TESC Promotes TGF-α/EGFR-FOXM1-Mediated Tumor Progression in Cholangiocarcinoma.

Cholangiocarcinoma is a relatively uncommon but highly lethal malignancy. Improving outcomes in patients depends on earlier diagnosis and appropriate treatment; however, no satisfactory diagnostic biomarkers or targeted therapies are currently available. To address this shortcoming, we analyzed the transcriptomic datasets of cholangiocarcinoma from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases and found that TESC is highly expressed in cholangiocarcinoma. Elevated cellular levels of TESC are correlated with larger tumor size and predict a poor survival outcome for patients. Knockdown of TESC via RNA interference suppresses tumor growth. RNA-sequencing analysis showed that silencing of TESC decreases the level of FOXM1, leading to cell cycle arrest. Correlation analysis revealed that the cellular level of TESC is correlated with that of FOXM1 in cholangiocarcinoma patients. We further observed that upon TGF-α induction, TESC is upregulated through the EGFR-STAT3 pathway and mediates TGF-α-induced tumor cell proliferation. In vivo experiments revealed that knockdown of TESC significantly attenuates tumor cell growth. Therefore, our data provide novel insight into TESC-mediated oncogenesis and reveal that TESC is a potential biomarker or serves as a therapeutic target for cholangiocarcinoma.

Klf10 induces cell apoptosis through modulation of BI-1 expression and Ca2+ homeostasis in estrogen-responding adenocarcinoma cells.

Estrogen stimulates cell growth and inhibits apoptosis through estrogen receptor-mediated mechanisms in many cell types. Remarkably, there is another dimension to estrogen action by which apoptosis is induced in breast cancer cells. While these mechanisms are not yet completely understood, finding the molecules involved has paved the way for the development of a new drug group. Using ChIP-chip, we have demonstrated that Klf10, a Krüppel-like zinc finger transcription factor, which was induced in response to estrogen, directly modulates the transcription of BI-1 (Bax inhibitor-1; also called the testis-enhanced gene transcript, TEGT). Eventually, the estrogen induced Klf10 and then suppresses BI-1 transcription. The estrogen/Klf10/BI-1 interrelationship was further confirmed using BI-1 promoter and EMSA assays. The estrogen-elicited reduction of BI-1 promoter activity was significantly reversed when the Klf10 binding element was mutated to abolish Klf10 binding. A si-Klf10 antisense-oligo nucleotide was also able to restore BI-1 promoter activity to its pre-estrogen-treatment level. BI-1 is known to regulate stress via the endoplasmic reticulum; in this context down-regulation of BI-1 is able to cause Ca(2+) release and trigger an apoptosis pathway in breast cancer. In our study, Klf10 not only suppressed cellular BI-1 expression but also increased the cytosolic Ca(2+) concentration, eventually causing apoptotic cell death. Based on these results, we suggest the pathway by which estrogen induces apoptosis is possibly through an up-regulation of Klf10 that decreases BI-1 and finally increases the concentration of cytoplasmic calcium.

The human papillomavirus-16 (HPV-16) oncoprotein E7 conjugates with and mediates the role of the transforming growth factor-beta inducible early gene 1 (TIEG1) in apoptosis.

The human papillomavirus (HPV) oncoprotein E7 is a major transforming protein. The E7 protein does not possess intrinsic enzymatic activity, but rather functions through direct and indirect interactions with cellular proteins, several of which are well known cellular tumor suppressors. Using the yeast two-hybrid system, we found that transforming growth factor-beta inducible early gene 1 (TIEG1), a member of the Krüppel-like family (KLF) that has been implicated as a putative tumor suppressor, interacts and forms a specific complex with HPV-16 E7. TIEG1 has been shown to mimic the effects of TGF-beta in various carcinoma cells and plays a critical role in the apoptotic cascade. Our results indicate that E7 binds to the C-terminus of TIEG1 and induces its degradation via the ubiquitin pathway. E7 not only increased the ubiquitination of TIEG1 but also influenced the ability of TIEG1 to affect apoptosis. Our results suggest that suppression of TIEG1-mediated signaling by E7 may contribute to HPV-associated carcinogenesis.

