Involvement of p53-dependent apoptosis signal in antitumor effect of Colchicine on human papilloma virus (HPV)-positive human cervical cancer cells.

Colchicine, a plant-derived alkaloid with relatively low toxicity on normal human epidermal keratinocytes (HEKn), has selective inhibitory effect on the growth of CaSki (HPV16-positive) and HeLa (HPV18-positive) human cervical cancer cell lines via the induction of apoptosis. Colchicine (2.5, 5.0 and 10.0 ng/ml) significantly reduced the expression of human papilloma virus (HPV) 16 E6/E7 mRNA and protein in CaSki and HeLa cells. Moreover, reduced expression of E6 and E7 induced by Colchicine resulted in the up-regulation of tumor suppressor proteins, p53 and Rb, as well as down-regulation of phospho Rb (pRb) protein. In addition, Bax, cytosolic cytochrome c and cleaved caspase-3 protein were increased while Bcl-2 protein was decreased significantly by 48 h of Colchicine treatment. These results implied that Colchicine could be explored as a potent candidate agent for the treatment and prevention of HPV-associated cervical cancer without deleterious effects.

Altered Rb, p16, and p53 expression is specific for porocarcinoma relative to poroma.

BACKGROUND: Porocarcinomas are rare aggressive carcinomas that harbor tumor suppressor mutations and must be distinguished from benign entities such as poromas. METHODS: To determine whether altered expression of these genes was diagnostically informative, we examined p53, Rb, and p16 staining patterns in 15 poromas and 16 porocarcinomas. RESULTS: Poromas consistently displayed diffuse strong expression of Rb in all but one case that displayed focal loss (1/15, 7%), and no evidence of aberrancy in p53 or p16. Porocarcinomas displayed diffuse or focal loss of Rb expression in 9/16 (56%) cases, diffuse loss or overexpression of p53 in 8/15 (53%), and diffuse loss or overexpression of p16 in 6/14 (43%). Diffuse aberrancy in p53 and Rb expression correlated with tumor mutations in TP53 and RB1, respectively, whereas focal Rb loss was associated with wild type RB1. Diffuse p16 overexpression correlated with Rb loss rather than CDKN2A mutation. For porocarcinomas with all three stains evaluable, 10/13 (77%) displayed aberrancy in at least one marker. CONCLUSIONS: Our findings suggest that altered p53, p16, and/or Rb expression is relatively specific to porocarcinoma in comparison with poroma. Technical limitations of this panel, including possible focal Rb loss, must be kept in mind, especially in limited samples.

The ING1a model of rapid cell senescence.

Replicative capacity of normal human cells decreases as telomeric sequence is lost at each division. It is believed that when a subset of chromosomes reach a critically short length, an ATM-initiated and p53-mediated transcriptional response inhibits cell growth, promoting cell senescence. In addition to loss of telomeric sequence, senescence can be induced by other stresses including ionizing radiation, oxidative damage, chemical crosslinkers like the chemotherapeutic agent cisplatin, as well as overactivation of oncogenes and tumor suppressors. Our group found that the expression of an isoform of the INhibitor of Growth 1 gene called ING1a increases approximately 10-fold as fibroblasts approach senescence and that forced expression rapidly induces a senescent phenotype in primary diploid fibroblasts, epithelial and endothelial cells that resembles replicative senescence by most physical and biochemical measures. ING1a induces these changes through strongly inhibiting endocytosis to block mitogen signaling by inducing the expression of intersectin 2, a key scaffolding protein of the endosomal pathway. This, in turn increases the expression of Rb and of p57Kip2 and p16INK4a that serve to maintain Rb is an active, growth inhibitory state. The ING1a model is currently being used to better understand the mechanism(s) responsible for activating Rb to enforce the senescent state.

SUV39H1/DNMT3A-dependent methylation of the RB1 promoter stimulates PIN1 expression and melanoma development.

