Transcription Profiling And Molecular Mechanism Of The LKB1Tumor Suppressor In Cervical Cancer And Regulation Of Multidrug Resistance Of Cervical Adenocarcinoma By CIP2A

Part One:Transcription profiling and molecular mechanism of the LKB1tumor suppressor in cervical cancerCervical cancer is the second most common cancer in women worldwide with an annual incidence of530,000cases. Although the incidence of cervical cancer in developed countries has decreased due to improved screening methods, it is still a main mortality factor for women in developing countries.Cervical cancer has a viral infectious etiology. Infection with high-risk types of human papillomavirus (HPVs) is strongly-associated with the development of cervical cancer. This fact provides a great opportunity for primary prevention of cervical cancer by prophylactic vaccination to prevent cervical HPV infection. Although HPV vaccine has become available, they are less likely to be effective for those who have already been infected by HPV and those who are immunosuppressed.Papillomaviruses are small DNA nonenvelopedviruses that replicate in epidermal or mucosal epithelial cells. HPVs can be classified into high-or low-risk types depending on their clinical associations. Persisting high-risk HPV infections are the most significant risk factor for development of cervical cancer. HPV types16and18are responsible for about70%of cervical cancers worldwide. The two oncogenes E6and E7of high-risk HPVs possess proliferation-stimulating activity and play a critical role during malignant transformation of cervical cancer. E6and E7interact with p53and pRB respectively and promote their degradation. Though HPVs play significant roles in the progression to cervical neoplasia, only a small percentage of high-risk HPV-infected women develop cancer eventually and it often takes decades after the initial infection, suggesting that infection by high-risk HPVs is not sufficient for cervical carcinogenesis. In addition, some cervical cancers are negative for HPVs. Therefore, there must be other cofactors or genetic events involved in cervical carcinogenesis.The liver kinase LKB1(also known as serine threonine kinase, STK11) was originally identified as the causative gene of Peutz-Jeghers syndrome (PJS), an autosomal dominant disease. Patients with PJS have increased cancer predisposition in GI tract, breast, and gynecological organs. Importantly, somatic LKB1mutations are frequently found in significant number of sporadic non-small cell lung carcinomas (approximately30%)、colon cancer、breast cancer and cervical carcinomas (approximately20%). In recent years, LKB1has become more established as a tumor suppressor. However, the molecular mechanism of LKB1has not been fully understood. LKB1is a master upstream kinase that forms a complex with a pseudokinase STRAD and the scaffold protein MO25. This complex can phosphorylate at least14serine-threonine kinases with conserved threonine in the "T-loop" activation site. A well-known substrate of LKB1is AMP-Activated Protein Kinase (AMPK), which is the master regulator of cellular and organismal metabolism. AMPK subsequently activates the TSC2complex or mTOR-binding partner raptor, which negatively regulates mTOR signaling. LKB1is involved in response to diverse signals under physiological and pathological conditions via AMPK, e.g.:skeletal muscle contraction, hypoxia, starvation, H2O2, metformin and phenformin. In addition, LKB1exerts its effects on cell polarity, proliferation, migration, senescence, apoptosis, DNA damage response, differentiation and aerobic glycolysis.Mutation of LKB1in cervical cancer was originally studied in1999. The frequency of LKB1mutation in cervical cancer is between2%to20%according to different reports. But the expression level of LKB1protein in cervical cancer is not known.At present, the molecular mechanism of LKB1as a tumor suppressor in cervical cancer is mainly focused on LKB1/AMPK pathway. It was reported that overexpression of LKB1in HeLa and SiHa could inhibit the growth of cells depending on its kinase activity. Moreover, LKB1can inhibit the anchorage-independent growth of HeLa not SiHa. AMPK activators, such as:AICAR, Metformin etc., are able to inhibit the growth of cervical cancer cell lines which have intact LKB1/AMPK/mTOR pathway. However, there are still a lot of questions about LKB1as a tumor suppressor. Are there more downstream targets of LKB1? Any new pathways involved into tumor-suppressing activity of LKB1? The expression of LKB1protein in normal cervix and cervical cancer?Detailed study of the molecular mechanisms of LKB1will help find more key molecules, provide new insights and targets for the treatment of cervical cancer.Given the importance