KLF10 loss in the pancreas provokes activation of SDF-1 and induces distant metastases of pancreatic ductal adenocarcinoma in the KrasG12D p53flox/flox model.

Krüppel-like transcription factor 10 (KLF10), also named as TIEG1, plays essential roles in mediating transforming growth factor beta (TGFß) signaling and has been shown to function as a tumor suppressor in multiple cancer types. However, its roles in mediating cancer progression in vivo have yet to be fully characterized. Here, we have employed two well-characterized Pdx-1CreLSL-KrasG12D and Pdx-1CreLSL-KrasG12Dp53L/L pancreatic cancer models to ablate KLF10 expression and determine the impact of KLF10 deletion on tumor development and progression. We show that loss of KLF10 cooperates with KrasG12D leading to an invasive and widely metastatic phenotype of pancreatic ductal adenocarcinoma (PDAC). Mechanistically, loss of KLF10 in PDAC is shown to increase distant metastases and cancer stemness through activation of SDF-1/CXCR4 and AP-1 pathways. Furthermore, we demonstrate that targeting the SDF-1/CXCR4 pathway in the context of KLF10 deletion substantially suppresses PDAC progression suggesting that inhibition of this pathway represents a novel therapeutic strategy for PDAC treatment.

Drug-Eluting Bead Transarterial Chemoembolization as Bridge Therapy for Hepatocellular Carcinoma Before Living-Donor Liver Transplantation.

BACKGROUND: The majority of hepatocellular carcinoma (HCC) patients is diagnosed in late stages and therefore becomes ineligible for potentially curative treatment such as resection or liver transplantation. Transarterial chemoembolization (TACE) with drug-eluting beads (DC beads, Biocompatibles, Farnham, United Kingdom) has been proven with less side effects and better efficacy than conventional TACE, especially among patients with poor liver function. PURPOSE: The aim of this study is to evaluate outcomes of HCC patients who received TACE with DC beads, which resulted to eligibility for liver transplantation. METHODS AND MATERIALS: From January 2012 to June 2015, 60 patients with HCC received pre-liver transplantation evaluation whose cases were managed with TACE using DC beads at Kaohsiung Chang Gung Memorial Hospital were included in the study. DC beads loaded with doxorubicin were used. RESULTS: Forty percent of the patients had complete tumor response. Thirty-three percent of the patients had partial tumor response, of which 15% showed stable disease, 11.7% exhibited disease progression including 3 with portal vein thrombosis, 1 with both hepatic vein and portal vein thrombosis, and 3 with increase in tumor size. Twenty-three patients were beyond University of California, San Francisco (UCSF) criteria initially. The successful downstage rate was 73.9% (17 of 23). Thirty-seven patients fit the USCF criteria initially. The 3-, 6- and 12-month drop rates of these patients were 0%, 3.9%, and 16.8%, respectively. Twenty-four (40%) patients successfully underwent liver transplantation. Three patients (12.5%) demonstrated recurrent HCC after liver transplantation. CONCLUSION: TACE with DC bead can effectively induce tumor necrosis and appears to be a successful approach as bridge therapy for patients with advanced HCC and poor liver function.

Leydig cells contribute to the inhibition of spermatogonial differentiation after irradiation of the rat.

Irradiation with 6 Gy produces a complete block of spermatogonial differentiation in LBNF1 rats that would be permanent without treatment. Subsequent suppression of gonadotropins and testosterone (T) restores differentiation to the spermatocyte stage; however, this process requires 6 weeks. We evaluated the role of Leydig cells (LCs) in maintenance of the block in spermatogonial differentiation after exposure to radiation by specifically eliminating functional LCs with ethane dimethane sulfonate (EDS). EDS (but not another alkylating agent), given at 10 weeks after irradiation, induced spermatogonial differentiation in 24% of seminiferous tubules 2 weeks later. However, differentiation became blocked again at 4 weeks as LCs recovered. When EDS was followed by treatment with GnRH antagonist and flutamide, sustained spermatogonial differentiation was induced in >70% of tubules within 2 weeks. When EDS was followed by GnRH antagonist plus exogenous T, which also inhibits LC recovery but restores follicle stimulating hormone (FSH) levels, the spermatogonial differentiation was again rapid but transient. These results confirm that the factors that block spermatogonial differentiation are indirectly regulated by T, and probably FSH, and that adult and possibly immature LCs contribute to the production of such inhibitory factors. We tested whether insulin-like 3 (INSL3), a LC-produced protein whose expression correlated with the block in spermatogonial differentiation, was indeed responsible for the block by injecting synthetic INSL3 into the testes and knocking down its expression in vivo with siRNA. Neither treatment had any effect on spermatogonial differentiation. The Leydig cell products that contribute to the inhibition of spermatogonial differentiation in irradiated rats remain to be elucidated.

