Endoscopic ultrasonography for microvascular imaging without contrast enhancement in the differential diagnosis of pancreatic lesions.

OBJECTIVES: Detective flow imaging endoscopic ultrasonography (DFI-EUS) is a recent imaging modality developed for detecting fine vessels without the need for ultrasound contrast agents. The aim of the present study was to evaluate the utility of DFI-EUS for solid pancreatic lesions and to compare the diagnostic ability for pancreatic cancer (PC) between DFI-EUS, directional power Doppler (eFLOW) EUS, and contrast-enhanced harmonic (CH)-EUS. METHODS: Patients with a pancreatic lesion who underwent DFI-EUS, eFLOW-EUS, and CH-EUS between March 2019 and November 2023 were retrospectively enrolled. Final diagnoses were confirmed by pathologic examination of EUS-guided tissue acquisition and/or resected specimens. Lesions were categorized into the three patterns of poor, mild, and rich vascularity on DFI-EUS and eFLOW-EUS, and hypo-, iso-, and hypervascular on CH-EUS. PC was defined as a poor pattern on DFI-EUS and eFLOW-EUS, and a hypovascular pattern on CH-EUS. RESULTS: The final diagnoses of 90 examined tumors were PC (n = 57), inflammatory mass (n = 6), autoimmune pancreatitis (n = 13), neuroendocrine tumor (n = 9), and others (n = 5). The sensitivity, specificity, and accuracy for diagnosis of PC were 93%, 82%, and 88%, respectively, on DFI-EUS, 97%, 42%, and 77% on eFLOW-EUS, and 95%, 89%, and 92% on CH-EUS. The accuracy of DFI-EUS was significantly superior to eFLOW-EUS (P = 0.005), but no significant difference was found between DFI-EUS and CH-EUS. CONCLUSION: DFI-EUS is more sensitive for depicting vasculature than eFLOW-EUS, and has higher diagnostic sensitivity for PC. Evaluation of vascularity on DFI-EUS is useful for the differential diagnosis of pancreatic lesions without the need for intravenous contrast agent.

Multicenter study comparing EUS-guided hepaticogastrostomy and ERCP for malignant biliary obstruction in patients with accessible papillae.

BACKGROUND: One advantage of endoscopic ultrasound-guided hepaticogastrostomy (EUS-HGS) is that it is difficult for reflux cholangitis, caused by duodenal pressure increasing due to duodenal obstruction, to occur. In addition, since stent deployment is performed away from the malignant stricture site, longer stent patency than with endoscopic retrograde cholangiopancreatography (ERCP) may be obtained. However, no study has previously compared EUS-HGS and ERCP for patients without duodenal obstruction or surgically altered anatomy. The aim of the present study was to compare clinical outcomes between EUS-HGS and ERCP in normal anatomy patients without duodenal obstruction. METHOD: In the ERCP group, patients who initially underwent biliary drainage were included. In the EUS-HGS group, patients who underwent EUS-HGS due to failed biliary cannulation were included. Patients with an inaccessible papilla, such as with surgically altered anatomy or duodenal obstruction, were excluded. RESULTS: A total of 314 patients who underwent ERCP and EUS-HGS were enrolled in this study. Of the 314 patients, 289 underwent biliary stenting under ERCP guidance, and 25 patients underwent biliary stenting under EUS-HGS. After propensity score-matching analysis, the adverse event rate tended to be lower in the EUS-HGS group than in the ERCP group. Although overall survival was not significantly different between the EUS-HGS and ERCP groups (p = .228), stent patency was significantly longer in the EUS-HGS group (median 366.0 days) than in the ERCP group (median 76.5 days). CONCLUSIONS: EUS-HGS had a lower adverse event rate, shorter procedure time, and longer stent patency than ERCP in cases of normal anatomy without duodenal obstruction.

Endoscopic ultrasound-guided hepaticogastrostomy vs. antegrade metal stent placement keeping an access route in patients with malignant biliary obstruction.