of LKB1in tumor suppression and the potential role in development of cervical cancer, we characterized the biological activities of LKB1, performed the immunohistochemical (IHC) study for LKB1in cervical cancers and initiated the first microarray experiment to identify LKB1-regulated genes. We used the bioinformatics to analyze the differentially expressed genes and verified the data at the mRNA and protein levels. More importantly, we identified inositol polyphosphate4-phosphatase type Ⅱ, INPP4B from PI3K pathway was upregulated after LKB1overexpression. And it was further confirmed that INPP4B is an important downstream target of LKB1. INPP4B is also a potential tumor suppressor and the regulation of LKB1on INPP4B is not reported untill now.1. Characterization of LKB1expressing cervical cancer cellsTo examine the effects of LKB1expression on cervical cancer cells, we transfected a plasmid encoding LKB1into HeLa cells, which contains a deletion in the gene for LKB1, followed with short period of G418selection. Upon transfection of the plasmid encoding LKB1, it becomes readily detectable by western blotting. We then examined the proliferation of LKB1expressing HeLa cells using the CCK8assay. The data showed that,HeLa cells expressing LKB1proliferate significantly slower than the vector control cells. We also examined cell cycle profiles in HeLa cells transfected with LKB1by BrdU assay. We found that overexpression of LKB1significantly reduced the number of cells in S-phase. These results demonstrate that, after transient transfection of LKB1in HeLa, proliferation and percentage in S phase significantly decreased, so expression of LKB1inhibits cervical cancer cell proliferation. Next we also examined p-AMPK of HeLa cells stably expressing LKB1. We can detect the expression of LKB1and increase of p-AMPK in HeLa-GFP-LKB1cell. The data indicated that LKB1can activate AMPK and the pathway was intact.2. LKB1expression in cervical cancerTo characterize the expression of LKB1protein in cervical cancer, we performed immunohistochemical analysis on a series of cervical carcinoma tissues as well as normal controls. Paraffin-embedded tissue blocks were obtained (78cervical cancer:25cervical adenocarcinoma and53cervical squamous carcinoma samples, and25normal cervical tissues) from Qilu Hospital of Shandong University. For cervical adenocarcinoma tissues,56%showed positive LKB1stain while the other44%stained negative. For squamous cell carcinoma of the cervix,60.4%(32/53) of the samples were negative for LKB1, only39.6%(21/53) showed positive stain. Among the cervical cancer tissues, more than50%of samples did not express LKBl. The results were consistent with the notion for LKB1as a tumor suppressor. The observation of weak or no LKB1protein expression in normal cervix was consistent with the reports of LKB1expression in normal epithelia of colorectum and pancreas.3. Transcriptional profiling of LKB1and INPP4B as a LKB1-regulated targetAlthough it’s well known that LKB1can activate at least14downstream kinases, molecular mechanism of LKB1was still not fully understood. Therefore we performed Microarray analysis to study transcriptional profiling of LKB1. We took advantage of the HeLa cell line stably expressing LKB1, to advoid the alteration of a large number of genes by transfection. We identified222LKB1-regulated genes by microarray,117of them were down-regulated while105were up-regulated after LKBl overexpression. We then performed real-time PCR (qRT-PCR) experiment to confirm the results obtained by microarray. LKB1and the other six randomly selected genes PLK2, ABCC2, GLP2R, KYNU, MAL2and AKAP12were verified at mRNA level. Thus the qRT-PCR data suggest that probe sets used in the microarray experiments are likely to accurately measure the levels of transcripts.Next we performed bioinformatic analysis of genes differentially expressed in LKB1expressing HeLa cells. We first analyzed the differentially expressed genes using gene ontology (GO) system. In the GO database, the genes are classified into three ontologies, i.e., biological process, molecular function and cellular component. Based on the GO annotation, the222genes whose expression was significantly altered in LKB1expressing HeLa cells can be classified into40,23,20functional groups in the biological process ontology, molecular function ontology and cellular component ontology, respectively. Most LKB1-regulated genes were involved in signal transduction, protein binding and relocated on the membrane. Then we searched the differentially expressed genes against KEGG pathway maps. Eight biological pathways were identified to be significantly regulated. Two pathways involved in metabolism, i.e., arginine and proline