This study has presented novel applications for an amino- bisphosphonate, ZA, currently used clinically to ameliorate bone metastasis. The strategy forthcoming involves treatment of either CIN or established cervical cancer so as to elicit stasis or regression rather than progression and growth. We demonstrate that ZA inhibits angiogenesis in premalignant lesions (CIN-3) and in cervical tumors by targeting both macrophage expres- sion and proteolytic activity of an MMP, MMP-9. Therapeutic regimes using ZA impaired progression of premalignant lesions to invasive carcinoma and antagonized the growth of preexist- ing cervical tumors. These results are likely to be relevant to human cervical carcinogenesis. MMP-9 has been shown to be upregulated in CIN-3 lesions and cervical cancers in humans (refs. 7, 8, 36 and our unpublished observations), and elevated expression and activity correlate with poor prognosis for subse- quent metastasis and tumor recurrence (37). Notably, MMP-9 and the related MMP-2 have been detected in both epithelial and stromal compartments in human cervical lesions (8, 36). We found MMP-9 expression exclusively in the stroma in the mouse model in infiltrating macrophages. MMP-9 is similarly expressed by macrophages (and other inflammatory cells), infiltrating both stroma and epithelia of human CIN-2/3 and cervical cancers (E. Giraudo and D. Hanahan, unpublished data); the reasons that MMP-9–expressing macrophages do not infiltrate the neoplas- tic epithelial compartments in the mouse model are presently unclear. We performed a genetic test to assess the importance of this and a related protease by analyzing the cervical cancer phe-
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The expressions of DNMT1 and CCNA1 mRNA in the process of cervical carcinogenesis was measured by qPCR (Figure 6). The amplification product was single and had a strong specificity. As shown in Table 5, the positive rates of DN- MT1 mRNA in the SCC, HSIL, and LSIL groups were 73.33%, 63.33% and 43.33%, respective- ly, which were significantly higher than they were in the control group (16.67%) (P<0.05). The DNMT1 mRNA in the SCC group was obvi- ously higher than it was in the LSIL group (P=0.018), but not significantly higher than it was in the HSIL group (P=0.405). As shown in Figure 7, the relative expression of DNMT1 mRNA significantly increased with the aggrava- tion of cervical lesions (P<0.05). This result agreed with the immunohistochemistry and Western blot results of the DNMT1 protein. On the contrary, the positive expression rates of the CCNA1 mRNA were 23.33%, 63.33%, 93.33%, and 100.00% in the SCC, HSIL, LSIL, and control groups (Table 6), respectively. The CCNA1 mRNA in the SCC and HSIL groups was significantly lower than it was in the LSIL and control groups (P<0.05). The relative expres- sion of CCNA1 mRNA was reduced in the pro- cess of cervical carcinogenesis (Figure 8). There was no significant decrease in the rela- tive expression of CCNA1 mRNA from the con- trol group to the LSIL group (P=0.65), and a significant decrease from the LSIL group to the HSIL group (P<0.05), and the HSIL group to the SCC group (P<0.05).
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To investigate the association between polymorphisms (SNP) in the p53 and murine double minute 2 homolog (MDM2) promoter 309 in cervical carcinogenesis. SNP at p53 codon 72 poly- morphisms and MDM2 promoter 309 (T/G) together with human papillomavirus (HPV) types were examined in a total of 187 cervical smear samples using real time PCR. 27 cases with HPV types 16 and/or 18 had significantly higher frequency of the TG + GG genotype and G allele than 56 with other types of high-risk HPV (P = 0.0136). 48 cases with HPV types 52 and/or 58 had signifi- cantly higher frequency of the TG + GG genotype and G allele than 56 with other types of high-risk HPV (P = 0.001). Our studies have demonstrated that the frequency of G allele in MDM2 promoter 309 increased from LSIL to HSIL and that there was an increased OR for G allele in HSIL cases with high-risk HPV types including 52 and 58. It is known that geographically different oncogenic HPV types 52 and 58 are more prevalent than 16 and 18 in a Japanese population.