BACKGROUND: Few studies have compared endoscopic ultrasound (EUS)-guided hepaticogastrostomy (HGS) with EUS-guided antegrade metal stent placement (AGMS). The purpose of this study was to compare times to recurrent biliary obstruction (TRBO) in patients who underwent HGS using metal stents (MS) and those who underwent AGMS keeping access routes with plastic stents (AGMS-AR). METHODS: This study retrospectively evaluated consecutive patients who underwent HGS or AGMS between September 2016 and December 2022. TRBO, overall survival (OS), and adverse event (AE) rates were compared in the two groups. The risk factors for RBO were determined using a multivariable Cox proportional hazards model. RESULTS: This study included 32 patients in the HGS group and 30 in the AGMS-AR group. Technical success rate was significantly higher in the HGS than in the AGMS-AR group (100 vs. 80%; P = 0.009). The technical success rate without tract dilation was significantly higher in the AGMS-AR than in the HGS group (83 vs. 38%; P < 0.001). RBO rates were significantly higher in the HGS than in the AGMS-AR group (53 vs. 17%; P = 0.024), whereas AE rates did not differ significantly. TRBO differed significantly in the HGS and AGMS-AR groups (159 days vs. not reached, P = 0.011), whereas OS did not differ significantly. Multivariable analysis revealed that HGS was an independent risk factor for RBO (hazard ratio, 6.48, P = 0.014). CONCLUSION: TRBO was significantly longer in patients who underwent AGMS with PS than HGS. AGMS with PS may be effective after the failure of ERCP in patients with malignant biliary obstruction.

Extracellular vesicles as novel uro-oncology biomarkers: insights toward clinical applications.

PURPOSE OF REVIEW: We discussed the challenges associated with the clinical application of extracellular vesicles and summarized their potential impact on oncological clinical practice in urology. RECENT FINDINGS: Despite extensive research on extracellular vesicles, their clinical applications remain limited; this is likely to be because of small study cohorts, a lack of large-scale analyses, and the impact of variable extraction and storage methods on analysis outcomes. However, promising results have emerged from clinical trials targeting urinary extracellular vesicles in prostate cancer using ExoDx Prostate Test. The ExoDx Prostate Test has demonstrated its efficacy in diagnosing prostate cancer in previous studies and is the only FDA-approved kit for this purpose. Moreover, recent trials have investigated the use of the ExoDx Prostate Test to determine the optimal timing for biopsies in prostate cancer patients undergoing active surveillance. SUMMARY: We summarized recent studies on the potential of extracellular vesicles in the management of urological cancers. Particularly, the diagnosis of prostate cancer using the ExoDx Prostate Test has yielded positive results in several clinical trials. Additionally, while there are other studies suggesting its efficacy, most of these are based on retrospective analyses. These findings warrant further large-scale studies to optimize extracellular vesicle-based diagnostic and monitoring strategies. Although further research is required, extracellular vesicles would be attractive for early detection and surveillance.

Safety of skipping the tract dilation step for EUS-guided biliary drainage in patients with unresectable malignant biliary obstruction (with video).

BACKGROUND: Thin delivery system stents can be inserted directly without the need for a tract dilation step and are expected to reduce bile leakage during endoscopic ultrasound-guided biliary drainage (EUS-BD). The present study retrospectively compared the safety and efficacy of EUS-BD using a thin metal stent (< 7.5 Fr) with those of EUS-BD using a conventional stent (≥ 7.5 Fr). METHODS: The present study enrolled 112 patients who underwent EUS-BD using metal stents for unresectable malignant biliary obstruction between April 2016 and July 2022. The primary endpoint was the rate of adverse events (AEs). The secondary endpoints were clinical success rate, procedure time, procedure success rate in the absence of the tract dilation step, recurrent biliary obstruction rate, time to biliary obstruction, and overall survival. Risk factors associated with early AEs were also evaluated. RESULTS: The rate of early AEs was significantly lower (12% vs. 35%, P = 0.013) and the procedure success without the tract dilation step was significantly higher (82% vs. 33%, P < 0.001) in the thin than in the conventional delivery system stent group. None of the other secondary endpoints differed significantly between the two groups. Multivariate analysis showed that employing the tract dilation step during EUS-BD was a significant independent risk factor for early AEs (skipping vs. employing; HR, 9.66; 95% CI, 1.13-83.0, P = 0.028). CONCLUSION: Employing the tract dilation step during EUS-BD was a significant risk factor for early AEs. Metal stents with a delivery diameter < 7.5 Fr can be inserted directly without the tract dilation step, resulting in lower early AE rates.

