Induction of dormancy in hypoxic human papillomavirus-positive cancer cells.
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ABSTRACT:
SUBMITTER: Hoppe-Seyler K
PROVIDER: S-EPMC5307428 | biostudies-other | 2017 Feb
REPOSITORIES: biostudies-other
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Project description:Although the effect of hypoxia on p53 in human papillomavirus (HPV)-positive cancer cells has been studied for decades, the impact of p53 regulation on downstream targets and cellular adaptation processes during different periods under hypoxia remains elusive. Here, we show that, despite continuous repression of HPV16 E6/E7 oncogenes, p53 did not instantly recover but instead showed a biphasic regulation marked by further depletion within 24 h followed by an increase at 72 h. Of note, during E6/E7 oncogene suppression, lysosomal degradation antagonizes p53 reconstitution. Consequently, the transcription of p53 responsive genes associated with senescence (e.g., PML and YPEL3) cannot be upregulated. In contrast, downstream genes involved in autophagy (e.g., DRAM1 and BNIP3) were activated, allowing the evasion of senescence under hypoxic conditions. Hence, dynamic regulation of p53 along with its downstream network of responsive genes favors cellular adaptation and enhances cell survival, although the expression of the viral E6/E7-oncogenes as drivers for proliferation remained inhibited under hypoxia.
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Project description:Penile squamous cell cancer (PSCC) is the most frequent penile malignant disease. Infections with human papillomaviruses (HPV) are a major etiologic driver of PSCC. However, the molecular details of the underlying carcinogenesis are understudied because of rare clinical specimens and missing cell lines. Here, we investigated if the expression of high-risk HPV16 oncogenes causes an augmentation of the Wnt pathway using unique HPV-positive penile cancer (PeCa) cell lines in monolayer and organotypic 3D raft cultures as well as tissue micro arrays containing clinical tissue specimens. The HPV oncoproteins enhanced the expression of Leucine-rich repeat-containing G-protein coupled receptor 6 (LGR6) and the HPV-positive PeCa cells expressed a signature of Wnt target and stemness-associated genes. However, the notable lack of nuclear β-catenin in vitro and in situ raised the question if the enhanced expression of Wnt pathway factors is tantamount to an active Wnt signaling. Subsequent TOP-flash reporter assays revealed Wnt signaling as absent and not inducible by respective Wnt ligands in PeCa cell lines. The HPV-positive PeCa cells and especially HPV-positive PeCa specimens of the tumor core expressed the Wnt antagonist and negative feedback-regulator Dickkopf1 (DKK1). Subsequent neutralization experiments using PeCa cell line-conditioned media demonstrated that DKK1 is capable to impair ligand-induced Wnt signaling. While gene expression analyses suggested an augmented and active canonical Wnt pathway, the respective signaling was inhibited due to the endogenous expression of the antagonist DKK1. Subsequent TMA stainings indicated Dkk1 as linked with HPV-positivity and metastatic disease progression in PeCa suggesting potential as a prognostic marker.
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Project description:Human papillomaviruses (HPVs) are small, double-stranded DNA viruses that are significant risk factors in the development of cancer, and HPV accounts for approximately 5% of all worldwide cancers. Recent studies using data from The Cancer Genome Atlas (TCGA) have demonstrated that elevated levels of estrogen receptor alpha (ER?) are associated with improved survival in oropharyngeal cancers, and these elevated receptor levels were linked with human papillomavirus-positive cancers (HPV+cancers). There has been a dramatic increase in HPV-related head and neck squamous cell carcinomas (HPV+HNSCCs) over the last 2 decades, and therapeutic options for this ongoing health crisis are a priority; currently, there are no antiviral therapeutics available for combatting HPV+cancers. During our TGCA studies on head and neck cancer, we had also discovered the overexpression of ER? in HPV+cancers. Here, we demonstrate that 17?-estradiol (estrogen) attenuates the growth/cell viability of HPV+cancers in vitro, but not HPV-negative cancer cells. In addition, N/Tert-1 cells (foreskin keratinocytes immortalized with human telomerase reverse transcriptase [hTERT]) containing human papillomavirus 16 (HPV16) have elevated levels of ER? and growth sensitivity after estrogen treatment compared with parental N/Tert-1 cells. Finally, we demonstrate that there are potentially two mechanisms contributing to the attenuation of HPV+ cell growth following estrogen treatment. First, estrogen represses the viral transcriptional long control region (LCR) downregulating early gene expression, including E6/E7. Second, expression of E6 and E7 by themselves sensitizes cells to estrogen. Overall, our results support the recent proposal that estrogen could be exploited therapeutically for the treatment of HPV-positive oral cancers.IMPORTANCE Human papillomaviruses cause around 5% of all human cancers, yet there are no specific antiviral therapeutic approaches available for combatting these cancers. These cancers are currently treated with standard chemoradiation therapy (CRT). Specific antiviral reagents are desperately required, particularly for HPV+HNSCC whose incidence is increasing and for which there are no diagnostic tools available for combatting this disease. Using data from The Cancer Genome Atlas (TCGA), we and others determined that the estrogen receptor alpha (ER?) is overexpressed in HPV+HNSCC and that elevated levels are associated with an improved disease outcome. This has led to the proposal that estrogen treatment could be a novel therapeutic approach for combatting HPV+cancers. Here, we demonstrate that estrogen attenuates the growth of HPV+epithelial cells using multiple mechanisms, supporting the idea that estrogen has potential as a therapeutic agent for the treatment of HPV+HNSCC.