Epigenetic alterations, unlike genetic mutations, may be reversed by inhibiting the associated enzymes, and need to be evaluated as therapeutic modalities for HPV-associated lesions and cancers. Currently, two main classes of epigenetic drugs, methylation in- hibitors and HDAC inhibitors, are in clinical trials for the treatment of cancer. Phase I study of Hydral- azine in cervical cancer patients showed that differ- ent doses of hydralazine treatment was well-tolerated and effective to demethylate and reactivate the ex- pression of eight tumor suppressor genes without af- fecting global DNA methylation . Trichosanthin (TCS), a bioactive component isolated from a Chinese medicinal herb has been shown to have the capacity of re- storing the expression of methylation-silenced tumor sup- pressor genes . Valproic acid (VPA), an effective inhibitor of histone deacetylases, has been shown to modulate multiple cellular pathways including cell cycle arrest, apoptosis, angiogenesis, metastasis. The antitumor effect of VPA in cervical cancer may be caused due to ei- ther hyperacetylation of p53 protein protecting it from degradation by E6 and increasing p53 activity or via inhib- ition of Akt1 and Akt2 gene expression which resulted in Akt deactivation and apoptotic cell death . Apicidin, a cyclic peptide HDAC inhibitor, was found to selectively downregulate DNA methyltransferase 1  whereas tri- chostatin A (TSA), a classical HDAC inhibitor, was shown to inhibit DNA methyltransferase 3A . In addition, the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) synergistically induces apoptosis in HeLa cer- vical cancer cells with bortezomib by activating caspase-3 and increasing the ratio of bax/bcl-2 ex- pression . Strategies targeting epigenetic aberra- tions appear promising therapeutic modalities for regulating cervical carcinogenesis.
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During HPV life cycle, the viral replication and the ex- pression of viral genes are closely tied to the differenti- ation program of host epithelial cell . In this process, CK network appears to be reorganized and regulated by viral proteins . Viral E4 protein is thought to partici- pate in CK disruption, virus release and transmission . Different viral protein expression according to the layer of CIN may be related with different CK expression patterns depending on the layer and the grade of CIN [9, 16]. In- deed the expression of E2 and E7, represented by p16 is mutually exclusive and shows topological distinction and association with specific CKs in CIN . Comprehensive expression of CK subtypes had been described in CIN and cancers , but the association between HPV status and CK subtypes, especially CK7 and CK19 in cervical cancer has not been studied. Thus, in this study, we analyzed the expression of CK7, CK19, and p16, and physical status of HPV in CIN3 and SCC to investigate their possible associ- ation in cervical carcinogenesis.
The tumor suppressor protein p53 is encoded by the TP53 gene the function of which is to induce cell cycle arrest and apoptosis with sev- eral target genes, including p21, BAX and Fas . The half-life of p53 is very short, and is mainly regulated by ubiquitination and protea- somal degradation. Several E3 ubiquitin ligas- es have been shown to directly poly-ubiquiti- nate the p53 protein and induce its proteasom- al degradation, including MDM2, RNF31 and COP1 [19, 20]. E3 ubiquitin ligases that indi- rectly modify p53 are highly expressed in can- cers and thought to be involved in carcinogen- esis by suppressing p53 function . In the present study, we observed the up-regulation of the p53 protein level after silencing RNF113A in cervical cancer cell lines, which indicates that RNF113A may also play a role as a modula- tor of P53. p53 acts as a tumor suppressor in many tumor types to induce apoptosis. Our
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model was applied to calculate the association of methylated CDKN2A with cervical cancer. Our results indicated that the frequency of methylated CDKN2A in cervical cancers was significantly higher than in controls (OR = 23.67, 95% CI = 15.54–36.06, Figure 2). Similar results could be observed by switching to the M–H model to recalculate the pooled OR (Figure 2), and a sensitivity analysis was performed (Table 2), suggesting the stability and credibility of our results. However, the Begg’s test implied the presence of publication bias (P = 0.02, Figure 3). To adjust for this, a trim-and-fill method was implemented (Figure 4). After filling eight missing studies the pooled OR was similar to our previous results, indicating the stability of our results. Furthermore, we applied an N fs to assess the efficacy of the meta-analysis (N fs0.05 = 1,744, N fs0.01 = 859), which indicated that our results were robust.
