Hidemichi Watari

Survival effect of para-aortic lymphadenectomy in endometrial cancer (SEPAL study): a retrospective cohort analysis

BACKGROUND In response to findings that pelvic lymphadenectomy does not have any therapeutic benefit for endometrial cancer, we aimed to establish whether complete, systematic lymphadenectomy, including the para-aortic lymph nodes, should be part of surgical therapy for patients at intermediate and high risk of recurrence. METHODS We selected 671 patients with endometrial carcinoma who had been treated with complete, systematic pelvic lymphadenectomy (n=325 patients) or combined pelvic and para-aortic lymphadenectomy (n=346) at two tertiary centres in Japan (January, 1986-June, 2004). Patients at intermediate or high risk of recurrence were offered adjuvant radiotherapy or chemotherapy. The primary outcome measure was overall survival. FINDINGS Overall survival was significantly longer in the pelvic and para-aortic lymphadenectomy group than in the pelvic lymphadenectomy group (HR 0.53, 95% CI 0.38-0.76; p=0.0005). This association was also recorded in 407 patients at intermediate or high risk (p=0.0009), but overall survival was not related to lymphadenectomy type in low-risk patients. Multivariate analysis of prognostic factors showed that in patients with intermediate or high risk of recurrence, pelvic and para-aortic lymphadenectomy reduced the risk of death compared with pelvic lymphadenectomy (0.44, 0.30-0.64; p<0.0001). Analysis of 328 patients with intermediate or high risk who were treated with adjuvant radiotherapy or chemotherapy showed that patient survival improved with pelvic and para-aortic lymphadenectomy (0.48, 0.29-0.83; p=0.0049) and with adjuvant chemotherapy (0.59, 0.37-1.00; p=0.0465) independently of one another. INTERPRETATION Combined pelvic and para-aortic lymphadenectomy is recommended as treatment for patients with endometrial carcinoma of intermediate or high risk of recurrence. If a prospective randomised or comparative cohort study is planned to validate the therapeutic effect of lymphadenectomy, it should include both pelvic and para-aortic lymphadenectomy in patients of intermediate or high risk of recurrence. FUNDING Japanese Foundation for Multidisciplinary Treatment of Cancer, and the Japan Society for the Promotion of Science.

Phosphorylation of Steroidogenic Acute Regulatory Protein (StAR) Modulates Its Steroidogenic Activity

Steroidogenic acute regulatory protein (StAR) plays a critical role in steroid hormone synthesis. StAR is thought to increase the delivery of cholesterol to the inner mitochondrial membrane where P450scc resides. Tropic hormones acting through the intermediacy of cAMP rapidly increase pregnenolone synthesis, and this rapid steroidogenic response is believed to be due to StAR’s action. The StAR protein contains two consensus sequences for phosphorylation catalyzed by protein kinase A that are conserved across all species in which the amino acid sequence of the StAR protein has been determined. We demonstrated that human StAR expressed in COS-1 cells exists in at least four species detectable by two-dimensional gel electrophoresis followed by Western blotting. The two more acidic species disappeared after treatment of the cell extracts with alkaline phosphatase.32P was incorporated into StAR protein immunoprecipitated from COS-1 cell extracts, and a 10-min treatment with 8-bromo-cAMP increased 32P incorporation into the StAR preprotein. StAR protein generated by in vitrotranscription/translation was phosphorylated by the protein kinase A catalytic subunit in the presence of [γ-32P]ATP. Mutation of potential sites for protein kinase A-mediated phosphorylation at serine 57 and serine 195 to alanines, individually, reduced 32P incorporation from labeled ATP into StAR preprotein produced by in vitro transcription/translation when incubated with protein kinase A catalytic subunit. 32P labeling of StAR protein expressed in COS-1 cells was also reduced when serine 57 or serine 195 were mutated to alanines. A double mutant in which both serine 57 and serine 195 were changed to alanines displayed markedly reduced 32P incorporation. To determine the functional significance of StAR phosphorylation, we tested the steroidogenic activity of the wild-type StAR and mutated StAR proteins in COS-1 cells expressing the human cholesterol side chain cleavage enzyme system. Mutation of the conserved protein kinase A phosphorylation site at serine 57 had no effect on pregnenolone synthesis. However, mutation of the serine residue at 195 resulted in an approximately 50% reduction in pregnenolone production. The S195A mutant construct did not yield the more acidic species of StAR detected in two-dimensional Western blots, indicating that the mutation affected the ability of the protein to be post-translationally modified. Mutation of the corresponding serine residues in murine StAR (Ser56 and Ser194) to alanines yielded results that were similar to those obtained with human StAR; the S56A mutant displayed a modest reduction in steroidogenic activity, whereas the S194A mutant had approximately 40% of the activity of murine wild-type StAR. In contrast to the human S195A mutation, conversion of serine 195 to an aspartic acid residue had no effect on steroidogenic activity, consistent with the idea that a negative charge at this site modulates StAR function. Our observations suggest that phosphorylation of serine 194/195 increases the biological activity of StAR and that this post- or co-translational event accounts, in part, for the immediate effects of cAMP on steroid production.

