The Role Of Protein Kinase D1Regulated Signaling Pathway In The Carcinogenesis And Progression Of Non-small Cell Lung Cancer

Malignant tumor is a serious threat to human health and disease. As the Number one cause of all deaths in China, it accounts for25%of all deaths. Globally, the morbidity of lung cancer is increasing rapidly and remains the leading cause of cancer death. In the past30years, the mortality for lung cancer increased for465%. It was reported that the morbidity of lung cancer is increasing26.9%every year. Smoking, air pollution is strongly associated with lung cancer. Lung cancer comprises non-small cell lung cancer(NSCLC) and small cell lung cancer (SCLC), in which NSCLC accounts for85%of all cases and is identified as the most commom and fatal lung cancer. Despite the development of early diagnosis menthods, around70%of patients therefore present with advanced stage IIIB or IV disease. Surgery, radical radiotherapy and chemotherapy is the major treatment method but5-year cure rates have only barely improved. With these problems in mind, it is timely that a new approach to treatment is emerging with targeted therapy. Mutations in the epidermal growth factor receptor (EGFR) have been identified in NSCLC, and overexpression of EGFR and its ligands has made it an attractive target for various antitumor strategies. EGFR inhibitors such as gefitinib and erotinib are gradually applied in advanced NSCLC patients. However, EGFR-TKI inhibitors represent better efficacy for specific population, and the tolerance of these drugs urgently need to be resolved. Therefore, further understanding of the related cancer cell signaling pathway and exploring novel therapy target is necessary.Protein kinase D (PKD) is a novel serine-threonine protein kinase family within the CAMK group. PKD is activated by a number of different agents, including GPCR agonists, growth factors and phorbol esters. The PKD family contains3members that are homologous in structure and function, namely, PKD1, PKD2,and PKD3. PKD1is the founding and the most studied member of the family. The PKD1gene, located on human chromosome14q11, is broadly expressed in many organs, including the thyroid, brain, heart, and lungs. PKD1has been shown to play important roles in a variety of cellular functions that regulate intracellular signal transduction pathways, cell survival, proliferation, motility, invasion, angiogenesis, and apoptosis. PKD1also plays a critical role in cardiac cell functioning and maintenance of cardiovascular health. Thus, the deregulation of PKD1has been connected with the development of cancers, cardiovascular hypertrophy and other diseases. Recently, PKD1has been shown to be downregulated in prostate cancer, breast cancer, gastric cancer and colon cancer. However, the overexpression of PKD1has been shown to play a role in the development of pancreatic cancer and skin cancers. Because PKD1functions as a critical kinase that integrates extracellular signals into intracellular processes by modulating a multitude of signaling pathways, the regulation of PKD1levels and/or activity through pharmacological or genetic intervention might aid in cancer treatment.PKD1modulates a variaty of cancer-related signaling pathways and therefore regulates multiple biological functions which are critical for the functions of the cancer cells. PKD1mediates the activation of MAPK, JNK and NFκB signaling pathways in response to GPCR agonists. However, the exact functions of PKD1in the signaling pathways regulation is still unknown. In the present study, we aim to explore the regulation of PI3K/Akt and HDAC mediated by PKD1. Also, we examine the expression of PKD1in NSCLC tissues and explore the function and mechanism of PKD1in NSCLC. These results raise the possibility that tumor-specific delivery of the PKD1gene or PKD1activators may have potential therapeutic value in NSCLC patients. Objectives:1. To identify the activity of PI3K/Akt regualted by PKD1;2. To explore the phosphorylations of HDAC4,5,7mediated by PKD1and its impacts for cell cycle and cell proliferation;3. To examine the expression patterns and functions of PKD1in NSCLC;4. To explore the possible mechanism of PKD1functions in NSCLC.Section Ⅰ PKD1Mediates Negative Feedback of PI3K/Akt Activation in Response to G Protein-Coupled ReceptorsObjectives:1. To examine the impacts of PKD1inhibitors and siRNAs to PI3K/Akt;2. To explore the mechanism of PKD1regulating PI3K/Akt;3. To validate the regulation pathway in transgenic animal models.Methods:1. Stimulation of intestinal epithelial IEC-18cells with angiotensin Ⅱ (ANG Ⅱ), a mitogenic agonist that activates Gq-coupled receptors endogenously expressed by these cells. Cultures of IEC-18cells were treated with increasing concentrations of the selective PKD family inhibitor kb NB142-70for1h and then challenged with50nM ANG Ⅱ. Akt phosphorylation at Thr308and Ser473was examined by western blot.2. IEC-18cells were treated with another PKD family inhibitor CRT0066101or transfected with siRNAs. Akt phosphorylation at Thr308and Ser473was examined by western blot.3. Cultures of IEC-18cells were treated without or with kb NB142-70or CRT0066101, stimulated with ANG Ⅱ and lysed. The p85aregulatory subunit of PI3K was immunoprecipitated from the lysates and the resulting immunoprecipitates were analyzed by immunoblotting with a motif-specific antibody that detects Ser/Thr phosphorylated by PKD family members. We then examined whether PKD1stimulates binding of p85ato PTEN in intestinal epithelial cells. 