metabolism and inositol phosphate metabolism were represented, which was consistent with a role of LKB1in metabolism. Notably, the phosphatidylinositol signaling system is also identified. It is known that the PI3K-AKT pathway and LKB1-AMPK pathway converge at mTOR. In addition, LKB1involved in axon guidance pathway and the hematopoietic cell lineage pathway.We found inositol polyphosphate4-phosphatase type II (INPP4B) in PI3K pathway was up-regulated after LKB1overexpression. We confirmed the increase of INPP4B at mRNA level and protein level by qRT-PCR and Western blot. Western blot results demonstrated that INPP4B increased1.4fold in HeLa stably-expressing LKBl and1.9fold in HeLa cells transfected with LKB1and followed with short period of G418selection. Moreover, knock-down of LKB1in the cervical carcinoma CaSki cells, which have an intact LKB1-AMPK pathway, reduced the steady state level of INPP4B. We further examined the level of p-Akt in HeLa stably-expressing LKB1and found the significant decrease in the level of p-Akt compared to the control cell of HeLa-vector. These results provide further evidence for INPP4B upregulation by LKB1and being a target for LKB1. INPP4B upregulation and decrease in level of p-Aktin HeLa stably-expressing LKB1suggest that LKB1regulates PI3K/Akt pathway negatively by upregulation of INPP4B and maybe this is a new signaling pathway for inhibition of cell growth by LKB1.In summary, we characterized the biological activities of LKB1in cervical cancer, performed transcriptional profiling of LKB1and found INPP4B as a new target in this study. The results showed that, transiently transfected LKB1inhibits the growth of cervical cancer cell, loss of LKB1expression was found in more than50%cervical cancer samples and identified LKB1-regulated222genes and8pathways. We performed bioinformatics analysis and therefore identified a target of LKB1in PI3K signaling pathway, INPP4B. INPP4B upregulation and decrease in level of p-Aktin HeLa stably-expressing LKB1suggest that LKB1regulates PI3K/Akt pathway negatively by upregulation of INPP4B and maybe this is a new signaling pathway for inhibition of cell growth by LKB1. This study sheds light on the novel signaling pathways regulated by LKB1and provides new therapeutic targets in cervical cancer in the future. Part Two:Regulation of multidrug resistance of cervical adenocarcinoma by CIP2ACervical cancer is one of the most common cancer in women worldwide. It is a major problem for women’s health and is the second mortality factor among female tumors in the developing countries. In recent years, the incidence of cervical cancer worldwide rises and patients become younger. Epidemiological studies suggest that infection of high-risk human papillomavirus (hr-HPV) is a risk factor for cervical cancer and cervical intraepithelial neoplasia.The incidence of cervical cancer worldwide has decreased due to improved screening methods and HPV prophylactic vaccines. However, prevention and early screening for cervical cancer has not been popularized in developing countries, due to economic factors and other medical conditions. Cervical cancer is still a mortality factor to women’s health. It’s well known that surgery and radiation therapy are the main treatments for cervical cancer, surgical treatment for early disease, radiation therapy for advanced. In the past, cervical cancer was considered to be not sensitive to chemotherapy and used in patients with advanced and recurrent tumors as part of a multimodality therapy.However, people performed a lot of basic and clinical research about chemotherapy in cervical cancer, and found that5-year survival rate was significantly improved in patients, thus establishing chemotherapy as an important part in cervical cancer treatment. Chemotherapy can be used in patients with tumor micrometastases, subclinical metastasis and recurrence. During the past10years, multimodality therapy has been used including surgery, radiation, and adjuvant and neoadjuvant chemotherapy for management of cervical cancer.Although the importance of chemotherapy in cervical cancer has been established, the crucial factor that influences the chemotherapeutic response is the limited sensitivity of carcinoma of the cervix to cytotoxic agents and acquired drug resistance frequently following chemotherapeutic regimens. During the formation of the drug resistance, the worst is the development of multidrug resistance which can severely impede the efficacy of chemotherapy treatment. There are two categories of mechanisms of multidrug resistance for cervical cancer cells:①natural resistance, including the overexpression of P-glycoprotein and change in DNA topoisomerase enzymes activity;②cquired resistance, including