10 cases with stable/progressive disease, indicating that S-1 chemosensitivity is dramatically effective in oral cancer patients with low DCN expression compared with high DCN expression. Our findings suggest that DCN is a key regulator for chemoresistant mechanisms, and is a predictive immunomarker of the response to S-1 NAC and patient prognosis.

Canonical correlation analysis for RNA-seq co-expression networks.

Digital transcriptome analysis by next-generation sequencing discovers substantial mRNA variants. Variation in gene expression underlies many biological processes and holds a key to unravelling mechanism of common diseases. However, the current methods for construction of co-expression networks using overall gene expression are originally designed for microarray expression data, and they overlook a large number of variations in gene expressions. To use information on exon, genomic positional level and allele-specific expressions, we develop novel component-based methods, single and bivariate canonical correlation analysis, for construction of co-expression networks with RNA-seq data. To evaluate the performance of our methods for co-expression network inference with RNA-seq data, they are applied to lung squamous cell cancer expression data from TCGA database and our bipolar disorder and schizophrenia RNA-seq study. The preliminary results demonstrate that the co-expression networks constructed by canonical correlation analysis and RNA-seq data provide rich genetic and molecular information to gain insight into biological processes and disease mechanism. Our new methods substantially outperform the current statistical methods for co-expression network construction with microarray expression data or RNA-seq data based on overall gene expression levels.

[Study on the expression and signification of TLR4/NO pathway in cervical tumorigenesis with high risk HPV infection].

OBJECTIVE: To explore the role of toll-like receptor (TLR)/ nitric oxide (NO) pathway in cervical tumor with high risk human papillomavirus (hrHPV) infection. METHODS: (1)Study was based on 36 women with nonmalignantcervical tissue as control group and 36 women with squamous cell cervical cancer (SCC), all with hrHPV infection which were assessed by using 14 types hrHPV E6/E7 mRNA real-time PCR kit. The amount of NO was detected by Griess reaction, the expression of inducible nitric oxide synthase(iNOS) was detected by immunohistochemistry (IHC). The mRNA expression of TLR3, TLR4, TLR7, TLR8, TLR9, nuclear factor-κB (NF-κB) p65 and iNOS in control and SCC epithelium which was captured by laser capture microdissection (LCM) were determined.(2)The expressions of TLR4 in CaSki, HeLa and C33a were detected by cell immunofluorescence method. The mRNA and protein expression of TLR/NO pathway transduction molecules including TLR4,NF-κBp65 and iNOS in CaSki, HeLa and C33a cell lines were detected by real-time PCR and western blot. RESULTS: (1)The level of NO was much higher in SCC group than that in control group [(42.92±0.36)µmol/L vs(15.49±0.24)µmol/L; P < 0.05 ]. iNOS was detected in 75% (27 cases ) of patients with squamous cervical carcinoma, while only 6% (2 cases) of normal controls were confirmed with positve result (P < 0.05). TLR/NO pathway maybe activated in SCC, for the mRNA levels of TLR3, TLR4, TLR7, TLR8, NF-κBp65 and iNOS increased significantly when compared to control group (all P < 0.05), and the greatest change in the expression level of TLR in SCC was spotted on TLR4(7.41±0.39 vs 1.86±0.21). (2)The results of immunofluorescence showed that TLR4 was located at plasma membrance of hrHPV positive HeLa and CaSki cells, while the integral optical density of TLR4 in HeLa cells (3 599±427) or CaSki cells (2 080±456) were higher than that in C33a cells (730± 96; P < 0.05). The mRNA and protein level of TLR4, NF-κBp65 and iNOS in HeLa and CaSki cells were higher than those of C33a cells (P < 0.05). CONCLUSION: TLR4/NO pathway is highly expressed in cervical cancer with hrHPV infection, while the pathway may be involved in cervical tumorigenesis with hrHPV infection.

Nicotine induces the up-regulation of the α7-nicotinic receptor (α7-nAChR) in human squamous cell lung cancer cells via the Sp1/GATA protein pathway.

