Satoko Sudo

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.

Characterization of Novel Splice Variants of LGR7 and LGR8 Reveals That Receptor Signaling Is Mediated by Their Unique Low Density Lipoprotein Class A Modules

The relaxin and insulin-like peptide 3 receptors, LGR7 and LGR8, respectively, are unique members of the leucine-rich repeat-containing G-protein-coupled receptor (LGR) family, because they possess an N-terminal motif with homology to the low density lipoprotein class A (LDLa) modules. By characterizing several LGR7 and LGR8 splice variants, we have revealed that the LDLa module directs ligand-activated cAMP signaling. The LGR8-short variant encodes an LGR8 receptor lacking the LDLa module, whereas LGR7-truncate, LGR7-truncate-2, and LGR7-truncate-3 all encode truncated secreted proteins retaining the LGR7 LDLa module. LGR8-short and an engineered LGR7 variant missing its LDLa module, LGR7-short, bound to their respective ligands with high affinity but lost their ability to signal via stimulation of intracellular cAMP accumulation. Conversely, secreted LGR7-truncate protein with the LDLa module was able to block relaxin-induced LGR7 cAMP signaling and did so without compromising the ability of LGR7 to bind to relaxin or be expressed on the cell membrane. Although the LDLa module of LGR7 was N-glycosylated at position Asn-14, an LGR7 N14Q mutant retained relaxin binding affinity and cAMP signaling, implying that glycosylation is not essential for optimal LDLa function. Using real-time PCR, the expression of mouse LGR7-truncate was detected to be high in, and specific to, the uterus of pregnant mice. The differential expression and evolutionary conservation of LGR7-truncate further suggests that it may also play an important role in vivo. This study highlights the essential role of the LDLa module in LGR7 and LGR8 function and introduces a novel model of GPCR regulation.

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.

The hedgehog-patched signaling pathway and function in the mammalian ovary: A novel role for hedgehog proteins in stimulating proliferation and steroidogenesis of theca cells

The expression of hedgehog (Hh) genes, their receptor, and the co-receptor in mice, rat, and bovine ovaries were investigated. RT-PCR of ovarian transcripts in mice showed amplification of transcripts for Indian (Ihh) and desert (Dhh) Hh, patched 1 (Ptch1), and smoothened (Smo) genes. Semi-quantitative RT-PCR and northern blot analyses showed that whole ovarian Ihh and Dhh transcripts decreased 4-24 h after hCG versus 0-48 h after pregnant mares serum gonadotrophin treatment in mice, whereas mouse Ptch1 and Smo transcripts were expressed throughout the gonadotropin treatments. Quantitative real-time RT-PCR (qRT-PCR) revealed that the expression of the Hh-patched signaling system with Ihh mRNA abundance in granulosa cells was greater, whereas Smo and Ptch1 mRNA abundance was less in theca cells of small versus large follicles of cattle. In cultured rat and bovine theca-interstitial cells, qRT-PCR analyses revealed that the abundance of Gli1 and Ptch1 mRNAs were increased (P<0.05) with sonic hedgehog (SHH) treatment. Additional studies using cultured bovine theca cells indicated that SHH induces proliferation and androstenedione production. IGF1 decreased Ihh mRNA abundance in bovine granulosa cells. The expression and regulation of Ihh transcripts in granulosa cells and Ptch1 mRNA in theca cells suggest a potential paracrine role of this system in bovine follicular development. This study illustrates for the first time Hh activation of Gli1 transcriptional factor in theca cells and its stimulation of theca cell proliferation and androgen biosynthesis.

A retrospective analysis of postoperative complications with or without para-aortic lymphadenectomy in endometrial cancer.