DYRK1A stabilizes HPV16E7 oncoprotein through phosphorylation of the threonine 5 and threonine 7 residues.

HPV16, a high-risk tumorigenic virus, has been identified as one of the causative agents for the development of cervical cancer. Subsequent to viral infection, the constitutive expression of the viral oncoproteins E6 and E7 plays a number of critical roles in maintaining the transformed phenotype. Here we demonstrate that a cellular kinase, dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A), interacts with and phosphorylates HPV16E7 in vitro and in vivo. Using substitution mutations, we identified that DYRK1A specifically phosphorylates HPV16E7 at Thr5 and Thr7, which are located within the N-terminal CRI domain. This interaction greatly increases the steady-state level of HPV-16E7 by interfering with the protein's 26S proteosome-dependent degradation. The half-life of E7 was extended significantly by replacing Thr5 and Thr7 with a phosphorylation mimetic residue, aspartic acid. In addition, DYRK1A-induced phosphorylation protected E7 from degradation and influenced E7's function when modulating pRb degradation. We propose a new mechanism whereby DYRK1A phosphorylates Thr5 and Thr7 within HPV16E7. This phosphorylation then interferes with the degradation of HPV16E7, extending the protein half-life of HPV16E7 and increasing the colony-formation efficacy of HPV16E7. Our findings suggest that DYRK1A increases the transforming potential of HPV16-infected cells because of the greater stability of HPV16E7.

USP11 stabilizes HPV-16E7 and further modulates the E7 biological activity.

HPV-16E7 is a major transforming protein, which has been implicated in the development of cervical cancer. The stability of E7 is thus important to ensure its fully functional status. Using the yeast two-hybrid system, we found that USP11 (ubiquitin-specific protease 11), a member of a protein family that cleaves polyubiquitin chains and/or ubiquitin precursors, interacts and forms a specific complex with HPV-16E7. Our results indicate that the USP11 can greatly increase the steady state level of HPV-16E7 by reducing ubiquitination and attenuating E7 degradation. In contrast, a catalytically inactive mutant of USP11 abolished the deubiquitinating ability and returned E7 to a normal rate of degradation. Moreover, USP11 not only protected E7 from ubiquitination but also influenced E7 function as a modulator of cell growth status. These results suggest that USP11 plays an important role in regulating the levels of E7 protein and subsequently affects the biological function of E7 as well as its contribution to cell transformation by HPV-16E7.

Increased expression of Dyrk1a in HPV16 immortalized keratinocytes enable evasion of apoptosis.

Immortalization is a critical event in virus-related oncogenesis. No enough information, however, is currently available to elucidate the changes that occur in cellular molecules during immortalization. To identify potential cellular markers or regulators involving in immortalization, a paired-cell model of primary foreskin keratinocytes (FK) and HPV16 immortalized foreskin keratinocytes were established. Using mRNA differential display, RT-PCR and Northern blot methods, we have identified and confirmed that Dyrk1a (dual-specificity tyrosine-phosphorylated and regulated kinase 1A) is present and increased in HPV16 immortalized cells, but is absent in primary keratinocytes. Moreover, transfection of E7 siRNA oligo into immortalized cells leads to a diminishing E7 expression and the eventual disappearance of Dyrk1a. Similar results of Dyrk1a expressional differences could also be seen when tissue specimens were compared using LCM/real-time PCR and immunohistochemistry analysis; malignant cervical lesions contain significantly more DYRK1A than normal tissue. It was also demonstrated that raised DYRK1A could rearrange the cellular localization of FKHR (forkhead in rhabdomyosarcoma), an apoptosis activator, and suppress BAD. Importantly, this phenomenon can be reversed when endogenous Dyrk1a was knocked down in immortalized cells by RNA interference. These results suggest that the raised Dyrk1a in HPV16 immortalized keratinocytes and cervical lesions may serve as a candidate antiapoptotic factor in the FKHR regulated pathway and initiate immortalization and tumorigenesis gradually.