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circ_0084927 was selected as the circRNA of interest in CC We analyzed the GSE102686 data series using the GEO2R algorithm and identified 21 differentially expressed genes (DEGs). The top five most significantly up-regu- lated circRNAs included circ_0084927, circ_0106385, circ_0099591, circ_0081723, and circ_0084912 (Fig. 1a). The expression of these five circRNAs was evaluated in the obtained tissue samples. The results of qRT-PCR showed that excluding circ_0106385, circ_0084927, circ_0099591, circ_0081723, and circ_0084912 were sig- nificantly up-regulated in CC tissues than were in paired healthy cervical tissues (Fig. 1b–f). We then selected circ_0084927, which had the -highest expression level, as the research object. Further analysis of circ_0084927 expression revealed that compared to the normal cervi- cal epithelial cell line (HcerEpic), circ_0084927 was sig- nificantly up-regulated in CC cell lines, including HeLa, CaSki, SW756, and C-33A. In particular, Hela and C-33A cell lines showed more than twofold circ_0084927 expression of the HcerEpic cell line (Fig. 1g). For this reason, they were selected for follow-up studies. It was observed that circ_0084927 was a closed circular RNA generated from and contained exons 7, 8 and 9 of its host gene, ESRP1 (Fig. 1h). To further characterize circ_0084927, we performed RNase R degradation exper- iments on HeLa and C-33A cells. Our analysis showed that RNase R greatly reduced linear_0084927 expression. Yet it had little effect on circ_0084927 expression in both cell lines (Fig. 1i). In addition, subcellular fractionation location analysis of HeLa and C-33A cells suggested that circ_0084927 and linear_0084927 were mainly located in the cytoplasm (Fig. 1j).
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Underlining the physiological relevance of our findings, both USP7 and MDC1 were significantly overexpressed in a subset of patients with cervical cancer, and the levels of these 2 factors positively correlated with each other. It is known that 99% of cervical cancers are associated with HPV infection, the incorporation of which results in a defective G 1 /S check- point (28, 61). The activation of the repair machinery and G 2 /M checkpoint thus becomes essential for protecting cancer cells from both endogenous replication stress and exogenous geno- toxic insults. These features enable cervical cancer to be more clinically sensitive to DNA-damaging agents. However, resis- tance to DNA-damaging therapy alone exists, as evidenced by the poor survival rates among patients with advanced HPV- positive cervical cancer (28, 61). Therefore, therapeutic modula- tion of the DDR is an attractive strategy to increase the response to chemotherapy or radiotherapy of patients suffering from cervical cancer. Targeting USP7 may thus provide an effec- tive treatment for patients with cervical cancer who undergo DNA-damaging therapy, particularly in light of recent efforts to develop a specific small-molecule antagonist toward the cata- lytic activity of USP7 (47, 74) or attenuate ubiquitin binding (48, 75). Indeed, we reveal that GNE-6640 and GNE-6776, which preferentially compete with K48-linked ubiquitin chains for binding to USP7, destabilize MDC1, thereby rendering cervical cancer cells more vulnerable to IR. In a way, this provides direct evidence for the idea that USP7 and MDC1 are viable therapeu- tic targets for the treatment of cervical cancer. In support of this, a recent study indicated that USP7 inhibition sensitizes p53- defective, chemotherapy-resistant chronic lymphocytic leuke- mia (CLL) cells to clinically achievable doses of chemotherapeu- tic agents in a murine xenograft model (76). Thus, USP7 could be a promising therapeutic target, at least for the treatment of malignancies with DDR defects in which the p53-dependent apoptosis pathway is compromised. In support of the patholog- ical significance of MDC1 dysregulation as well as our current work, recent studies suggest that silencing MDC1 enhances the radiosensitivity of human nasopharyngeal cancer in xenograft models (77) and that patients with oral squamous cell carcinoma with weak expression of MDC1 protein benefit significantly when treated with surgery followed by radiation therapy (78).