Nicotine, the addictive component of cigarettes, promotes lung cancer proliferation via the α7-nicotinic acetylcholine receptor (α7-nAChR) subtype. The present manuscript explores the effect of nicotine exposure on α7-nAChR levels in squamous cell carcinoma of the lung (SCC-L) in vitro and in vivo. Nicotine (at concentrations present in the plasma of average smokers) increased α7-nAChR levels in human SCC-L cell lines. Nicotine-induced up-regulation of α7-nAChR was confirmed in vivo by chicken chorioallantoic membrane models. We also observed that the levels of α7-nAChR in human SCC-L tumors (isolated from patients who are active smokers) correlated with their smoking history. Nicotine increased the levels of α7-nAChR mRNA and α7-nAChR transcription in human SCC-L cell lines and SCC-L tumors. Nicotine-induced up-regulation of α7-nAChR required GATA4 and GATA6. ChIP assays showed that nicotine induced the binding of GATA4 or GATA6 to Sp1 on the α7-nAChR promoter, thereby inducing its transcription and increasing its levels in human SCC-L. Our data are clinically relevant because SCC-L patients smoked for decades before being diagnosed with cancer. It may be envisaged that continuous exposure to nicotine (in such SCC-L patients) causes up-regulation of α7-nAChRs, which facilitates tumor growth and progression. Our results will also be relevant to many SCC-L patients exposed to nicotine via second-hand smoke, electronic cigarettes, and patches or gums to quit smoking.

Identification of candidate biomarkers for early detection of human lung squamous cell cancer by quantitative proteomics.

To discover novel biomarkers for early detection of human lung squamous cell cancer (LSCC) and explore possible mechanisms of LSCC carcinogenesis, iTRAQ-tagging combined with two dimensional liquid chromatography tandem MS analysis was used to identify differentially expressed proteins in human bronchial epithelial carcinogenic process using laser capture microdissection-purified normal bronchial epithelium (NBE), squamous metaplasia (SM), atypical hyperplasia (AH), carcinoma in situ (CIS) and invasive LSCC. As a result, 102 differentially expressed proteins were identified, and three differential proteins (GSTP1, HSPB1 and CKB) showing progressively expressional changes in the carcinogenic process were selectively validated by Western blotting. Immunohistochemistry was performed to detect the expression of the three proteins in an independent set of paraffin-embedded archival specimens including various stage tissues of bronchial epithelial carcinogenesis, and their ability for early detection of LSCC was evaluated by receiver operating characteristic analysis. The results showed that the combination of the three proteins could perfectly discriminate NBE from preneoplastic lesions (SM, AH and CIS) from invasive LSCC, achieving a sensitivity of 96% and a specificity of 92% in discriminating NBE from preneoplatic lesions, a sensitivity of 100% and a specificity of 98% in discriminating NBE from invasive LSCC, and a sensitivity of 92% and a specificity of 91% in discriminating preneoplastic lesions from invasive LSCC, respectively. Furthermore, we knocked down GSTP1 in immortalized human bronchial epithelial cell line 16HBE cells, and then measured their susceptibility to carcinogen benzo(a)pyrene-induced cell transformation. The results showed that GSTP1 knockdown significantly increased the efficiency of benzo(a)pyrene-induced 16HBE cell transformation. The present data first time show that GSTP1, HSPB1 and CKB are novel potential biomarkers for early detection of LSCC, and GSTP1 down-regulation is involved in human bronchial epithelial carcinogenesis.

Enhancement of phototoxicity of curcumin in human oral cancer cells using silica nanoparticles as delivery vehicle.

We report results on the use of organically modified silica nanoparticles (SiNp) as a vehicle for the delivery of curcumin in human oral cancer cells for improvement of uptake and phototoxicity. Nanoformulated drug (curcumin-SiNp complex) was prepared by postloading curcumin in SiNp, and the complex was soluble in aqueous solution. Cellular uptake studied by fluorescence microscopy and spectroscopy showed that curcumin accumulation was higher when cells were incubated with curcumin-SiNp complex as against free curcumin. Studies carried out on incubation time-dependent cytotoxicity, inhibition of NF-κB activity, suppression of NF-κB-regulated proteins involved in invasion (MMP-9), angiogenesis (VEGF), and inflammation (TNF-α) showed that curcumin-SiNp leads to significant improvement over free curcumin in dark as well as on exposure to light.