Introduction: Although para-aortic lymphadenectomy (PALX) has not been accepted as a standard treatment for patients with endometrial cancer, it is possible that systematic lymphadenectomy including PALX has therapeutic significance for patients with intermediate-/high-risk endometrial cancer. On the other hand, a consensus regarding the safety of PALX has not been reached. The aim of this study was to compare the incidence rates of postoperative complications after pelvic lymphadenectomy (PLX) with or without PALX in patients with uterine corpus cancer. Methods: A retrospective chart review was carried out for all patients with endometrial cancer treated at 2 tertiary centers between 1998 and 2004. Surgery at one institute included both PLX and PALX, whereas PLX alone was routinely performed at the other institute. A total of 142 patients underwent PLX + PALX and 138 patients underwent PLX alone. We evaluated postoperative complications including intraoperative injury, ileus, lymphedema, lymphocyst, and thrombosis. Results: There was no fatal accident associated with surgery. Lymphedema was the most frequent complication. Comparing the PLX + PALX group and the PLX group, there were no significant differences in the rate of cases of lymphedema (23.2% vs 28.3%), lymphocyst (9.2% vs 9.4%), and thrombosis (4.9% vs 2.2%). The rate of cases of mild/moderate ileus in the PLX + PALX group was significantly higher than that in the PLX group (10.5% vs 2.9%; P = 0.011). However, no significant difference in the rates of cases of severe ileus was found between the 2 groups (1.4% vs 0.7%). There were also no significant differences between the 2 groups in the rates of intraoperative organ injury (2.8% vs 2.2%) and secondary operation for postoperative complications (4.9% vs 4.3%). Conclusions: Para-aortic lymphadenectomy can be performed with an acceptable morbidity under the conditions in which it is performed by experienced surgeons, and measures to prevent complications are properly taken.

Lack of LGR8 gene mutation in Finnish patients with a family history of cryptorchidism

Cryptorchidism is the most frequent congenital anomaly of the urogenital tract in the male. Although in Western countries 1-2% of males at the age of 3 months are diagnosed with this condition, its aetiology is still unknown. Animal models suggest a possible genetic basis for this disorder. Recently, the INSL3 (Leydig insulin-like peptide) gene and its cognate receptor, LGR8, were found to be important in testicular descent by regulating gubernacular development. Male mice null for either INSL3 or LGR8 genes exhibited bilateral cryptorchidism. Because earlier studies indicated that mutation of the INSL3 gene is not associated with the development of human cryptorchidism, this study analysed whether mutations in the LGR8 gene could be associated with this disorder. Sequencing of 18 exons of the LGR8 gene in 23 cryptorchid Finnish patients and a group of 33 control subjects allowed the identification of three nucleotide changes in exons 12 and 17, showing single base substitutions from A to G at positions 957, 993, and 1810 of LGR8. Among the three changes, only the 1810 A to G substitution is associated with an amino acid change from isoleucine to valine (Ile604Val) located in the fifth transmembrane domain of this seven-transmembrane receptor. This change was more frequent in a control group of normal fertile adult males and infant boys than in the group of cryptorchid males. The change is not associated with altered receptor signalling, thus suggesting the presence of a polymorphism unrelated to the cryptorchid phenotype. These data indicate that mutations involving the human LGR8 gene do not represent a frequent cause of cryptorchidism in the Finnish population.

Identification of KLF17 as a novel epithelial to mesenchymal transition inducer via direct activation of TWIST1 in endometrioid endometrial cancer

Krüppel-like factor 17 (KLF17), a member of the KLF transcription factor family, has been shown to inhibit the epithelial-mesenchymal transition (EMT) and tumor growth. However, the expression, the cellular function and the mechanism of KLF17 in endometrioid endometrial cancer (EEC; a dominant type of endometrial cancer) remain elusive. Here, we report that among the KLF family members, KLF17 was consistently upregulated in EEC cell lines compared with immortalized endometrial epithelial cells. Overexpression of KLF17 in EEC cell lines induced EMT and promoted cell invasion and drug resistance, resulting in increased expression of TWIST1. In contrast, KLF17 suppression reversed EMT, diminished cell invasion, restored drug sensitivity and suppressed TWIST1 expression. Luciferase assays, site-directed mutagenesis and transcription factor DNA-binding analysis demonstrated that KLF17 transactivates TWIST1 expression by directly binding to the TWIST1 promoter. Knockdown of TWIST1 prevented KLF17-induced EMT. Consistent with these results, both KLF17 and TWIST1 levels were found to be elevated in EECs compared with normal tissues. KLF17 expression positively correlated with tumor grade but inversely correlated with estrogen and progesterone receptor expression. Thus, KLF17 may have an oncogenic role during EEC progression via initiating EMT through the regulation of TWIST1.