Melanoma is among the most aggressive and treatment-resistant human cancers. Aberrant histone H3 methylation at Lys 9 (H3K9) correlates with carcinogenic gene silencing, but the significance of suppressor of variegation 3-9 homolog 1 (SUV39H1), an H3K9-specific methyltransferase, in melanoma initiation and progression remains unclear. Here, we show that SUV39H1-mediated H3K9 trimethylation facilitates retinoblastoma ( RB) 1 promoter CpG island methylation by interacting with DNA methyltransferase 3A and decreasing RB mRNA and protein in melanoma cells. Reduced RB abundance, in turn, impairs E2F1 transcriptional inhibition, leading to increased peptidyl-prolyl cis-trans isomerase never-in-mitosis A (NIMA)-interacting 1 (PIN1) levels, human keratinocyte neoplastic cell transformation, and melanoma tumorigenesis via enhanced rapidly accelerated fibrosarcoma 1(RAF1)-MEK-ERK signaling pathway activation. In a synergistic model with B16-F1 murine melanoma cells, SUV39H1 and PIN1 overexpression increased melanoma growth, which was abrogated by their inhibition in SUV39H1-overexpressing B16-F1 mice. SUV39H1 also positively correlated with PIN1 expression in human melanoma. Our studies establish SUV39H1 as an oncogene in melanoma and underscore the role of chromatin factors in regulating tumorigenesis.-Kim, G., Kim, J.-Y., Lim, S.-C., Lee, K. Y., Kim, O., Choi, H. S. SUV39H1/DNMT3A-dependent methylation of the RB1 promoter stimulates PIN1 expression and melanoma development.

The critical role of p16/Rb pathway in the inhibition of GH3 cell cycle induced by T-2 toxin.

T-2 toxin is a worldwide trichothecenetoxin and can cause various toxicities.T-2 toxin is involved in G1 phase arrest in several cell lines but molecular mechanism is still not clear. In present study, we used rat pituitary GH3 cells to investigate the mechanism involved in cell cycle arrest against T-2 toxin (40 nM) for 12, 24, 36 and 48 h as compared to control cells. GH3 cells showed a considerable increase in reactive oxygen species (ROS) as well as loss in mitochondrial membrane potential (△Ym) upon exposure to the T-2 toxin. Flow cytometry showed a significant time-dependent increase in percentage of apoptotic cells and gel electrophoresis showed the hallmark of apoptosis oligonucleosomal DNA fragmentation. Additionally, T-2 toxin-induced oxidative stress and DNA damage with a time-dependent significant increased expression of p53 favors the apoptotic process by the activation of caspase-3 in T-2 toxin treated cells. Cell cycle analysis by flow cytometry revealed a time-dependent increase ofG1 cell population along with the significant time-dependent up-regulation of mRNA and protein expression of p16 and p21 and significant down-regulation of cyclin D1, CDK4, and p-RB levels further verify the G1 phase arrest in GH3 cells. Morphology of GH3 cells by TEM clearly showed the damage and dysfunction to mitochondria and the cell nucleus. These findings for the first time demonstrate that T-2 toxin induces G1 phase cell cycle arrest by the involvement of p16/Rb pathway, along with ROS mediated oxidative stress and DNA damage with p53 and caspase cascade interaction, resulting in apoptosis in GH3 cells.

Cell Cycle Protein Expression in Neuroendocrine Tumors: Association of CDK4/CDK6, CCND1, and Phosphorylated Retinoblastoma Protein With Proliferative Index.

OBJECTIVES: Dysregulation of the cell cycle has been observed and implicated as an etiologic factor in a range of human malignancies, but remains relatively unstudied in neuroendocrine tumors (NETs). We evaluated expression of key proteins involved in cell cycle regulation in a large cohort of NETs. METHODS: We evaluated immunohistochemical expression of CDKN1B, CDKN1A, CDKN2A, CDK2, CDK4, CDK6, cyclin D1, cyclin E1, and phosphorylated retinoblastoma protein (phospho-RB1) in a cohort of 267 patients with NETs. We then explored associations between cell cycle protein expression, mutational status, histologic features, and overall survival. RESULTS: We found that high expression of CDK4, CDK6, CCND1, and phospho-RB1 was associated with higher proliferative index, as defined by MKI67. We additionally observed a trend toward shorter overall survival associated with low expression of CDKN1B. This association seemed strongest in SINETs (multivariate hazards ratio, 2.04; 95% confidence interval, 1.06-3.93; P = 0.03). We found no clear association between CDKN1B mutation and protein expression. CONCLUSIONS: Our results suggest that dysregulation and activation of the CDK4/CDK6-CCND1-phospho-RB1 axis is associated with higher proliferative index in NETs. Investigation of the therapeutic potential of CDK4/CDK6 inhibitors in higher grade NETs is warranted.