APC haploinsufficiency coupled with p53 loss sufficiently induces mucinous cystic neoplasms and invasive pancreatic carcinoma in mice.

Adenomatous polyposis coli (APC), a tumor-suppressor gene critically involved in familial adenomatous polyposis, is integral in Wnt/ß-catenin signaling and is implicated in the development of sporadic tumors of the distal gastrointestinal tract including pancreatic cancer (PC). Here we report for the first time that functional APC is required for the growth and maintenance of pancreatic islets and maturation. Subsequently, a non-Kras mutation-induced premalignancy mouse model was developed; in this model, APC haploinsufficiency coupled with p53 deletion resulted in the development of a distinct type of pancreatic premalignant precursors, mucinous cystic neoplasms (MCNs), exhibiting pathomechanisms identical to those observed in human MCNs, including accumulation of cystic fluid secreted by neoplastic and ovarian-like stromal cells, with 100% penetrance and the presence of hepatic and gastric metastases in >30% of the mice. The major clinical implications of this study suggest targeting the Wnt signaling pathway as a novel strategy for managing MCN.

Increasing testicular temperature by exposure to elevated ambient temperatures restores spermatogenesis in adult Utp14b (jsd) mutant (jsd) mice.

Because mutations in the human UTP14C gene are associated with male infertility, we sought to develop a method for fertility restoration in azoospermic mice with a mutation in the orthologous Utp14b(jsd) (jsd) gene that have spermatogonial arrest. The method is based on our observation that elevation of testicular temperatures restores spermatogonial differentiation in jsd mutant mice. To non-surgically raise intrascrotal temperatures we placed these mice in incubators at different elevated ambient temperatures. Exposure of jsd/jsd mice to ambient temperatures of 34.5 °C or 35.5 °C for 24 days increased the proportion of tubules with spermatocytes from 0% in untreated controls to over 80%. As those higher temperatures interfere with spermatid differentiation, the mice were then transferred to incubators at 32-32.5 °C for the next 24 days. These environments allowed differentiation to progress, resulting in up to 42% of tubules having late spermatids and about half of the mutant mice having spermatozoa in testicular suspensions. When these spermatozoa were used in intracytoplasmic sperm injection, all gave rise to viable healthy offspring with normal weight gain and fertility. The successful restoration of fertility in Utp14b mutant mice suggests that transient testicular warming might also be useful for spermatogenesis recovery in infertile men with UTP14C gene mutations.

Hormone suppression with GnRH antagonist promotes spermatogenic recovery from transplanted spermatogonial stem cells in irradiated cynomolgus monkeys.

Hormone suppression given before or after cytotoxic treatment stimulates the recovery of spermatogenesis from endogenous and transplanted spermatogonial stem cells (SSC) and restores fertility in rodents. To test whether the combination of hormone suppression and transplantation could enhance the recovery of spermatogenesis in primates, we irradiated (7 Gy) the testes of 12 adult cynomolgus monkeys and treated six of them with gonadotropin-releasing hormone antagonist (GnRH-ant) for 8 weeks. At the end of this treatment, we transfected cryopreserved testicular cells with green fluorescent protein-lentivirus and autologously transplanted them back into one of the testes. The only significant effect of GnRH-ant treatment on endogenous spermatogenesis was an increase in the percentage of tubules containing differentiated germ cells (tubule differentiation index; TDI) in the sham-transplanted testes of GnRH-ant-treated monkeys compared with radiation-only monkeys at 24 weeks after irradiation. Although transplantation alone after irradiation did not significantly increase the TDI, detection of lentiviral DNA in the spermatozoa of one radiation-only monkey indicated that some transplanted cells colonized the testis. However, the combination of transplantation and GnRH-ant clearly stimulated spermatogenic recovery as evidenced by several observations in the GnRH-ant-treated monkeys receiving transplantation: (i) significant increases (~20%) in the volume and weight of the testes compared with the contralateral sham-transplanted testes and/or to the transplanted testes of the radiation-only monkeys; (ii) increases in TDI compared to the transplanted testes of radiation-only monkeys at 24 weeks (9.6% vs. 2.9%; p = 0.05) and 44 weeks (16.5% vs. 6.1%, p = 0.055); (iii) detection of lentiviral sequences in the spermatozoa or testes of five of the GnRH-ant-treated monkeys and (iv) significantly higher sperm counts than in the radiation-only monkeys. Thus hormone suppression enhances spermatogenic recovery from transplanted SSC in primates and may be a useful tool in conjunction with spermatogonial transplantation to restore fertility in men after cancer treatment.