Apoptosis and Molecular Targeting Therapy in Cancer

Apoptosis is the programmed cell death which maintains the healthy survival/death balance in metazoan cells. Defect in apoptosis can cause cancer or autoimmunity, while enhanced apoptosis may cause degenerative diseases. The apoptotic signals contribute into safeguarding the genomic integrity while defective apoptosis may promote carcinogenesis. The apoptotic signals are complicated and they are regulated at several levels. The signals of carcinogenesis modulate the central control points of the apoptotic pathways, including inhibitor of apoptosis (IAP) proteins and FLICE-inhibitory protein (c-FLIP). The tumor cells may use some of several molecular mechanisms to suppress apoptosis and acquire resistance to apoptotic agents, for example, by the expression of antiapoptotic proteins such as Bcl-2 or by the downregulation or mutation of proapoptotic proteins such as BAX. In this review, we provide the main regulatory molecules that govern the main basic mechanisms, extrinsic and intrinsic, of apoptosis in normal cells. We discuss how carcinogenesis could be developed via defective apoptotic pathways or their convergence. We listed some molecules which could be targeted to stimulate apoptosis in different cancers. Together, we briefly discuss the development of some promising cancer treatment strategies which target apoptotic inhibitors including Bcl-2 family proteins, IAPs, and c-FLIP for apoptosis induction.

The Mechanism of Action of Steroidogenic Acute Regulatory Protein (StAR) StAR ACTS ON THE OUTSIDE OF MITOCHONDRIA TO STIMULATE STEROIDOGENESIS

Steroidogenic acute regulatory protein (StAR) plays an essential role in steroidogenesis, facilitating delivery of cholesterol to cytochrome P450scc on the inner mitochondrial membrane. StAR is synthesized in the cytoplasm and is subsequently imported by mitochondria and processed to a mature form by cleavage of the NH2-terminal mitochondrial targeting sequence. To explore the mechanism of StAR action, we produced 6-histidine-tagged N-62 StAR (His-tag StAR) constructs lacking the NH2-terminal 62 amino acids that encode the mitochondrial targeting sequence and examined their steroidogenic activity in intact cells and on isolated mitochondria. His-tag StAR proteins stimulated pregnenolone synthesis to the same extent as wild-type StAR when expressed in COS-1 cells transfected with the cholesterol side-chain cleavage system. His-tag StAR was diffusely distributed in the cytoplasm of transfected COS-1 cells whereas wild-type StAR was localized to mitochondria. There was no evidence at the light or electron microscope levels for selective localization of His-tag StAR protein to mitochondrial membranes. In vitro import assays demonstrated that wild-type StAR preprotein was imported and processed to mature protein that was protected from subsequent trypsin treatment. In contrast, His-tag StAR was not imported and protein associated with mitochondria was sensitive to trypsin. Using metabolically labeled COS-1 cells transfected with wild-type or His-tag StAR constructs, we confirmed that wild-type StAR preprotein was imported and processed by mitochondria, whereas His-tag StAR remained largely cytosolic and unprocessed. To determine whether cytosolic factors are required for StAR action, we developed an assay system using washed mitochondria isolated from bovine corpora lutea and purified recombinant His-tag StAR proteins expressed in Escherichia coli. Recombinant His-tag StAR stimulated pregnenolone production in a dose- and time-dependent manner, functioning at nanomolar concentrations. A point mutant of StAR (A218V) that causes lipoid congenital adrenal hyperplasia was incorporated into the His-tag protein. This mutant was steroidogenically inactive in COS-1 cells and on isolated mitochondria. Our observations conclusively document that StAR acts on the outside of mitochondria, independent of mitochondrial import, and in the absence of cytosol. The ability to produce bioactive recombinant StAR protein paves the way for refined structural studies of StAR and StAR mutants.