the reduction in the intracellular accumulation of the drug, increase of the drug detoxification capacity of mercapto compounds (GSH, GST, MT) and increased DNA repair capacity.P-glycoprotein (P-gp) is a transmembrane glycoprotein and is encoded by multidrug resistance gene (MDR1). P-gp is a member of sub-family B of the ATP-binding cassette (ABC) transporter superfamily, The protein displays basal ATPase activity, which is often (but not always) stimulated by substrates, and it hydrolyzes ATP to power active transport, generating a drug concentration gradient across the membrane and extrudes the drug out of the cells leading to the multidrug resistance (MDR). Almost50%of cancers have increased P-gp expression after treatment of natural chemotherapeutic regimens. Overexpression of P-gp is related with MDR in cervical cancer.CIP2A is a newly identified oncoprotein that stabilizes c-Myc protein level by inhibiting the activity of protein phosphatase2A (PP2A) dephosphorylating c-Myc S62in cancer cells. We and other researchers have reported that CIP2A is overexpressed in many carcinomas such as gastric cancer, breast cancer, non small-cell lung cancer, acute myeloid leukemia, prostate cancer and cervical cancer. Overexpression of CIP2A promotes the malignant growth of cancer cells. Furthermore, CIP2A has been demonstrated as a major determinant mediating chemotherapeutic drug-induced apoptosis in hepatocellular carcinoma, breast cancer andleukemia cells. It is still unknown whether CIP2A is related with P-gp and involved in the multi-drug resistance of cervical cancer cells in vitro and in vivo.1. CIP2A knockdown increased the sensitivity of HeLa cells to chemotherapeutics After efficient depletion of CIP2A protein expression in HeLa cell,3chemotherapeutic agents, Dox, Cis and Pac, were used to observe the cytotoxic effect by MTT assay.Dox, Cis and Pac, dose-dependently inhibited cell proliferation of HeLa cells. Such inhibition was facilitated after CIP2A siRNA knockdown. The data indicates that CIP2A might be associated with MDR of HeLa cells.2. Expression of CIP2A and P-gp in cervical canerWe obtained103paraffin-embedded tissue blocks (43cervical adenocarcinoma,16CIN I,17CIN II,12CIN III and15normal cervical tissues) from Qilu Hospital for immunohistochemistry staining of CIP2A and P-gp. CIP2A and P-gp expression were absent in15normal cervical tissue samples and16CIN I and17CIN II samples. CIP2A was present in1of12CIN III samples (8.3%) and in16of43adenocarcinoma samples (37.2%). Meanwhile, P-gp expression was not detected in any CIN III samples but in13of43adenocarcinoma samples (30.2%). CIP2A was positively stained in cytoplasm and P-gp in the cell membrane. P-gp and CIP2A immunoreactivity were uniformly both in location and intensity on the same cancer specimens. Additionally, P-gp and CIP2A expression levels were positively correlated in cervical adenocarcinoma (r2=0.617, p<0.001). P-gp expression was associated with differentiation grade (p=0.029) of tumors but not age, tumor size, grade, tumor-node-metastasis stage or lymph-node metastasis. The data showed that CIP2A and P-gp expression might be linked in cervical adenocarcinoma.3. CIP2A contributes to MDR of HeLa cells via P-gpTo further confirm that upregulation of P-gp by CIP2A, we performed the following experiments:examined P-gp, CIP2A protein levels in HeLa cells treated with Dox, Cis and Pac; examined P-gp, CIP2A protein levels in HeLa/Dox cells and P-gp protein levels after CIP2A knockdown by specific siRNA in HeLa/Dox cells; conducted Rh123efflux assay after CIP2A knockdown by specific siRNA in HeLa, HeLa/Dox cells. The results demonstrated that protein levels of P-gp and CIP2A increase in drug-treated HeLa cells; the P-gp and CIP2A levels in the drug-resistant HeLa/Dox cells were remarkably elevated compared with the HeLa cell line, P-gp protein level was significantly decreased after CIP2A knockdown by specific siRNA in HeLa/Dox cells; Flow cytometry results revealed that when the CIP2A expression was knocked down by specific siRNA, the Rh123fluorescence signal in HeLa, HeLa/Doxcells was significantly increased and indicated that the function of P-gp was inhibited.CIP2A might enhance the P-gp function via up-regulating the P-gp expression, and thus induce the drug resistance of cervical adenocarcinoma cells.In summary, our study first demonstrated CIP2A is involved in multidrug resistance of cervical adenocarcinoma via upregulation of P-gp and CIP2A.CIP2Aand P-gp are positively related in cervical adenocarcinoma.CIP2A knockdown maybe increase the sensitivity of cervical adenocarcinoma cells to chemotherapeutics. Our study suggests, CIP2A is a good target for gene therapy for clinical application.