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This study had total 168 subjects (84 cases and 84 controls). Table 1 shows that socioeconomic status and dietary details of cases and controls. In the present study, 76.2% of the cases and 17.9% of the controls were belong to low socioeconomic status (LSS) (p<0.05). Protective association was found in univariate analysis of hygiene practices which explained that those cases (77.4%) and controls (32.1%), reuse the sanitary napkins or cloth during their menstrual cycle were at more risk. Moreover, passive smoking also play a significant role in cervical carcinogenesis in north Indian women and increased risk was observed among cases as compared to controls (OR =4.38, CI= 95%, p=0.000).This is because smoking is related in causing defects in DNA repair system in biological cell and leads to carcinogenesis, and DNA damage has been also found in cervical tissue of smokers. Besides this, women suffering from cc found 83.3% were educated up to middle school certificate and mostly are illiterate (47.6% in control), 15.5% were graduate (47.6% in control), 1.2% were professional (4.8% in control) and p-value found significant (p value= <0.05). Husband’s education also shows the same.
HPV is the leading risk factor for cervical cancer, and HPV infection has been shown to cause aberrant acety- lation. For instance, Jansma and colleagues have docu- mented that the oncoprotein E7 from human HPV strains mediates the interactions between CBP/p300 and pRb and promotes pRb acetylation, leading to disruption of cell cycle control . In this study, the three patients were HPV infected, and we found that viral carcinogenesis was predominantly over-represented in KEGG analysis. More importantly, our acetylome analysis and IP experiments demonstrated that CREBBP and p300 were up-acetylated in cervical cancer tissues compared with adjacent normal tissues. It seems reasonable to propose that HPV infection changes acetylation levels of many proteins in cervical cancer and contributes to cervical carcinogenesis.
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Since hBD3 expression is elevated in cervical cancer samples and correlates with disease stages, we speculated that hBD3 may contribute to the cervical carcinogenesis. Given the low endogenous level of hBD3, we tested the impact of hBD3 on the growth of cervical cancer cells by an overexpression approach. HeLa and CaSki cell lines that stably express hBD3 or eGFP (as negative control) were established by lenti-viral transduction and the growth of these cells were monitored using a real-time cell analysis (RTCA) system. The expression of the transgenes were verified by western blot and immunofluorescence (Figure 2E–2F and Supplementary Figure S1). As expected, eGFP- expressing HeLa or CaSki cells grew at a similar rate to the parental cells, indicating the lentivrial transduction and expression of the irrelevant eGFP gene had no apparent impacts on the growth of these cervical cancer cells. By contrast, hBD3 overexpression significantly enhanced the growth of both cell lines (Figure 2A–2B). This growth stimulating effect of hBD3 was associated with increased S phase population as determined by PI cell cycle analysis (Figure 2C–2D) and was accompanied by the induction of multiple cell cycle regulators including Cyclin A, D1/ D2, E, CDK2 and CDK4, although the identities of the key regulators varied between the two cell lines (Figure 2E–2F). Together, our results demonstrated that hBD3 promotes the G1/S progression and thus the proliferation of cervical cancer cells.