L-type amino acid transporter 1 inhibitor JPH203 prevents the growth of cabazitaxel-resistant prostate cancer by inhibiting cyclin-dependent kinase activity.

L-type amino acid transporter 1 (LAT1, SLC7A5) is an amino acid transporter expressed in various carcinomas, and it is postulated to play an important role in the proliferation of cancer cells through the uptake of essential amino acids. Cabazitaxel is a widely used anticancer drug for treating castration-resistant prostate cancer (CRPC); however, its effectiveness is lost when cancer cells acquire drug resistance. In this study, we investigated the expression of LAT1 and the effects of a LAT1-specific inhibitor, JPH203, in cabazitaxel-resistant prostate cancer cells. LAT1 was more highly expressed in the cabazitaxel-resistant strains than in the normal strains. Administration of JPH203 inhibited the growth, migration, and invasive ability of cabazitaxel-resistant strains in vitro. Phosphoproteomics using liquid chromatography-mass spectrometry to comprehensively investigate changes in phosphorylation due to JPH203 administration revealed that cell cycle-related pathways were affected by JPH203, and that JPH203 significantly reduced the kinase activity of cyclin-dependent kinases 1 and 2. Moreover, JPH203 inhibited the proliferation of cabazitaxel-resistant cells in vivo. Taken together, the present study results suggest that LAT1 might be a valuable therapeutic target in cabazitaxel-resistant prostate cancer.

ERCP using balloon-assisted endoscopes versus EUS-guided treatment for common bile duct stones in Roux-en-Y gastrectomy.

BACKGROUND AND AIMS: We compared ERCP using a balloon-assisted endoscope (BE-ERCP) with EUS-guided antegrade treatment (EUS-AG) for removal of common bile duct (CBD) stones in patients with Roux-en-Y (R-Y) gastrectomy. METHODS: Consecutive patients who had previous R-Y gastrectomy undergoing BE-ERCP or EUS-AG for CBD stones in 16 centers were retrospectively analyzed. RESULTS: BE-ERCP and EUS-AG were performed in 588 and 59 patients, respectively. Baseline characteristics were similar, except for CBD diameter and angle. The technical success rate was 83.7% versus 83.1% (P = .956), complete stone removal rate was 78.1% versus 67.8% (P = .102), and early adverse event rate was 10.2% versus 18.6% (P = .076) in BE-ERCP and EUS-AG, respectively. The mean number of endoscopic sessions was smaller in BE-ERCP (1.5 ± .8 vs 1.9 ± 1.0 sessions, P = .01), whereas the median total treatment time was longer (90 vs 61.5 minutes, P = .001). Among patients with biliary access, the complete stone removal rate was significantly higher in BE-ERCP (93.3% vs 81.6%, P = .009). Negative predictive factors were CBD diameter ≥15 mm (odds ratio [OR], .41) and an angle of CBD <90 degrees (OR, .39) in BE-ERCP and a stone size ≥10 mm (OR, .07) and an angle of CBD <90 degrees (OR, .07) in EUS-AG. The 1-year recurrence rate was 8.3% in both groups. CONCLUSIONS: Effectiveness and safety of BE-ERCP and EUS-AG were comparable in CBD stone removal for patients after R-Y gastrectomy, but complete stone removal after technical success was superior in BE-ERCP.

Utility of contrast-enhanced harmonic endoscopic ultrasonography for prediction of pathological response after neoadjuvant chemotherapy in patients with pancreatic cancer.