Rat models of post-irradiation recovery of spermatogenesis: interstrain differences.

Recently, we reported large differences between rat strains in spermatogenesis recovery at 10 weeks after 5-Gy irradiation suggesting that there are interstrain as well as interspecies differences in testicular radiation response. To determine whether these interstrain differences in sensitivity might be a result of the particular dose and time-point chosen, we performed dose-response and time-course studies on sensitive Brown-Norway (BN) and more resistant spontaneously hypertensive rats (SHR) and Sprague-Dawley (SD) rats. Type A spermatogonia were observed in atrophic tubules at 10 weeks after irradiation in all strains indicating that tubular atrophy was caused by a block in their differentiation, but the doses to produce the block ranged from 4.0 Gy in BN to 10 Gy in SD rats. Although the numbers of type A spermatogonial were unaffected at doses below 6 Gy, higher doses reduced their number, indicating that stem cell killing also contributed to the failure of recovery. After 10 weeks, there was no further recovery and even a decline in spermatogonial differentiation in BN rats, but in SHR rats, sperm production returned to control levels by 20 weeks after 5.0 Gy and, after 7.5 Gy, differentiation resumed in 60% of tubules by 30 weeks. Suppression of testosterone and gonadotropins after irradiation restored production of differentiated cells in nearly all tubules in BN rats and in all tubules in SHR rats. Thus, the differences in recovery of spermatogenesis between strains were a result of both quantitative differences in their sensitivities to a radiation-induced, hormone-dependent block of spermatogonial differentiation and qualitative interstrain differences in the progression of post-irradiation recovery. The progression of recovery in SHR rats was similar to the prolonged delays in recovery of human spermatogenesis after cytotoxic agent exposure and thus may be a system for investigating a phenomenon also observed in men.

Anticonvulsant effect of BmK IT2, a sodium channel-specific neurotoxin, in rat models of epilepsy.

BACKGROUND AND PURPOSE: The sodium channel is a primary target for treating central nervous system disorders such as epilepsy. In this study the anticonvulsant effect of BmK IT2, a sodium channel-specific neurotoxin, was evaluated in different animal models of epilepsy. EXPERIMENTAL APPROACH: Experiments were performed on freely moving rats made epileptic by administration of either pentylenetetrazole (PTZ) or pilocarpine. BmK IT2 (0.05-0.5 microg in 2 microl) was microinjected into the CA1 area and its effects on PTZ-induced widespread, seizure-like behaviour and cortex epileptiform EEG, as well as on pilocarpine-induced seizure-like behaviour and c-Fos expression were studied. KEY RESULTS: Intrahippocampal application of BmK IT2 dose-dependently inhibited PTZ-induced seizure-like behaviour, and reduced the numbers and duration of the high amplitude and frequency discharges (HAFDs) of the epileptiform EEG component induced by PTZ. Similarly, in the pilocarpine-induced status epilepticus (SE) model, BmK IT2 significantly prolonged the latency to onset of the SE, reduced the severity of SE and suppressed hippocampal c-Fos expression during SE. CONCLUSIONS AND IMPLICATIONS: BmK IT2 showed anticonvulsant activity as it inhibited the widespread seizures induced by PTZ and pilocarpine-induced SE in rats. This activity might be due to the modulation of sodium channels in the hippocampus. Hence, BmK IT2 could be used as a novel tool to explore the molecular and pathological mechanisms of epilepsy with regard to the involvement of sodium channels.