Ubiquitin B in Cervical Cancer: Critical for the Maintenance of Cancer Stem-Like Cell Characters

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate. Ubiquitin, which is a small, highly conserved protein expressed in all eukaryotic cells, can be covalently linked to certain target proteins to mark them for degradation by the ubiquitin-proteasome system. Previous studies highlight the essential role of Ubiquitin B (UbB) and UbB-dependent proteasomal protein degradation in histone deacetylase inhibitor (HDACi) -induced tumor selectivity. We hypothesized that UbB plays a critical role in the function of cervical cancer stem cells. We measured endogenous UbB levels in mammospheres in vitro by real-time PCR and Western blotting. The function of UbB in cancer stem-like cells was assessed after knockdown of UbB expression in prolonged Trichostatin A-selected HeLa cells (HeLa/TSA) by measuring in vitro cell proliferation, cell apoptosis, invasion, and chemotherapy resistance as well as by measuring in vivo growth in an orthotopic model of cervical cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human cervical cancer xenografts after UbB silencing. We found that HeLa/TSA were resistant to chemotherapy, highly expressed the UbB gene and the stem cell markers Sox2, Oct4 and Nanog. These cells also displayed induced differentiation abilities, including enhanced migration/invasion/malignancy capabilities in vitro and in vivo. Furthermore, an elevated expression of UbB was shown in the tumor samples of chemotherapy patients. Silencing of UbB inhibited tumorsphere formation, lowered the expression of stem cell markers and decreased cervical xenograft growth. Our results demonstrate that UbB was significantly increased in prolonged Trichostatin A-selected HeLa cells and it played a key role in the maintenance of cervical cancer stem-like cells.     

Angiopoietin 1 Promotes Tumor Angiogenesis and Tumor Vessel Plasticity of Human Cervical Cancer in Mice

Angiopoietins have been increasingly implicated to play important roles in blood vessel formation, remodeling, maturation, and maintenance. However, their roles in tumor angiogenesis and hence tumor growth and metastasis still remain uncertain. In this work, angiopoietin 1 expression was amplified in human cervical cancer HeLa cells by stable transfection or recombinant human adenovirus-mediated gene transfer. We show that increased angiopoietin 1 expression promoted in vivo growth of human cervical cancers in mice by promoting tumor angiogenesis and inhibiting tumor cell apoptosis. Furthermore, we also show for the first time that overexpression of angiopoietin 1 also leads to increased tumor vessel plasticity with a large number of vessels lacking periendothelial supporting cells. These results indicate that angiopoietin 1 promotes tumor angiogenesis and tumor vessel plasticity of human cervical cancer in mice.     

Tanshinone I attenuates proliferation and chemoresistance of cervical cancer in a KRAS‐dependent manner

Chemoresistance is a common occurrence during advanced or recurrent cervical cancer therapy when treated by conventional treatment, platinum‐based chemotherapy. This study aimed to investigate the effect and underlying mechanism of tanshinone I on attenuating proliferation and chemoresistance of cervical cancer cells. In cervical cancer cells, cell proliferation was examined by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), cell count, and soft‐agar colony‐formation assay. rVista analysis and luciferase reporter assay were used to explore the upstream regulator of KRAS, and the expression levels of key genes were also detected. Western blot analysis showed that tanshinone I significantly suppressed KRAS expression and inhibited AKT phosphorylation. rVista analysis and luciferase reporter assay demonstrated that ELK1 can binds directly to KRAS promoter and positively regulates KRAS expression. MTT assay showed that KRAS or ELK1 overexpression significantly attenuated the suppressive effects of tanshinone I on HeLa cells proliferation. In addition, tanshinone I recovered the cisplatin sensitivity of HeLa CR cells, whereas KRAS or ELK1 overexpression significantly inhibited this phenomenon. Our results suggested that tanshinone I had anticancer effects on cervical cancer cells via inhibiting ELK1 and downregulating KRAS‐AKT axis, which subsequently suppressed the proliferation and cisplatin resistance of cervical cancer cells.     