CHFR protein regulates mitotic checkpoint by targeting PARP-1 protein for ubiquitination and degradation.

The mitotic checkpoint gene CHFR (checkpoint with forkhead-associated (FHA) and RING finger domains) is silenced by promoter hypermethylation or mutated in various human cancers, suggesting that CHFR is an important tumor suppressor. Recent studies have reported that CHFR functions as an E3 ubiquitin ligase, resulting in the degradation of target proteins. To better understand how CHFR suppresses cell cycle progression and tumorigenesis, we sought to identify CHFR-interacting proteins using affinity purification combined with mass spectrometry. Here we show poly(ADP-ribose) polymerase 1 (PARP-1) to be a novel CHFR-interacting protein. In CHFR-expressing cells, mitotic stress induced the autoPARylation of PARP-1, resulting in an enhanced interaction between CHFR and PARP-1 and an increase in the polyubiquitination/degradation of PARP-1. The decrease in PARP-1 protein levels promoted cell cycle arrest at prophase, supporting that the cells expressing CHFR were resistant to microtubule inhibitors. In contrast, in CHFR-silenced cells, polyubiquitination was not induced in response to mitotic stress. Thus, PARP-1 protein levels did not decrease, and cells progressed into mitosis under mitotic stress, suggesting that CHFR-silenced cancer cells were sensitized to microtubule inhibitors. Furthermore, we found that cells from Chfr knockout mice and CHFR-silenced primary gastric cancer tissues expressed higher levels of PARP-1 protein, strongly supporting our data that the interaction between CHFR and PARP-1 plays an important role in cell cycle regulation and cancer therapeutic strategies. On the basis of our studies, we demonstrate a significant advantage for use of combinational chemotherapy with PARP inhibitors for cancer cells resistant to microtubule inhibitors.

[Expression and clinical significance of E-cadherin and ß-catenin in the vulvar squamous cell carcinoma tissues].

OBJECTIVE: To investigate the expression of E-cadherin and ß-catenin in the Vulvar squamous cell carcinoma (VSCC) tissues. METHODS: A total of 63 documented paraffin blocks of VSCC (n=41), vulvar intraepithelial neoplasia (n=22), vulvar negative cutting edge tissues (n=10) diagnosed in department of pathology of Shengjing Hospital of China Medical University from January 2005 to April 2012 were enrolled. EliVision immunohistochemical staining was used to detect the expression of E-cadherin and ß-catenin in the three groups. Then to do a statistical analysis among the expression of them with patients' menopause status, pathological grade, clinical stage and lymph node metastasis. Spearman correlation analysis was used to analyse the expression of E-cadherin and ß-catenin in the vulvar lesion tissues. RESULTS: The abnormal immunoreactivity for E-cadherin [46%(19/41), 64% (14/22)] and ß-catenin [61% (25/41), 68% (15/22)] in VSCC and VINII-III were found, which were significantly different from that in normal epithelium samples (P<0.05). The abnormal expression of E-cadherin and ß-catenin have no statistically significant difference between VSCC group and VINII-III group (P>0.05). The abnormal expression of E-cadherin and ß-catenin were collected with tumor pathological grade and lymph node metastasis status (all P<0.05). The abnormal expression of E-cadherin and ß-catenin have no statistically significant difference between menopause and the surgical stage of patients (all P>0.05). The abnormal expression of E-cadherin and ß-catenin have a significant positive correlation in the same sample in the VSCC tissue (r=0.543, P=0.000). The abnormal expression of E-cadherin and ß-catenin have no correlation in the VINII-III tissue (r=0.295, P=0.182). CONCLUSIONS: The abnormal expression of E-cadherin and ß-catenin may occurs frequently in the VSCC. The abnormal expression of E-cadherin and ß-catenin have correlation with vulvar cancer pathological grade and lymph node metastasis, which may be important mechanisms promoting the invasion and metastasis of VSCC.