Rb Loss and KRAS Mutation Are Predictors of the Response to Platinum-Based Chemotherapy in Pancreatic Neuroendocrine Neoplasm with Grade 3: A Japanese Multicenter Pancreatic NEN-G3 Study.

Purpose: Patients with pancreatic neuroendocrine neoplasm grade-3 (PanNEN-G3) show variable responses to platinum-based chemotherapy. Recent studies indicated that PanNEN-G3 includes well-differentiated neuroendocrine tumor with G3 (NET-G3). Here, we examined the clinicopathologic and molecular features of PanNEN-G3 and assessed the responsiveness to chemotherapy and survival.Experimental Design: A total of 100 patients with PanNEN-G3 were collected from 31 institutions, and after central review characteristics of each histologic subtype [NET-G3 vs. pancreatic neuroendocrine carcinoma (NEC-G3)] were analyzed, including clinical, radiological, and molecular features. Factors that correlate with response to chemotherapy and survival were assessed.Results: Seventy patients analyzed included 21 NETs-G3 (30%) and 49 NECs-G3 (70%). NET-G3 showed lower Ki67-labeling index (LI; median 28.5%), no abnormal Rb expression (0%), and no mutated KRAS (0%), whereas NEC-G3 showed higher Ki67-LI (median 80.0%), Rb loss (54.5%), and KRAS mutations (48.7%). Chemotherapy response rate (RR), platinum-based chemotherapy RR, and prognosis differed significantly between NET-G3 and NEC-G3. Chemotherapeutic outcomes were worse in NET-G3 (P < 0.001). When we stratified PanNEN-G3 with Rb and KRAS, PanNENs-G3 with Rb loss and those with mutated KRAS showed significantly higher RRs to platinum-based chemotherapy than those without (Rb loss, 80% vs. normal Rb, 24%, P = 0.006; mutated KRAS, 77% versus wild type, 23%, P = 0.023). Rb was a predictive marker of response to platinum-based chemotherapy even in NEC-G3 (P = 0.035).Conclusions: NET-G3 and NEC-G3 showed distinct clinicopathologic characteristics. Notably, NET-G3 does not respond to platinum-based chemotherapy. Rb and KRAS are promising predictors of response to platinum-based chemotherapy for PanNEN-G3, and Rb for NEC-G3. Clin Cancer Res; 23(16); 4625-32. ©2017 AACR.

Growth arrest of vascular smooth muscle cells in suspension culture using low-acyl gellan gum.

The proliferation of vascular smooth muscle cells (SMCs) causes restenosis in biomaterial vascular grafts. The purposes of this study were to establish a suspension culture system for SMCs by using a novel substrate, low-acyl gellan gum (GG) and to maintain SMCs in a state of growth inhibition. When SMCs were cultured in suspension with GG, their proliferation was inhibited. Their viability was 70% at day 2, which was maintained at more than 50% until day 5. In contrast, the viability of cells cultured in suspension without GG was 5.6% at day 2. By cell cycle analysis, the ratio of SMCs in the S phase when cultured in suspension with GG was lower than when cultured on plastic plates. In SMCs cultured in suspension with GG, the ratio of phosphorylated retinoblastoma (Rb) protein to Rb protein was decreased and p27Kip1 expression was unchanged in comparison with SMCs cultured on plastic plates. In addition, SMCs could be induced to proliferate again by changing the culture condition from suspension with GG to plastic plates. These results suggest that our established culturing method for SMCs is useful to maintain SMCs in a state of growth inhibition with high viability.

Mechanism of action of EBV, Bcl-2, p53, c-Myc and Rb in non-Hodgkin's lymphoma.