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Project description:BackgroundCervical cancer caused by human papillomavirus (HPV) infections is one of the most common cancers affecting women worldwide. Current preventative HPV vaccines on the market are composed of HPV L1 protein produced either in the yeast such as Gardasil or in the insect cells such as Cervarix. The duration of efficacy and cross-protection remain highly desirable for the improvement of current prophylactic HPV vaccine. Given that HPV carries out infection and replicates in mammalian cells, L2 protein, which is not included in the current licensed vaccines, is included in the third generation of HPV vaccine in pursuing of providing broader prevention. We hypothesize that a virus-like particle (VLP) consisting of HPV L1 plus L2 proteins generated in mammalian cells will present conformations more closely to native HPV, thus it will provide more durable and broader efficacy of prevention.MethodsWe took advantage of 293TT cells to produce VLP containing L1 and L2 proteins of HPV16 and HPV18, respectively.ResultsVLP particles of uniformed size and morphology were observed, and potent and broadly neutralizing antibodies were induced in mice and rabbits. In addition, compared to bivalent HPV vaccine of Cervarix, our HPV L1-L2 VLPs elicited higher titer of anti-sera, and the anti-sera also presented comparable neutralization potency against HPV16 and HPV18 infections even a much less potent adjuvant was used in our case.ConclusionOur VLPs were capable of eliciting stronger and more broadly neutralizing activities against various HPV subtypes and were potential candidate HPV vaccines.
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Project description:Human cancer cell lines are the most frequently used preclinical models in the study of cancer biology and the development of therapeutics. Although anatomically diverse, human papillomavirus (HPV)-driven cancers have a common etiology and similar mutations that overlap with but are distinct from those found in HPV-negative cancers. Building on prior studies that have characterized subsets of head and neck squamous cell carcinoma (HNSCC) and cervical squamous cell carcinoma (CESC) cell lines separately, we performed genomic, viral gene expression, and viral integration analyses on 74 cell lines that include all readily-available HPV-positive (9 HNSCC, 8 CESC) and CESC (8 HPV-positive, 2 HPV-negative) cell lines and 55 HPV-negative HNSCC cell lines. We used over 700 human tumors for comparison. Mutation patterns in the cell lines were similar to those of human tumors. We confirmed HPV viral protein and mRNA expression in the HPV-positive cell lines. We found HPV types in three CESC cell lines that are distinct from those previously reported. We found that cell lines and tumors had similar patterns of viral gene expression; there were few sites of recurrent HPV integration. As seen in tumors, HPV integration did appear to alter host gene expression in cell lines. The HPV-positive cell lines had higher levels of p16 and lower levels of Rb protein expression than did the HPV-negative lines. Although the number of HPV-positive cell lines is limited, our results suggest that these cell lines represent suitable models for studying HNSCC and CESC, both of which are common and lethal.
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Project description:Breast cancer cells frequently home to the bone marrow, where they may enter a dormant state before forming a bone metastasis. Several members of the interleukin-6 (IL-6) cytokine family are implicated in breast cancer bone colonization, but the role for the IL-6 cytokine leukaemia inhibitory factor (LIF) in this process is unknown. We tested the hypothesis that LIF provides a pro-dormancy signal to breast cancer cells in the bone. In breast cancer patients, LIF receptor (LIFR) levels are lower with bone metastases and are significantly and inversely correlated with patient outcome and hypoxia gene activity. Hypoxia also reduces the LIFR:STAT3:SOCS3 signalling pathway in breast cancer cells. Loss of the LIFR or STAT3 enables otherwise dormant breast cancer cells to downregulate dormancy-, quiescence- and cancer stem cell-associated genes, and to proliferate in and specifically colonize the bone, suggesting that LIFR:STAT3 signalling confers a dormancy phenotype in breast cancer cells disseminated to bone.