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Abstract: Overexpression of aquaporins (AQPs) has been reported in several human cancers. Extracellular signal- regulated kinases 1/2 (Erk1/2) are associated with tumorigenesis and cancer progression and may upregulate AQPs expression. In this study, we examined cervical tissue samples to establish the relationship between Erk1/2 and AQPs in cervical carcinoma by RT-PCR, Western blot and immunohistochemistry. We also examined the relation- ship between AQP8, Erk1/2 and clinicopathological variables in patients with cervical cancer. Our results showed that Erk1/2 was differentially expressed at the level of transcription and was most highly expressed in CIN samples (P < 0.05). At the level of translation, significant differences were seen in the expression of AQP8, Erk1/2 and P- Erk1/2 (P < 0.05). Expression was highest in CIN samples, where 80.9%, 76.6%, and 66% of samples were positive for AQP8, Erk1/2 and P-Erk1/2, respectively. Expression in cervical carcinoma samples was higher than in normal cervical tissues (P < 0.01). AQP8 expression was associated with the depth of invasion of cervical cancer cells, and the expression of Erk1/2 and P-Erk1/2 was increased in earlier clinical stages and in lymphatic metastasis. AQP8 expression was positively correlated with Erk1/2 expression in cervical cancer. In conclusions, increased AQP8, Erk1/2 and P-Erk1/2 expression may play a role in transformation of CIN into cervical cancer, and in early invasion and lymphatic metastasis of cervical cancer. These proteins could potentially be used as molecular markers for early diagnosis of cervical carcinoma.
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producing ovarian tumours are at risk for developing endometrial carcinoma (54). Patients with Lynch Syndrome and Cowden syndrome are also at an increased risk of developing endometrial carcinoma (55). The average age of diagnosis is around 63 years (56). The most common presenting symptom is post-menopausal bleeding. Postmenopausal bleeding is defined as uterine bleeding after permanent cessation of menstruation resulting from loss of ovarian follicular activity (57) . The other symptoms include vaginal discharge and pelvic pain. In few cases, abnormal cells may be found in routine cervical cytology (PAP smear) (58). The initial imaging done in patients with abnormal uterine bleeding is pelvic ultrasonography. A cut off value >11mm endometrial thickness is associated with an increased risk of cancer by 6.7% (59).
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In order to assess the alteration in lncRNA expression in rat lung carcinogenesis induced by NNK, we selected 3 pairs of rat lung tumor samples and matched normal lung tissues and 2 blood samples from the control and NNK treatment group in the 95th week, and determined the lncRNA expression profiles using Arraystar rat lncRNA microarray. After normalization for raw signal intensities in Quantile method by GeneSpring GX v11.5, and filtering for low intensity lncRNAs, the quality of lncRNA data was assessed by box plot and scatter plot. As shown in Figure 1A, after normalization, the distributions of intensities in all samples were almost the same. The scatter-plot was then used to assess the variation in lncRNA expression between the two compared arrays (Figure 1B and 1C). To identify differentially expressed lncRNAs which were significantly different, we performed Volcano Plot filtering between the compared groups (the filtering threshold was a fold change ≥ 2 and a p value ≤ 0.05). According to these screening factors, the differential expression profiles between lung tumor tissues and matched normal lung tissues are shown in Figure 1D. Compared with normal lung tissues, 757 lncRNAs were significantly up-regulated and 519 lncRNAs were significantly down-regulated in lung tumor tissues induced by NNK. The lncRNA differential expression profiles
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were not evaluated in an analogous manner because encompassing Emca1 also contains a determinant of sus- only a subset of this population was genotyped at mark- ceptibility to E2-induced mammary carcinogenesis in ers in the Ept intervals. crosses between the ACI and unrelated BN rat strain (M. Linkage analysis revealed no significant evidence for Tochacek, B. Schaffer and J. D. Shull, unpublished linkage of a locus modulating E2-induced pituitary tu- data).