OBJECTIVE: The aim of the present study is to explore the mechanism of action of several proteins, including Epstein-Barr virus (EBV), B-cell lymphoma (Bcl)-2, p53, c-Myc and retinoblastoma (Rb), in Non-Hodgkin's lymphoma (NHL). PATIENTS AND METHODS: Between July 2010 and July 2015, samples of 142 patients with pathologically confirmed NHL which presented at our institution were included in the observation group. In addition, samples from 55 patients with hyperplastic lymphadenitis presented during the same period were enrolled as control group. The expressions of EBV (+), p53(+), Bcl-2(+), Rb(-) and c-Myc(+) were determined and compared PATIENTS AND METHODS: Between July 2010 and July 2015, samples of 142 patients with pathologically confirmed NHL which presented at our institution were included in the observation group. In addition, samples from 55 patients with hyperplastic lymphadenitis presented during the same period were enrolled as control group. The expressions of EBV (+), p53(+), Bcl-2(+), Rb(-) and c-Myc(+) were determined and compared among different subtypes and stages of NHLs of observation group. Besides, the correlation of EBV with p53, Bcl-2, Rb and c-Myc were investigated in NHLs of observation group. RESULTS: In the observation group, the expression rates of EBV(+), p53(+), Bcl-2(+), Rb(-), and c-Myc(+) were significantly higher than those, respectively, in the control group (p < 0.05). No significant correlation was observed between EBV expression and the expressions of p53, Bcl-2, Rb and c-Myc in the observation group (p > 0.05). The expression rates of p53(+) and Bcl-2(+) were significantly higher in aggressive and highly-aggressive NHLs than in indolent NHLs of the observation group (p < 0.05). The expressions of EBV(+), p53(+), Bcl-2(+), Rb(-), and c-Myc(+) were significantly higher in stage III-IV NHLs than in stage I-II NHLs (p < 0.05). CONCLUSIONS: The expressions of EBV(+), p53(+), Bcl-2(+), Rb(-), and c-Myc(+) are closely associated with NHL pathogenesis. Expressions of these proteins are higher in later stages of NHLs, and expressions of p53(+) and Bcl-2(+) are higher in more aggressive NHLs.

RBEL1 is required for osteosarcoma cell proliferation via inhibiting retinoblastoma 1.

Osteosarcoma is the most common type of primary malignant tumor of the bone. However, mechanisms underlying osteosarcoma cell proliferation are poorly understood. The present study shows that RBEL1, a newly identified Rab-like GTPase, may be a key regulator of osteosarcoma cell proliferation. Knockdown of RBEL1 in osteosarcoma cells resulted in impaired colony formation and cell proliferation. Cell cycle analysis suggested that RBEL1 depletion induced G1-S arrest in osteosarcoma cells. Furthermore, it was demonstrated that retinoblastoma 1 (Rb) was upregulated and activated following RBEL1 knockdown. In addition, Rb inhibitory downstream targets, such as cyclin A2, cyclin D1, c-Myc and cyclin-dependent kinase 2, were downregulated. Rb knockdown reversed RBEL1 depletion-induced tumor suppressive effects. In conclusion, the present results suggest that RBEL1 modulates cell proliferation and G1­S transition by inhibiting Rb in osteosarcoma. These results suggest a potential therapeutic target in osteosarcoma.

From HPV-positive towards HPV-driven oropharyngeal squamous cell carcinomas.

The incidence of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC), which is both biologically and clinically distinct from tobacco- and alcohol-related OPSCC, is dramatically increasing. The finding that individuals with HPV-positive local/regionally advanced OPSCC have a significantly better prognosis than their negative counterparts have led to efforts to de-escalate treatment in those patients to avoid serious side effects and to improve their long-term quality of life, while maintaining treatment efficacy. Identifying diagnostic tests that are able to distinguish cancers etiologically associated with HPV is thus becoming a pressing challenge for researchers. The purpose of this review is to provide an overview of the diagnostic tools presently available to evaluate HPV status in patients with OPSCC and, in particular, to discuss their strengths and weaknesses in identifying those infections that are the real driving force in the oropharyngeal carcinogenesis process.

miR-449a enhances radiosensitivity through modulating pRb/E2F1 in prostate cancer cells.