CLIC4 is a tumor suppressor for cutaneous squamous cell cancer.

Chloride intracellular channel (CLIC) 4 is a member of a redox-regulated, metamorphic multifunctional protein family, first characterized as intracellular chloride channels. Current knowledge indicates that CLICs participate in signaling, cytoskeleton integrity and differentiation functions of multiple tissues. In metabolically stressed skin keratinocytes, cytoplasmic CLIC4 is S-nitrosylated and translocates to the nucleus where it enhances transforming growth factor-ß (TGF-ß) signaling by protecting phospho-Smad 2 and 3 from dephosphorylation. CLIC4 expression is diminished in multiple human epithelial cancers, and the protein is excluded from the nucleus. We now show that CLIC4 expression is reduced in chemically induced mouse skin papillomas, mouse and human squamous carcinomas and squamous cancer cell lines, and the protein is excluded from the nucleus. The extent of reduction in CLIC4 coincides with progression of squamous tumors from benign to malignant. Inhibiting antioxidant defense in tumor cells increases S-nitrosylation and nuclear translocation of CLIC4. Adenoviral-mediated reconstitution of nuclear CLIC4 in squamous cancer cells enhances TGF-ß-dependent transcriptional activity and inhibits growth. Adenoviral targeting of CLIC4 to the nucleus of tumor cells in orthografts inhibits tumor growth, whereas elevation of CLIC4 in transgenic epidermis reduces de novo chemically induced skin tumor formation. In parallel, overexpression of exogenous CLIC4 in squamous tumor orthografts suppresses tumor growth and enhances TGF-ß signaling. These results indicate that CLIC4 suppresses the growth of squamous cancers, that reduced CLIC4 expression and nuclear residence detected in cancer cells is associated with the altered redox state of tumor cells and the absence of detectable nuclear CLIC4 in cancers contributes to TGF-ß resistance and enhances tumor development.

Elevated progesterone receptor membrane component 1/sigma-2 receptor levels in lung tumors and plasma from lung cancer patients.

Cancer is one of the leading causes of death, and there is an urgent need for new biomarkers and therapeutic targets. The progesterone receptor membrane component 1 (Pgrmc1) protein is upregulated in multiple types of cancer, and Pgrmc1 is required for tumor cell proliferation, motility and tumor formation in vivo. Furthermore, a small molecule inhibitor of Pgrmc1 suppressed the growth of lung, breast and cervical cancer cell lines. Recently, Pgrmc1 was identified as the sigma-2 receptor, a putative type of opioid receptor, and sigma-2 receptors are induced in cancers. However, Pgrmc1 shares no homology with known opioid or hormone receptors but is related to cytochrome b(5), and Pgrmc1 binds to heme and has reducing activity. In this study, we have analyzed Pgrmc1 levels in clinical tumor samples from squamous cell lung cancers (SCLC) and lung adenocarcinomas compared to corresponding nonmalignant tissue. Pgrmc1 levels increased significantly (p ≤ 0.05) in 12/15 SCLC samples and was elevated in poorly differentiated tumors. Pgrmc1 was highly expressed in SCLC cell lines, and SCLC cell survival was inhibited by siRNA knockdown of Pgrmc1 or the Pgrmc1 inhibitor AG-205. In adenocarcinomas, 6/15 tumors significantly had elevated Pgrmc1 levels, which correlated with patient survival. Pgrmc1 localizes to secretory vesicles in cancer cells, and Pgrmc1 was secreted by lung cancer cells. Furthermore, Pgrmc1 was significantly elevated in the plasma of lung cancer patients compared to noncancer patients. Together, the results demonstrate that Pgrmc1 is a potential tumor and serum biomarker, as well as a therapeutic target, for lung cancer.