Mutant p53 gain-of-function induces epithelial–mesenchymal transition through modulation of the miR-130b–ZEB1 axis

The tumor suppressor gene p53 has been implicated in the regulation of epithelial–mesenchymal transition (EMT) and tumor metastasis by regulating microRNA (miRNA) expression. Here, we report that mutant p53 exerts oncogenic functions and promotes EMT in endometrial cancer (EC) by directly binding to the promoter of miR-130b (a negative regulator of ZEB1) and inhibiting its transcription. We transduced p53 mutants into p53-null EC cells, profiled the miRNA expression by miRNA microarray and identified miR-130b as a potential target of mutant p53. Ectopic expression of p53 mutants repressed the expression of miR-130b and triggered ZEB1-dependent EMT and cancer cell invasion. Loss of an endogenous p53 mutation increased the expression of miR-130b, which resulted in reduced ZEB1 expression and attenuation of the EMT phenotype. Furthermore, re-expression of miR-130b suppressed mutant p53-induced EMT and ZEB1 expression. Importantly, the expression of miR-130 was significantly reduced in EC tissues, and patients with higher expression levels of miR-130b survived longer. These data provide a novel understanding of the roles of p53 gain-of-function mutations in accelerating tumor progression and metastasis through modulation of the miR-130b–ZEB1 axis.

Emerging Therapeutic Biomarkers in Endometrial Cancer

Although clinical trials of molecular therapies targeting critical biomarkers (mTOR, epidermal growth factor receptor/epidermal growth factor receptor 2, and vascular endothelial growth factor) in endometrial cancer show modest effects, there are still challenges that might remain regarding primary/acquired drug resistance and unexpected side effects on normal tissues. New studies that aim to target both genetic and epigenetic alterations (noncoding microRNA) underlying malignant properties of tumor cells and to specifically attack tumor cells using cell surface markers overexpressed in tumor tissue are emerging. More importantly, strategies that disrupt the cancer stem cell/epithelial-mesenchymal transition-dependent signals and reactivate antitumor immune responses would bring new hope for complete elimination of all cell compartments in endometrial cancer. We briefly review the current status of molecular therapies tested in clinical trials and mainly discuss the potential therapeutic candidates that are possibly used to develop more effective and specific therapies against endometrial cancer progression and metastasis.

Downregulation of miRNA-31 induces taxane resistance in ovarian cancer cells through increase of receptor tyrosine kinase MET

Ovarian cancer is one of the most aggressive female reproductive tract tumors. Paclitaxel (PTX) is widely used for the treatment of ovarian cancer. However, ovarian cancers often acquire chemotherapeutic resistance to this agent. We investigated the mechanism of chemoresistance by analysis of microRNAs using the ovarian cancer cell line KFr13 and its PTX-resistant derivative (KFr13Tx). We found that miR-31 was downregulated in KFr13Tx cells, and that re-introduction of miR31 re-sensitized them to PTX both in vitro and in vivo. miR-31 was found to bind to the 3′-UTR of mRNA of MET, and the decrease in MET correlated to higher sensitivity to PTX. Furthermore, co-treatment of KFr13Tx cells with MET inhibitors sensitized the tumor cells to PTX both in vitro and in vivo. In addition, lower levels of miR31 and higher expression of MET in human ovarian cancer specimens were significantly correlated with PTX chemoresistance and poor prognosis. This study demonstrated miR31-dependent regulation of MET for chemoresistance of ovarian cancer, raising the possibility that combination therapy with a MET inhibitor and PTX will increase PTX efficacy.

Unveiling the mechanism of action and regulation of the steroidogenic acute regulatory protein

Stimulation of steroid-producing cells of the gonads and adrenals with trophic hormone (LH, and ACTH, respectively) produces a marked increase in steroid hormone synthesis within minutes. The rate-limiting step in this acute steroidogenic response is the transport of cholesterol from the outer to the inner mitochondrial membrane, where the first committed step in steroid synthesis is performed by the side-chain cleavage enzyme system (P450scc), resulting in the production of pregnenolone. This process of cholesterol translocation is blocked by inhibitors of protein synthesis (i.e. cycloheximide) indicating that the effect of trophic hormones, acting through the intermediacy of cAMP, most likely involves the de novo synthesis of a protein that is rapidly inactivated. The recently identified steroidogenic acute regulatory protein (StAR) appears to be the most likely candidate for the labile protein: (1) StAR is synthesized in response to cAMP and the StAR preprotein disappears rapidly in the presence of inhibitors of protein synthesis; (2) StAR has an N-terminal targeting sequence that directs the protein to the mitochondria; and (3) StAR protein is expressed almost exclusively in steroid-producing cells, its presence is correlated with steroid hormone production, and lack of functional StAR causes the autosomal recessive disease congenital lipoid adrenal hyperplasia (lipoid CAH), characterized by markedly impaired gonadal and adrenal steroid hormone synthesis. We have demonstrated that StAR is a target for serine phosphorylation mediated by protein kinase A (PKA), a process that is essential to maximizing StAR activity. StAR import by mitochondria is not essential to its steroidogenesis enhancing activity, and more likely, represents a means of rapidly inactivating StAR. Truncation mutations and site-directed mutations in StAR demonstrated that the C-terminus of the protein contains the functionally important domains. Further, we have demonstrated potent steroidogenic activity of recombinant StAR protein on isolated mitochondria from bovine corpus luteum using protein that lacks the mitochondrial targeting sequence. These observations confirm that StAR import is not essential for its steroidogenic activity and suggest that StAR acts directly on the outer mitochondrial membrane in the absence of intermediary cytosolic factors. More recently, we have found that StAR functions as a cholesterol transfer protein that does not require a protein receptor or co-factor, suggesting that StAR acts directly on lipids of the outer mitochondrial membrane to promote cholesterol translocation.