miR-449a, a novel tumor suppressor, is deregulated in various malignancies, including prostate cancer. Overexpression of miR-449a induces cell cycle arrest, apoptosis, and senescence, but its role in response to ionizing radiation and underlying molecular mechanism are still unknown. Here, we report that miR-449a enhances radiation-induced G2/M phase arrest and apoptosis through modulating pRb/E2F1 and sensitizes prostate cancer cells to X-ray radiation. In wild-type Rb PC-3 cells, overexpression of miR-449a enhances radiation-induced G2/M arrest and apoptosis and promotes the sensitivity to X-ray radiation. While mutant Rb DU-145 cells are resistant to the X-ray radiation despite in the presence of miR-449a. The cell cycle distribution of DU-145 cells is not significantly altered by miR-449a in the response to ionizing radiation. Furthermore, elevated miR-449a downregulates cell cycle regulator CDC25A and oncogene HDAC1. By targeting genes involved in controlling pRb/E2F1 activity, miR-449a regulates cell cycle progression and apoptosis and consequently enhances the radiosensitivity of PC-3 cells. Thus, miR-449a, as a miRNA component of the Rb pathway, promotes the radiosensitivity of PC-3 cells through regulating pRb/E2F1.

Differential regulation and synthetic lethality of exclusive RB1 and CDKN2A mutations in lung cancer.

Genetic alterations in lung cancer are distinctly represented in non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC). Mutation of the RB1 and CDKN2A genes, which are tightly associated with cell cycle regulation, is exclusive to SCLC and NSCLC cells, respectively. Through the systematic analysis of transcriptome and proteome datasets for 318 cancer cell lines, we characterized differential gene expression and protein regulation in RB1-mutant SCLC and CDKN2A-mutant NSCLC. Many of the genes and proteins associated with RB1-mutant SCLC cell lines belong to functional categories of gene expression and transcription, whereas those associated with CDKN2A-mutant NSCLC cell lines were enriched in gene sets of the extracellular matrix and focal adhesion. These results indicate that the loss of RB1 and CDKN2A function induces distinctively different signaling cascades in SCLC and NSCLC cells. In addition, knockdown of the RB1 gene in CKDN2A-mutant cell lines (and vice versa) synergistically inhibits cancer cell proliferation. The present study on the exclusive role of RB1 and CDKN2A mutations in lung cancer subtypes demonstrates a synthetic lethal strategy for cancer regulation.

The anti-diabetic drug metformin inhibits pancreatic cancer cell proliferation in vitro and in vivo: Study of the microRNAs associated with the antitumor effect of metformin.

Recent studies suggest that metformin, which is a commonly used oral anti-hyperglycemic agent of the biguanide family, may reduce cancer risk and improve prognosis, yet the detailed mechanisms by which metformin affects various types of cancers, including pancreatic cancer, remain unknown. The aim of the present study was to evaluate the effects of metformin on human pancreatic cancer cell proliferation in vitro and in vivo, and to study microRNAs (miRNAs) associated with the antitumor effect of metformin. We used the human pancreatic cancer cell lines Panc1, PK1 and PK9 to study the effects of metformin on human pancreatic cancer cells. Athymic nude mice bearing xenograft tumors were treated with or without metformin. Tumor growth was recorded after 5 weeks, and the expression of cell cycle-related proteins was determined. In addition, we used miRNA microarray tips to explore the differences in the levels of miRNAs in Panc1 cells and xenograft tumors treated with metformin or without. Metformin inhibited the proliferation of Panc1, PK1 and PK9 cells in vitro. This inhibition was accompanied by a strong decrease in G1 cyclins (particularly in cyclin D1) and retinoblastoma protein (Rb) phosphorylation. In addition, metformin reduced the phosphorylation of epidermal growth factor receptor (EGFR), particularly the phosphorylation of EGFR at Tyr845, and insulin-like growth factor 1 receptor (IGF-1R) in vitro and in vivo. miRNA expression was markedly altered by the treatment with metformin in vitro and in vivo. Our results revealed that metformin inhibits human pancreatic cancer cell proliferation and tumor growth, possibly by suppressing the cell cycle-related molecules via alteration of miRNAs.

Loss of the retinoblastoma tumor suppressor correlates with improved outcome in patients with lung adenocarcinoma treated with surgery and chemotherapy.