Multivariate prognostic analysis of adenocarcinoma of the uterine cervix treated with radical hysterectomy and systematic lymphadenectomy

Objective The aim of this study was to investigate the prognostic factors and treatment outcome of patients with adenocarcinoma of the uterine cervix who underwent radical hysterectomy with systematic lymphadenectomy. Methods A total of 130 patients with stage IB to IIB cervical adenocarcinoma treated with hysterectomy and systematic lymphadenectomy from 1982 to 2005 were retrospectively analyzed. Clinicopathological data including age, stage, tumor size, the number of positive node sites, lymphovascular space invasion, parametrial invasion, deep stromal invasion (>2/3 thickness), corpus invasion, vaginal infiltration, and ovarian metastasis, adjuvant therapy, and survival were collected and Cox regression analysis was used to determine independent prognostic factors. Results An estimated five-year survival rate of stage IB1 was 96.6%, 75.0% in stage IB2, 100% in stage IIA, and 52.8% in stage IIB. Prognosis of patients with one positive-node site is similar to that of those with negative-node. Prognosis of patients with multiple positive-node sites was significantly poorer than that of negative and one positive-node site. Multivariate analysis revealed that lymph node metastasis, lymphovascular space invasion, and parametrial invasion were independent prognostic factors for cervical adenocarcinoma. Survival of patients with cervical adenocarcinoma was stratified into three groups by the combination of three independent prognostic factors. Conclusion Lymph node metastasis, lymphovascular space invasion, and parametrial invasion were shown to be independent prognostic factors for cervical adenocarcinoma treated with hysterectomy and systematic lymphadenectomy.

Initial failure site according to primary treatment with or without para-aortic lymphadenectomy in endometrial cancer

OBJECTIVE The objective of this study was to compare the initial failure sites in patients with endometrial cancer who underwent surgical treatment including pelvic lymphadenectomy with or without para-aortic lymphadenectomy. METHODS A retrospective chart review was carried out for 657 endometrial cancer patients with no residual disease after initial treatments including lymphadenectomy at two tertiary centers between 1987 and 2004. Surgical treatment at one institute included pelvic lymphadenectomy (PLX) without para-aortic lymphadenectomy (PALX), while surgical treatment including PLX+PALX was routinely performed at the other institute. We identified patients with recurrence and evaluated initial failure sites. Rates of recurrence in the respective sites were compared according to the type of lymphadenectomy. RESULTS Of the 657 patients, 103 (15.7%) suffered recurrence. There was no significant difference between the rate of intrapelvic recurrence in the PLX alone group and that in the PLX+PALX group (4.7% vs. 2.9%, p=0.22). The rate of extrapelvic recurrence in the PLX alone group was significantly higher than that in the PLX+PALX group (16.1% vs. 6.2%, p<0.0001), and the rate of para-aortic node (PAN) recurrence in the PLX alone group was also significantly higher than that in the PLX+PALX group (5.1% vs. 0.6%, p=0.0004). In the analysis of patients who received adjuvant chemotherapy, the rate of PAN recurrence in the PLX alone group was significantly higher than that in the PLX+PALX group (9.5% vs. 1.3%, p=0.0036). CONCLUSION PAN recurrence was a failure pattern peculiar to the PLX alone group. Adjuvant chemotherapy might not be able to replace surgical removal as a treatment for metastatic lymph nodes.

Survival and failure pattern of patients with endometrial cancer after extensive surgery including systematic pelvic and para-aortic lymphadenectomy followed by adjuvant chemotherapy.

We investigated the survival and the failure pattern of 288 patients with endometrial cancer treated with extensive surgery including systematic pelvic and para-aortic lymphadenectomy followed by cisplatin-based chemotherapy from 1982 to 2002. We correlated the failure pattern with various clinicopathologic factors to find the predictors of recurrence sites. The 5-year overall survival rates were 97.5% for stage I, 87.5% for stage II, 85.2% for stage III, and 12.5% for stage IV. Notably, the 5-year survival rate was 76.5% for patients with stage IIIC disease. Among patients with a low risk (n = 92) for recurrence who received no adjuvant chemotherapy, 2 (2.2%) showed recurrent disease. Among those with intermediate (n = 98) and high (n = 98) risks for recurrence who received adjuvant chemotherapy, 9 (9.2%) and 20 (20.4%) showed recurrent disease, respectively. The recurrence sites were described as follows: distant (n = 12), vaginal (n = 8), peritoneal (n = 7), pelvic (n = 2), and lymphatic (n = 2). Lymphatic failure was found beyond the area of lymphadenectomy. Architectural and nuclear grades; myometrial, lymph-vascular space, and cervical invasions; and lymph node metastasis were predictors of distant failure. Cervical invasion and lymph node metastasis were predictors of vaginal failure. For patients with stage I/II cancer, the architectural and nuclear grades were related to distant failure. Seven (63.6%) of 11 patients with a low or intermediate risk survived after relapse, whereas only 1 (4.8%) of 21 patients with a high risk survived after a recurrence. We conclude that we need to further test the efficacy of systemic adjuvant therapy using new chemotherapeutic regimens to prevent distant failure and to improve the survival of patients with endometrial cancer.