Lipopolysaccharide induces expression of genes encoding pro-inflammatory cytokines and the elastin-degrading enzyme, cathepsin S, in human cervical smooth-muscle cells.

Objective: Vaginal and amniotic infection with gram-negative bacteria is associated with preterm birth. We previously reported that human cervical smooth-muscle cells (CSMC) respond to pro-inflammatory cytokines by expressing enzymes that degrade the extracellular matrix. Our objective was to characterize the effects of lipopolysaccharide (LPS) from Escherichia coli (E coli), Bacteroides fragilis, (B frag) and Fusobacterium nucleatum (F nac) on the expression of pro-inflammatory cytokines and the elastindegrading enzyme, cathepsin S, in human CSMC. Methods: Human CSMC were exposed to LPS and the expression of mRNAs encoding pro-inflammatory cytokines and cathepsin S, and selected matrix metalloproteinases (MMPs) was analyzed by Northern blotting. The effect of cytokine-neutralizing antibodies on LPS-induced cathepsin S mRNA expression also was determined. Results: E coli LPS increased expression of cathepsin S 12.5-fold after 12 hours; MMP-1 and MMP-3 mRNAs also were increased 2.9- and 3.5-fold, respectively. Tumor necrosis factor (TNF)-α, interleukin (IL-1)α, and IL-1β mRNAs were markedly up-regulated after 3 hours of LPS treatment. B frag and F nuc LPS also induced TNF-α and cathepsin S mRNAs. E coli LPS caused a sevenfold increase in TNF-α secretion after 5 to 8 hours. Antihuman TNF-α monoclonal antibody, but not a monoclonal antibody to the low-density lipoprotein receptor,r educed the LPS-induced increase in cathepsin S mRNA by 27%, whereas neutralizing antibodies against IL-1α and IL-1β did not suppress the response. Human CSMC were shown to express the toll-like receptor (TLR-2) and TLR-4 genes, which mediate the action of LPS. TLR-2 mRNA was up-regulated by TNF-α. Conclusion: CSMC respond to LPS with increased expression of pro-inflammatory cytokines and cathepsin S. Increases in catehpsin S mRNA result only in part from the rapid induction of TNF-α gene expression. TNF-α may also augment the CSMC response to LPS by increasing expression of the LPS signaling receptor, TLR-2, which probably directly mediates LPS action. These observations provide a mechanism by which gram-negative bacteria can precipitate cervical changes associated with pretenn birth.

MicroRNA-106b modulates epithelial–mesenchymal transition by targeting TWIST1 in invasive endometrial cancer cell lines

Type II endometrial carcinoma is an aggressive subtype of endometrial cancer (EC). TWIST1, a helix‐loop‐helix transcription regulator, is known to induce epithelial–mesenchymal transition (EMT) and promote tumor metastasis. MicroRNAs (miRNAs) also serve as important regulators of EMT and metastasis by regulating EMT‐related genes. In this study, we sought to explore the role of TWIST1 in inducing EMT in representative type II EC cell lines, and to determine the miRNAs involved in regulating TWIST1 gene expression. Functional analysis suggested that TWIST1 contributes to the EMT phenotypes of EC cells, as evidenced by the acquisition of fibroblast‐like properties, enhanced invasiveness, and induction of an EN‐switch (downregulation of epithelial marker E‐cadherin and upregulation of mesenchymal marker N‐cadherin). Conversely, silencing of TWIST1 by siRNA inhibited cell invasion and the mesenchymal phenotype, which was accompanied by a reversion of the EN‐switch. We also observed a novel post‐transcriptional regulatory mechanism of TWIST1 expression mediated by miR‐106b via its direct interaction with TWIST1 mRNAs at the 3′‐untranslated region. Our data suggest that TWIST1 is a critical inducer of EMT in invasive EC cells and that miR‐106b could suppress EC cell invasion by downregulating TWIST1 expression.