The retinoblastoma tumor suppressor pathway is frequently inactivated in human cancer, enabling unrestrained proliferation. Most cancers, however, maintain expression of a wild-type (WT) retinoblastoma tumor suppressor protein (pRB). It is generally in a hyperphosphorylated state (ppRB) because of mutations in upstream regulators such as p16 and cyclin D. Hyperphosphorylated ppRB is considered inactive, although data are emerging that suggest it can retain some function. To test the clinical relevance of pRB status, we obtained archival tissue sections from 91 cases of lung adenocarcinoma resected between 2003 and 2008. All cases received platinum doublet chemotherapy, and the median survival was 5.9 years. All cases were assessed for pRB and ppRB using immunohistochemistry and quantified based on intensity of signal and proportion of positive cells. pRB expression was lost in 15% of lung adenocarcinoma cases. In tumors that did not express pRB, the survival rate was significantly improved (hazard ratio, 0.21; 95% confidence interval, 0.06-0.69; P = .01) in comparison to tumors that express pRB. pRB status was found to be an independent predictor of overall survival on multivariate analysis (hazard ratio, 0.22; 95% confidence interval, 0.07-0.73; P = .01) along with increased stage and age. pRB status did not alter baseline levels of apoptotic or proliferative markers in these tumors, but the DNA damage response protein 53BP1 was higher in cancers with high levels of pRB. In summary, loss of pRB expression is associated with improved survival in patients treated with surgical resection and chemotherapy. This may be useful in classifying patients at greatest benefit for aggressive treatment regimes.

Inactivation of Retinoblastoma Protein (Rb1) in the Oocyte: Evidence That Dysregulated Follicle Growth Drives Ovarian Teratoma Formation in Mice.

The origin of most ovarian tumors is undefined. Here, we report development of a novel mouse model in which conditional inactivation of the tumor suppressor gene Rb1 in oocytes leads to the formation of ovarian teratomas (OTs). While parthenogenetically activated ooctyes are a known source of OT in some mutant mouse models, enhanced parthenogenetic propensity in vitro was not observed for Rb1-deficient oocytes. Further analyses revealed that follicle recruitment and growth is disrupted in ovaries of mice with conditional inactivation of Rb1, leading to abnormal accumulation of secondary/preantral follicles. These findings underpin the concept that miscues between the germ cell and somatic compartments cause premature oocyte activation and the formation of OTs. Furthermore, these results suggest that defects in folliculogenesis and a permissive genetic background are sufficient to drive OT development, even in the absence of enhanced parthenogenetic activation. Thus, we have discovered a novel role of Rb1 in regulating the entry of primordial oocytes into the pool of growing follicles and signaling between the oocyte and granulosa cells during the protracted process of oocyte growth. Our findings, coupled with data from studies of other OT models, suggest that defects in the coordinated regulation between growth of the oocyte and somatic components in follicles are an underlying cause of OT formation.

Resibufogenin Induces G1-Phase Arrest through the Proteasomal Degradation of Cyclin D1 in Human Malignant Tumor Cells.

Huachansu, a traditional Chinese medicine prepared from the dried toad skin, has been used in clinical studies for various cancers in China. Resibufogenin is a component of huachansu and classified as bufadienolides. Resibufogenin has been shown to exhibit the anti-proliferative effect against cancer cells. However, the molecular mechanism of resibufogenin remains unknown. Here we report that resibufogenin induces G1-phase arrest with hypophosphorylation of retinoblastoma (RB) protein and down-regulation of cyclin D1 expression in human colon cancer HT-29 cells. Since the down-regulation of cyclin D1 was completely blocked by a proteasome inhibitor MG132, the suppression of cyclin D1 expression by resibufogenin was considered to be in a proteasome-dependent manner. It is known that glycogen synthase kinase-3ß (GSK-3ß) induces the proteasomal degradation of cyclin D1. The addition of GSK-3ß inhibitor SB216763 inhibited the reduction of cyclin D1 caused by resibufogenin. These effects on cyclin D1 by resibufogenin were also observed in human lung cancer A549 cells. These findings suggest that the anti-proliferative effect of resibufogenin may be attributed to the degradation of cyclin D1 caused by the activation of GSK-3ß.