The Zinc Finger Protein ZNF268 Is Overexpressed in Human Cervical Cancer and Contributes to Tumorigenesis via Enhancing NF-κB Signaling

Cervical cancer is one of the most common tumors affecting women''s health worldwide. Although human papillomavirus can be detected in nearly all cases, the mechanism of cervical carcinogenesis remains to be further addressed. Here, we demonstrated that ZNF268, a Krüppel-associated box-containing zinc finger protein, might contribute to the development of cervical cancer. We found that ZNF268b2, an isoform of ZNF268, was overexpressed in human squamous cervical cancer specimens. Knockdown of ZNF268 in cervical cancer cells caused cell cycle arrest at the G₀/G₁ phase, reduced colony formation, and increased sensitivity to TNFα-induced apoptosis. In addition, HeLa cell growth in xenograft nude mice was suppressed by ZNF268 knockdown, with increased apoptosis. Furthermore, ZNF268b2 was shown to increase NF-κB signaling in vitro and in vivo. Reconstitution of NF-κB activity restored proliferation in ZNF268 knockdown HeLa cells. Of note, we observed a high frequency of NF-κB activation in ZNF268-overexpressing cervical cancer tissues, suggesting a pathological coincidence of ZNF268b2 overexpression and NF-κB activation. Taken together, our results reveal a novel role of ZNF268b2 that contributes to cervical carcinogenesis in part through enhancing NF-κB signaling.     

Human papillomavirus (HPV) oncoprotein E6 facilitates Calcineurin-Nuclear factor for activated T cells 2 (NFAT2) signaling to promote cellular proliferation in cervical cell carcinoma

The calcineurin-NFAT signaling pathway regulates cell proliferation, differentiation, and development in diverse cell types and organ systems. Deregulation of calcineurin-NFAT signaling has been reported in leukaemias and few solid tumors such as breast and colon. In the present study, we found elevated calcineurin protein levels and phosphatase activity in cervical cancer cell lines and depletion of the same attenuated cell proliferation. Additionally, nuclear levels of NFAT2, a downstream target of calcineurin, viz, was found elevated in human papillomavirus (HPV) infected cells, HeLa and SiHa, compared to the HPV negative cells, HaCaT and C33A, indicative of its higher DNA binding activity. The nuclear levels of both NFAT1 and NFAT3 remain unaltered implicating they have little role in cervical carcinogenesis. Similar to the in vitro studies, the HPV infected human squamous cell carcinoma specimens showed higher NFAT2 levels compared to the normal cervical epithelium. Depletion of NFAT2 by RNAi attenuated growth of SiHa cells. Overexpression of HPV16 oncoproteins viz, E6 and E7 increased NFAT2 expression levels and DNA binding activity, while knockdown of E6 by RNAi decreased the same. Briefly, we now report an activation of calcineurin-NFAT2 axis in cervical cancer and a novel role of HPV oncoprotein in facilitating NFAT2 dependent cell proliferation.     

Lethality of PAK3 and SGK2 shRNAs to Human Papillomavirus Positive Cervical Cancer Cells Is Independent of PAK3 and SGK2 Knockdown

The p21-activated kinase 3 (PAK3) and the serum and glucocorticoid-induced kinase 2 (SGK2) have been previously proposed as essential kinases for human papillomavirus positive (HPV+) cervical cancer cell survival. This was established using a shRNA knockdown approach. To validate PAK3 and SGK2 as potential targets for HPV+ cervical cancer therapy, the relationship between shRNA-induced phenotypes in HPV+ cervical cancer cells and PAK3 or SGK2 knockdown was carefully examined. We observed that the phenotypes of HPV+ cervical cancer cells induced by various PAK3 and SGK2 shRNAs could not be rescued by complement expression of respective cDNA constructs. A knockdown-deficient PAK3 shRNA with a single mismatch was sufficient to inhibit HeLa cell growth to a similar extent as wild-type PAK3 shRNA. The HPV+ cervical cancer cells were also susceptible to several non-human target shRNAs. The discrepancy between PAK3 and SGK2 shRNA-induced apoptosis and gene expression knockdown, as well as cell death stimulation, suggested that these shRNAs killed HeLa cells through different pathways that may not be target-specific. These data demonstrated that HPV+ cervical cancer cell death was not associated with RNAi-induced PAK3 and SGK2 knockdown but likely through off-target effects.     