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Project description:BACKGROUND:Human papillomavirus (HPV) infection accounts for about 40-50% of all cases of penile carcinoma suggesting that other factors, including host genetic status, are involved in neoplastic transformation. In this perspective, STK11 gene, which has been found frequently mutated in HPV-related cervical carcinoma, has been analyzed in HPV-positive and HPV-negative invasive penile cancers to establish its mutational status and the possible correlation of HPV infection with specific genetic alterations. METHODS:Genomic DNAs extracted from 26 cases of penile squamous cell carcinoma were analyzed for genetic alterations in the exons 1 to 9 of STK11 gene by quantitative real-time PCR. Ratios of potentially deleted and non-deleted exons were indicative of specific loss of STK11 coding regions. DNA samples of 5 cancer cases were subjected to standard PCR amplification of STK11 exons 1 to 9 and analyzed for somatic mutations by direct nucleotide sequencing analysis. RESULTS:Heterozygous deletions of STK11 exon 1 and 2 were identified in 2 out of 14 HPV-positive (14.3%) and 1 out of 12 HPV-negative cases (8.3%). Complete nucleotide sequencing analysis of exons 1 to 9 showed a single nucleotide change upstream the exon 2 coding region in 1 out of 5 penile carcinoma samples. CONCLUSIONS:The present results suggest that single nucleotide mutations and/or deletions of STK11 gene are rare events in penile cancer. Moreover, no significant association was observed between STK11 alterations and HPV infection in these tumors.
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Project description:Obligately aerobic tubercle bacilli are capable of adapting to survive hypoxia by developing into a nonreplicating or dormant form. Dormant bacilli maintain viability for extended periods. Furthermore, they are resistant to antimycobacterials, and hence, dormancy might play a role in the persistence of tuberculosis infection despite prolonged chemotherapy. Previously, we have grown dormant Mycobacterium bovis BCG in an oxygen-limited Wayne culture system and subjected the bacilli to proteome analysis. This work revealed the upregulation of the response regulator Rv3133c and three other polypeptides (alpha-crystallin and two "conserved hypothetical" proteins) upon entry into dormancy. Here, we replaced the coding sequence of the response regulator with a kanamycin resistance cassette and demonstrated that the loss-of-function mutant died after oxygen starvation-induced termination of growth. Thus, the disruption of this dormancy-induced transcription factor resulted in loss of the ability of BCG to adapt to survival of hypoxia. Two-dimensional gel electrophoresis of protein extracts from the gene-disrupted strain showed that the genetic loss of the response regulator caused loss of the induction of the other three dormancy proteins. Thus, the upregulation of these dormancy proteins requires the response regulator. Based on these two functions, dormancy survival and regulation, we named the Rv3133c gene dosR for dormancy survival regulator. Our results provide conclusive evidence that DosR is a key regulator in the oxygen starvation-induced mycobacterial dormancy response.
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Project description: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.
| S-EPMC4304782 | biostudies-literature
Project description:Background:While hypoxia has been well-studied in various tumor microenvironments, its role in cancer cell dormancy is poorly understood, in part due to a lack of well-established in vitro and in vivo models. Hypoxic conditions under conventional hypoxia chambers are relatively unstable and cannot be maintained during characterization outside the chamber since normoxic response is quickly established. To address this challenge, we report a robust in vitro cancer dormancy model under a hypoxia-mimicking microenvironment using cobalt chloride (CoCl2), a hypoxia-mimetic agent, which stabilizes hypoxia inducible factor 1-alpha (HIF1?), a major regulator of hypoxia signaling. Methods:We compared cellular responses to CoCl2 and true hypoxia (0.1% O2) in different breast cancer cell lines (MCF-7 and MDA-MB-231) to investigate whether hypoxic regulation of breast cancer dormancy could be mimicked by CoCl2. To this end, expression levels of hypoxia markers HIF1? and GLUT1 and proliferation marker Ki67, cell growth, cell cycle distribution, and protein and gene expression were evaluated under both CoCl2 and true hypoxia. To further validate our platform, the ovarian cancer cell line OVCAR-3 was also tested. Results:Our results demonstrate that CoCl2 can mimic hypoxic regulation of cancer dormancy in MCF-7 and MDA-MB-231 breast cancer cell lines, recapitulating the differential responses of these cell lines to true hypoxia in 2D and 3D. Moreover, distinct gene expression profiles in MCF-7 and MDA-MB-231 cells under CoCl2 treatment suggest that key cell cycle components are differentially regulated by the same hypoxic stress. In addition, the induction of dormancy in MCF-7 cells under CoCl2 treatment is HIF1?-dependent, as evidenced by the inability of HIF1?-suppressed MCF-7 cells to exhibit dormant behavior upon CoCl2 treatment. Furthermore, CoCl2 also induces and stably maintains dormancy in OVCAR-3 ovarian cancer cells. Conclusions:These results demonstrate that this CoCl2-based model could provide a widely applicable in vitro platform for understanding induction of cancer cell dormancy under hypoxic stress.
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