4. we used transgenic mice that express elevated PKD1protein in the small intestine epithelium. Total PKD1and pAkt was examined by western blot.Results:1. Exposure of IEC-18cells to the selective PKD family inhibitor kb NB142-70potentiates GPCR-induced Akt activation. The selective PKD family inhibitor CRT0066101and knockdown of PKD1potentiate GPCR-induced Akt phosphorylation at Thr308and Ser473.2. Inhibition of PKD1increases Akt translocation to the plasma membrane in response to GPCR agonists. Inhibitors of class Ⅰ A PI3K and EGFR prevent the potentiation of Akt induced by suppression of PKD1activity.3. ANG Ⅱ markedly increased the phosphorylation of p85adetected by a PKD motif-specific antibody and enhanced the association of p85awith PTEN.4. Transgenic mice overexpressing PKD1showed a reduced phosphorylation of Akt at Ser473in intestinal epithelial cells compared to wild type littermates.Conclusions:1. PKD1activation mediates feedback inhibition of PI3K/Akt signaling.2. PKD1-mediated phosphorylation of p85α mediates negative feedback of PIP3accumulation and Akt phosphorylation in GPCR-stimulated cells, at least in part, by enhancing the stimulatory association of p85a with PTEN. Section Ⅱ Protein Kinase D1mediates Class Ⅱa Histone Deacetylase Phosphorylation and Nuclear Extrusion:Role in Mitogenic SignalingObjectives:To examine whether class Ⅱa histone deacetylases (HDACs) play a role in mitogenic signaling mediated by protein kinase D1(PKD1) Methods:1. Cell lysates were analyzed by Western blotting using antibodies thatrecognize class Ⅱa HDACs4,57and9. We then used an antibody that detects the phosphorylated state of HDAC4at Ser246, HDAC5at Ser259and HDAC7at Ser155and a second antibody that recognizes the phosphorylated state HDAC4at Ser632, HDAC5at Ser498and HDAC7at Ser486in cells stimulated with GPCR agonists(ANGII).2. We used the recently identified preferential PKD family inhibitors kb NB142-70and CRT0066101which act as potent PKD1inhibitors in intact IEC-18cells. Cultures of IEC-18cells were treated with increasing concentrations of kb NB142-70or CRT0066101for1h and then stimulated with ANGⅡ.3. The effect of ANG Ⅱ on endogenous HDAC5nucleocytoplasmic shuttling was examined by immunofluorescence analysis. Cultures of IEC-18cells were transfected with epitope (FLAG)-tagged HDAC5or an identical construct in which Ser259and Ser498were mutated to non-phosphorylatable Ala.4. Cultures of IEC-18cells in serum-free medium were stimulated with ANG Ⅱ in the absence or presence ofincreasing concentrations of the specific class Ⅱa HDAC inhibitor MC1568and DNA synthesis was assessed by measuring [3H]thymidine incorporation into acid-precipitable material.5. We then used transgenic mice that express elevated PKD1protein in the small intestine epithelium and display a marked increase in DNAsynthesizing cells in their intestinal crypts and a significant increase in the length and total number of cells per crypt.Results:1. Class Ⅱa HDAC4,5and7are prominently expressed in these cells. Simulation with angiotensin Ⅱ (ANG Ⅱ), a potent mitogen for IEC-18cells, induced a striking increase in the phosphorylation of HDAC4at Ser246and Ser632, HDAC5at Ser259and Ser498and HDAC7at Ser155.2. Treatment with the PKD family inhibitors kb NB142-70and CRT0066101or siRNA-mediated knockdown of PKD1prevented ANG Ⅱ-induced phosphorylation of HDAC4,5and7.3. PKD1-mediated phosphorylation of HDAC5induces its nuclear extrusion into the cytoplasm. In contrast, HDAC5with Ser259and Ser498mutated to Ala was localized to the nucleus in both unstimulated and stimulated cells.4. Treatment of IEC-18cells with specific inhibitors of class ⅡaHDACs, including MC1568and TMP269, prevented cell cycle progression, DNA synthesis and proliferation induced in response to GPCR/PKD1activation.5. The PKD1/class Ⅱa HDAC axis also functions in intestinal epithelial cell in vivo.Conclusions:Our results reveal a PKD1/classIIa HDAC axis in intestinal epithelial cells leading to mitogenic signaling. Section Ⅲ PKD1is downregulated in non-small cell lung cancer and mediates the feedback inhibition of mTORCl-S6K1axis in responseto phorbol esterObjectives:1. To determine the expression patterns and the role of PKD1in NSCLC;2. To elucidate the regulation of the mTORC1activity by PKD1.Methods:1. Thirty-four pairs of human NSCLC and matched normal bronchiolar epitheliums were enrolled and evaluated for PKD1expression by quantitative real-time PCR.2. Exposure of NSCLC A549and H520cells to the PKD family inhibitor kb NB 142-70(Kb), S6K1phosphorylation at Thr389and S6phosphorylation at Ser235/236was determined by western blot.3. We then used the PI3K inhibitors LY294002, BKM120and MEK inhibitors U0126, PD0325901to block the enhanced S6K1activity induced by the PKDl inhibition by Kb.Results:1. PKD1was downregulated in26of34cancer tissues in comparison with matched normal epitheliums. Moreover, patients with venous invasion or lymph node metastasis showed significant lower expression of PKD1.2. Exposure of NSCLC A549and H520cells to the PKD family inhibitor kb NB142-70(Kb), at concentrations that inhibited PKD1activation, strikingly potentiated S6K1phosphorylation at Thr389and S6phosphorylation at Ser235/236in response to phorbol ester (PMA).3. Knockdown of PKD1with siRNA also strikingly enhanced S6K1phosphorylation in response to PMA stimulation.4. exposure of cells to either Kb/PI3K inhibitor or Kb/MEK inhibitor strikingly inhibits S6K1and S6phosphorylation in response to PMA.Conclusions:Our results identify decreased expression of the PKD1as a marker for NSCLC and the loss of PKD1expression increases the malignant potential of NSCLC cells. This may be due to the function of PKD1as a negative regulator of mTORCl-S6K1. Re-expression or activation of PKDl might serve as a potential therapeutic target for NSCLC treatment.