NFBD1/MDC1 is a protein of oncogenic potential in human cervical cancer

A large nuclear protein of 2089 amino acids, NFBD1/MDC1 has recently been implicated in tumorigenesis and tumor growth. In this study, we investigated its expression in cervical cancers and explored its function using gene knockdown approaches. We report here that NFBD1 expression is substantial increased in 24 of 39 cases (61.5%) of cervical cancer tissues at the mRNA level and in 35 of 60 cases (58.3%) at the protein level compared with the case matched normal tissues. Tumors with higher grade of malignancy tend to have higher levels of NFBD1 expression. By infecting cells with retroviruses expressing NFBD1 shRNA, we successfully knocked down NFBD1 expression in cervical cancer cell lines HeLa, SiHa, and CaSki. NFBD1 knockdown cells display significant growth inhibition, cell cycle arrest, higher apoptotic rate, and enhanced sensitivity to adriamycin. Furthermore, NFBD1 knockdown also inhibits the growth of HeLa cells in nude mice. Western blot analyses further revealed that NFBD1 knockdown induced Bax, Puma, and Noxa while down-regulating Bcl-2; it also up-regulated cytochrome C and activated caspases 3 and 9. Therefore, the function of NFBD1 may be involved in the CDC25C-CyclinB1/CDC2 pathway at the G2/M checkpoint, and the cytochrome C/caspase 3 apoptotic pathway. Since expression of NFBD1 seems to be related to the oncogenic potential of cervical cancer, and suppression of its expression can inhibit cancer cell growth both in vitro and in vivo, NFBD1 may be a potential therapeutic target in human cervical cancer.     

Knockdown of SMYD3 by RNA interference inhibits cervical carcinoma cell growth and invasion in vitro

Elevated expression of SMYD3 is a frequent genetic abnormality in several malignancies. Few studies knocking down SMYD3 expression in cervical carcinoma cells have been performed to date. In this paper, we established an inducible short hairpin RNA expression system to examine its role in maintaining the malignant phenotype of HeLa cells. After being induced by doxycycline, SMYD3 mRNA and protein expression were both reduced, and significant reductions in cell proliferation, colony formation and migration/invasion activity were observed in the SMYD3-silenced HeLa cells. The percentage of cells in sub-G1 was elevated and DNA ladder formation could be detected, indicating potent induction of apoptosis by SMYD3 knockdown. These findings imply that SMYD3 plays crucial roles in HeLa cell proliferation and migration/invasion, and that it may be a useful therapeutic target in human cervical carcinomas.     

Exogenous interleukin-1 beta promotes the proliferation and migration of HeLa cells via the MEK/ERK signaling pathway

Objective

Interleukin-1 beta (IL-1β) is a crucial cytokine that has been implicated in cancer and metastasis development. However, its possible mechanistic role in cervical cancer remains unclear. This study aimed to investigate the functions of exogenous IL-1β in cervical cancer cell proliferation and migration.

Methods

HeLa cell proliferation and migration were measured using MTT and Transwell assays. A lentivirus-mediated packaging system was used to construct an IL-1β overexpressing cell line. MEK/ERK signal transduction was inhibited by pretreatment with the MEK inhibitor PD98059. qRT–PCR and Western blotting were used to test the expression of relevant genes.

Results

Exogenous IL-1β promoted the proliferation and migration of HeLa cells. In addition, overexpression of IL-1β in HeLa cells promoted cell proliferation. Mechanistically, exogenous IL-1β increased the phosphorylated MEK and ERK levels in HeLa cells and the expression of JUN, RELB, and NF-κB2. Alternatively, blockade of MEK inhibited the promoting proliferation effects of IL-1β and the expression of JUN, RELB, and NF-κB2.