Comparative analysis of Rb1, P16 and ER as diagnostic, prognostic and potential targets for therapeutic agents in ovarian epithelial tumors: an immunohistochemical study of 130 ovarian carcinomas.

BACKGROUND: Deregulation of CDK4/6, cyclin D/P16 and retinoblastoma (Rb) are known aberrations in certain malignancies. There has been a recent interest in exploring the combination of letrozole and CDK4/6 inhibitors in recurrent ER+ ovarian cancers. METHODS: This study aimed to determine the frequency of expression of Rb1, P16 and ER in ovarian epithelial tumors by immunohistochemistry. RESULTS: Co-expression of all 3 markers studied was seen in 10% of high grade serous carcinoma (HGSC) and low grade serous carcinoma (LGSC). Coordinate expression of Rb1+ and ER+ in HGSC and LGSC was seen in 67% of grade 1/2 vs. 44 % of grade three tumors (p < 0.05). The reverse was true with positive P16 staining in 73% of grade three vs. 32% of grade 1/2 tumors (p < 0.001). CONCLUSIONS: Coordinate pattern of Rb1+ and ER+ in HGSC and LGSC is 19 and 50%, respectively. Rb1 and P16 show inverse expression pattern according to tumor grade with more frequent Rb1 in low grade vs. more frequent P16 in grade 3 tumors. These data provide a rational basis for clinical trials that aim to target these proteins.

Combinations of indole-3-carbinol and silibinin suppress inflammation-driven mouse lung tumorigenesis by modulating critical cell cycle regulators.

Chronic inflammation is an important risk factor for lung cancer. Therefore, identification of chemopreventive agents that suppress inflammation-driven lung cancer is indispensable. We studied the efficacy of combinations of indole-3-carbinol (I3C) and silibinin (Sil), 20 µmol/g diet each, against mouse lung tumors induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and driven by lipopolysaccharide (LPS), a potent inflammatory agent and constituent of tobacco smoke. Mice treated with NNK + LPS developed 14.7±4.1 lung tumors/mouse, whereas mice treated with NNK + LPS and given combinations of I3C and Sil had 7.1±4.5 lung tumors/mouse, corresponding to a significant reduction of 52%. Moreover, the number of largest tumors (>1.0mm) was significantly reduced from 6.3±2.9 lung tumors/mouse in the control group to 1.0±1.3 and 1.6±1.8 lung tumors/mouse in mice given I3C + Sil and I3C alone, respectively. These results were paralleled by significant reductions in the level of proinflammatory and procarcinogenic proteins (pSTAT3, pIκBα and COX-2) and proteins that regulate cell proliferation (pAkt, cyclin D1, CDKs 2, 4, 6 and pRB). Further studies in premalignant bronchial cells showed that the antiproliferative effects of I3C + Sil were higher than the individual compounds and these effects were mediated by targeting cyclin D1, CDKs 2, 4 and 6 and pRB. I3C + Sil suppressed cyclin D1 by reducing its messenger RNA level and by enhancing its proteasomal degradation. Our results showed the potential lung cancer chemopreventive effects of I3C + Sil in smokers/former smokers with chronic pulmonary inflammatory conditions.

Emerging links between E2F control and mitochondrial function.

The family of E2F transcription factors is the key downstream target of the retinoblastoma tumor suppressor protein (pRB), which is frequently inactivated in human cancer. E2F is best known for its role in cell-cycle regulation and triggering apoptosis. However, E2F binds to thousands of genes and, thus, could directly influence a number of biologic processes. Given the plethora of potential E2F targets, the major challenge in the field is to identify specific processes in which E2F plays a functional role and the contexts in which a particular subset of E2F targets dictates a biologic outcome. Recent studies implicated E2F in regulation of expression of mitochondria-associated genes. The loss of such regulation results in severe mitochondrial defects. The consequences become evident during irradiation-induced apoptosis, where E2F-deficient cells are insensitive to cell death despite induction of canonical apoptotic genes. Thus, this novel function of E2F may have a major impact on cell viability, and it is independent of induction of apoptotic genes. Here, we discuss the implications of these findings in cancer biology.