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In this review we have presented evidence that carcino- genesis in the mammary gland, and in other organs, might result from transformation of stem and/or progenitor cells by the deregulation of self-renewal pathways. These pathways include Hedgehog, Notch, Wnt, and the transcription factor Bmi-1. The hypothesis that mammary carcinogenesis results from the deregulation of normal stem cell self-renewal pathways suggests that components of these pathways might provide attractive targets for therapeutic development. This is of great importance because current therapies may be limited in their effectiveness by virtue of the fact that they might selectively target the more differentiated cells in a tumor. Tumor stem cells, by virtue of their slow cell cycle kinetics, transporter proteins, and anti-apoptotic mechanisms, might be resistant to these treatments (reviewed in ). The targeting of self-renewal pathways might provide a more specific approach to the elimination of cancer stem cells. A potential challenge in this regard is the development of
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Results: Iron promoted proliferation, carcinogenesis and Warburg effect of osteosarcoma cells. Iron-induced reactive oxygen species (ROS) played an important role in these processes. Iron accumulated more in mitochondrion than in cytoplasm, suggesting mitochondrion-mediated iron accumulation was involved in the development of osteo- sarcoma. Moreover, iron upregulated the expression of mitoferrin 1 (SLC25A37) and mitoferrin 2 (SLC25A28). Knock- down of mitoferrin 1 (SLC25A37) and mitoferrin 2 (SLC25A28) decreased the production of ROS. In addition, iron increased the expression of Warburg key enzymes HK2 and Glut1, and affected AMPK/mTORC1 signaling axis. Conclusions: Mitochondrion-mediated iron accumulation promotes carcinogenesis and Warburg effect of osteosar- coma cells. Meanwhile, iron deprivation might be a novel effective strategy in the treatment of osteosarcoma. Keywords: Iron, Mitoferrin, Warburg, Reactive oxygen species, Osteosarcoma
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human cancers. However, no information is available regarding Orai1 expression during oral/oropharyngeal carcinogenesis. Our results showed that Orai1 is highly expressed in OSCC compared to precancerous and normal tissues in vivo. Moreover, precancerous oral epithelial cells express higher Orai1 protein than normal oral epithelial cells, suggesting that Orai1 plays an important role in cancer progression. To our knowledge, our finding is the first report showing a stepwise elevation of Orai1 in multistep carcinogenesis in vivo and further oncogenic transformation of immortalized cells by Orai1 overexpression. Consistent with previous observation , our immunohistochemistry study confirmed localization of Orai1 in the plasma membrane. We also observed diffused staining of Orai1 in both the cytoplasm and nucleus. Since the tissue sections were obtained from 3 dimensional tissue structures, there could be some overlap with another cell layer positioned in different orientation compared to monolayer-cultured cells. Indeed, we were able to detect predominant Orai1 staining to the plasma membrane in the monolayer-cultured cells of HOK-16B/ Orai1 (data not shown). Our study clearly demonstrated that Orai1 is required for tumorigenicity of OSCC in vivo. Inactivation of Orai1 by the dominant negative Orai1 mutant not only suppressed SOCE, but also abolished in vivo tumorigenic potential of OSCC. Conversely, ectopic Orai1 expression further transformed non-tumorigenic oral epithelial cells to tumorigenic cells. This finding is consistent with the results of a previous study reporting that Orai1 inhibition led to significant decreases in cancer growth in vivo and in vitro [32-34]. Our findings support the hypothesis that Orai1 is a novel molecular determinant of oral/oropharyngeal cancer progression.
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cervical cancer tissues was significantly lower than that in adjacent non-tumor tissues (P < 0.05). To identify the clinical relevance of lncRNA LET expression in cervical cancer, cor- relation between lncRNA LET expression and clinicopathological features such as age, tumor size, histology, tumor differentiation, FIGO stage, lymph node metastasis and depth of cervical invasion was examined. The mean expression level of LncRNA LET was used as a cutoff point to divide all patients into two groups: cervical cancer patients who express LncRNA LET at levels less than the cutoff value were assigned to the low expression group, and those with expression above the cutoff value were assigned to the high expression group. Our results showed that decreased lncRNA LET expression was significantly correlated with FIGO stage, lymph node metastasis, depth of cervical invasion (P < 0.05), but not associated with other clinicopathologic factors such as age, tumor size, histology, and tumor differen- tiation (P > 0.05).