Conclusions

Our data suggest that exogenous IL-1β regulates HeLa cell functions by regulating the MEK/ERK signaling pathway and by targeting JUN, RELB, and NF-κB2. Our study uncovered a potential association across IL-1β, cervical tumor development, and cancer progression.

     

Therapeutic efficacy of natural dipeptide carnosine against human cervical carcinoma cells

Natural substances have been attracted several researchers in the recent years, because of its potential antioxidant, anti‐inflammatory and anti‐cancer properties. We have investigated the effect of carnosine on cell viability, apoptosis, DNA damage, reactive oxygen species (ROS) and caspase 3 enzyme expression in human cervical carcinoma and Madin‐Darby Kidney Cells (MDCK) cells . Carnosine inhibited cancer cell growth up to 23%. ROS level was increased up to 30 and 31% in MDCK and HeLa cells respectively. Tunnel assay showed 42 and 14% of positive apoptotic cells in cancer and normal cells respectively. The alteration in mitochondrial and nuclear morphology was determined. The extended lace‐like network of normal mitochondria found in control cells. Carnosine treatment significantly altered the mitochondrial morphology of normal cervical carcinoma cell. Mitochondria were condensed clump structures in carnosine treated cancer cells. Carnosine reduced the number of colonies of cervical carcinoma cells. Caspase 3 expression was corresponded to the appearance of immunofluorescence in the cytoplasm. Caspase 3 expression was gradually increased in cervical carcinoma cells. In Silico, docking study was performed to recognize the binding activity of carnosine against a subunit of the caspase 3 , and carnosine was able to bind to the drug binding pocket of caspase 3. The glide energy is ?5.2 kcal/mol, suggesting the high binding affinity of carnosine to caspase 3. Taking all these data together, the natural dipeptide L‐carnosine could be a suitable antiproliferative agent in cervical carcinoma cells. Copyright © 2016 John Wiley & Sons, Ltd.     

CD155 knockdown promotes apoptosis via AKT/Bcl‐2/Bax in colon cancer cells

CD155, one of the nectin‐like molecule family members, is involved in cell adhesion and motility. CD155 is overexpressed in several human cancers, but its role in proliferation and apoptosis of colorectal cancer remains unclear. We found that CD155 was up‐regulated in colorectal cancer tissues. CD155 knockdown via shRNA lentiviruses inhibited colon cancers cell migration and invasion, with a reduction in the expression of FAK, Src and MMP‐2. CD155 down‐regulation also suppressed colon cancer cell proliferation, accompanied by changing expressions of some molecules related to cell cycle. Finally, CD155 knockdown increased the expression ratio between Bax and Bcl‐2, resulting in a significant increase in colon cancer cell apoptosis. Taken together, these results demonstrate that CD155 is involved in not only migration and invasion but also proliferation and survival abilities of colon cancer cells, suggesting that CD155 is one of key molecules promoting the growth and metastasis of colorectal cancer.     

HMQ‐T‐F2 exert antitumour effects by upregulation of Axin in human cervical HeLa cells

Looking for novel, effective and less toxic therapies for cervical cancer is of significant importance. In this study, we reported that HMQ‐T‐F2(F2) significantly inhibited cell proliferation and transplantable tumour growth. Mechanistically, HMQ‐T‐F2 inhibited HeLa cell growth through repressing the expression and nuclear translocation of β‐catenin, enhancing Axin expression, as well as downregulating the Wnt downstream targeted proteins. Knock‐down of a checkpoint β‐catenin by siRNA significantly attenuated HeLa cell proliferation. Furthermore, XAV939, an inhibitor of β‐catenin, was used to treat HeLa cells and the results demonstrated that HMQ‐T‐F2 inhibited proliferation and migration via the inhibition of the Wnt/β‐catenin pathway.