HPV-18 E7 conjugates to c-Myc and mediates its transcriptional activity.

Several reports in the literature have indicated that the E6 not only elevates the level of c-Myc level but that the protein also associates with the Myc complex and activates Myc-responsive genes. There would seem to be a mechanism by which this oncogene can modulate cell proliferation and differentiation. Furthermore, an increase in c-Myc levels has also observed during ectopic expression of HPV E7 alone. Using the yeast two-hybrid system, we further found that the c-Myc interacts and forms a specific complex with HPV-16E7. In this study, we have demonstrated that E7 does indeed interact with c-Myc and a sequential deletion analysis of E7 maps the c-Myc interaction site to the carboxyl-terminal region. We determined two HPV-18 E7 binding sites on c-Myc involving the amino acids regions 1-100 and 367-439. The interaction of the high-risk type HPV E7 with c-Myc can augment c-Myc transactivation activity but this does not occur with low-risk type HPV E7. Deletion within the Cys-X-X-Cys repeat motif at the C-terminus of HPV-18 E7 leads to a lost of association with c-Myc and also abolishes the enhancement of c-Myc's transactivation activity. Furthermore, the interaction of HPV-18 E7 with c-Myc functionally promotes c-Myc's DNA-binding ability. Using the hTERT promoter as a model, enhanced c-Myc binding ability to the hTERT promoter as measured by immunoprecipitation assay was observed and occurred in an E7 dose-dependent manner. Taken together, these results provide significant new insights into the association of c-Myc with E7 and the possible involvement of high-risk E7 in oncogenesis.

Transgenerational epigenetic imprinting of the male germline by endocrine disruptor exposure during gonadal sex determination.

Embryonic exposure to the endocrine disruptor vinclozolin at the time of gonadal sex determination was previously found to promote transgenerational disease states. The actions of vinclozolin appear to be due to epigenetic alterations in the male germline that are transmitted to subsequent generations. Analysis of the transgenerational epigenetic effects on the male germline (i.e. sperm) identified 25 candidate DNA sequences with altered methylation patterns in the vinclozolin generation sperm. These sequences were identified and mapped to specific genes and noncoding DNA regions. Bisulfite sequencing was used to confirm the altered methylation pattern of 15 of the candidate DNA sequences. Alterations in the epigenetic pattern (i.e. methylation) of these genes/DNA sequences were found in the F2 and F3 generation germline. Therefore, the reprogramming of the male germline involves the induction of new imprinted-like genes/DNA sequences that acquire an apparent permanent DNA methylation pattern that is passed at least through the paternal allele. The expression pattern of several of the genes during embryonic development were found to be altered in the vinclozolin F1 and F2 generation testis. A number of the imprinted-like genes/DNA sequences identified are associated with epigenetic linked diseases. In summary, an endocrine disruptor exposure during embryonic gonadal sex determination was found to promote an alteration in the epigenetic (i.e. induction of imprinted-like genes/DNA sequences) programming of the male germline, and this is associated with the development of transgenerational disease states.

Using siRNA technique to generate transgenic animals with spatiotemporal and conditional gene knockdown.

Based on the RNAi technique, we have developed a new approach that generates transgenic animals capable of mimicking human genetic diseases. The new system is a combination of siRNA with Cre-loxP and tetracycline-on. It has the characteristics of being stable, inheritable, and inducible, with the siRNA able to be transcribed tissue specifically. To support the ability of this new method to generate a model for a disease, we created an ABCA1-deficient mouse line that mimics Tangier disease under controlled conditions. Thus, it should now be possible to rapidly establish human genetic diseases as a whole animal model without the use of embryonic stem cell and gene targeting. This system also provides a tool for pathological and pharmacological studies of aspects peculiar to particular human genetic diseases.