OBJECTIVES: Contrast-enhanced harmonic endoscopic ultrasonography (CH-EUS) plays an important role in the diagnosis of pancreatic lesions. The aim of this study was to evaluate whether CH-EUS is useful for predicting the treatment efficacy of neoadjuvant chemotherapy (NAC) determined by pathological response. METHODS: Patients who underwent CH-EUS before chemotherapy and surgical resection were divided into two groups according to poor (group-P) or rich tumor vascularity (group-R) determined by enhancement pattern on early- and late-phase CH-EUS. The pathological response to chemotherapy was categorized according to Evans' classification. Pathological analysis showing tumor cell destruction (>50 %) defined a good response. RESULTS: Early-phase CH-EUS classified 44 patients into group-R and 50 into group-P, whereas late-phase CH-EUS classified 10 into group-R and 84 into group-P. Early-phase CH-EUS classification resulted in significantly higher numbers of patients with a good response in the rich group (n = 19) than in the poor group (n = 4; P = 0.0015). Multivariate analysis showed that assignment to the rich group was the strongest independent factor associated with chemosensitivity (P = 0.006, hazard ratio = 5.66, 95 % confidence interval: 1.17-19.27). In resectable patients, the enhancement pattern was the only independent factor associated with chemosensitivity (group-P vs. group-R, P = 0.003; HR [95 % CI], 14.59 [1.38-154.38]). Late-phase CH-EUS did not reveal a significant difference between group-P and group-R. CONCLUSIONS: Evaluation of vascular pattern on CH-EUS could be useful for predicting the efficacy of NAC in patients with pancreatic cancer. The enhancement pattern on CH-EUS could be a one of the useful features for determining NAC indications in resectable pancreatic cancer patients.

Novel Technique of Endoscopic Ultrasonography for the Differential Diagnosis of Gallbladder Lesions and Intraductal Papillary Mucinous Neoplasms: A Single-Center Prospective Study.

Detective flow imaging endoscopic ultrasonography (DFI-EUS) is an innovative imaging modality that was developed to detect fine vessels and low-velocity blood flow without contrast agents. We evaluate its utility for the differential diagnosis of gallbladder lesions and intraductal papillary mucinous neoplasms (IPMNs). We enrolled patients who underwent DFI-EUS, e-FLOW EUS, and contrast-enhanced EUS for gallbladder lesions or IPMNs. The detection of vessels using DFI-EUS and e-FLOW EUS was compared with that via contrast-enhanced EUS and pathological findings. The vessel pattern was also categorized as regular or irregular. Of the 33 lesions included, there were final diagnoses of 13 IPMNs and 20 gallbladder lesions. DFI-EUS was significantly superior to e-FLOW EUS for discriminating between mural nodules and mucous clots and between solid gallbladder lesions and sludge using the presence or absence of vessel detection in lesions (p = 0.005). An irregular vessel pattern with DFI-EUS was a significant predictor of malignant gallbladder lesions (p = 0.002). DFI-EUS is more sensitive than e-FLOW-EUS for vessel detection and the differential diagnosis of gallbladder lesions and IPMNs. Vessel evaluation using DFI-EUS may be a useful and simple method for differentiating between mural nodules and mucous clots in IPMN, between solid gallbladder lesions and sludge, and between malignant and benign gallbladder lesions.

Extracellular vesicles in bone homeostasis: key roles of physiological and pathological conditions.

Extracellular vesicles (EVs) are small particles with lipid bilayer membranes that are secreted by all cell types and are widely known as crucial intercellular communication mediators, shuttling biologically active molecules. The bone is a typically preferred site of cancer metastasis due to its unique cellular compositions and dynamics. Bone cell-derived EVs serve as regulators that orchestrate harmonious bone homeostasis. Cancer cells secrete specific EVs in a series of the bone metastatic process to dominate the bone microenvironment. Additionally, cancer cell-related EVs contribute to pre-metastatic niche formation, bone homeostasis disruption, and tumor bone progression and survival. Here, we investigated recent studies on EV-mediated crosstalk in the bone tumor microenvironment. Furthermore, this review aimed to elucidate the EV-based therapeutic perspectives for bone metastasis.

Contrast-Enhanced Harmonic Endoscopic Ultrasound for Diagnosis of the Aggressiveness of Pancreatic Neuroendocrine Neoplasm.

The purpose of this study is to clarify the associations between the enhancement patterns on contrast-enhanced harmonic endoscopic ultrasound (CH-EUS) and the aggressiveness and prognosis of pancreatic neuroendocrine neoplasms (PanNENs). Patients who underwent CH-EUS and were pathologically diagnosed with PanNEN were included in this study. Patients were divided into three groups according to contrast-enhancement patterns on early-phase and late-phase imaging: "Group A", vascular rich in both phases; "Group B", vascular rich and vascular poor in early and late phases, respectively; "Group C", vascular poor in both phases. Of 39 patients, 25 were assigned to Group A, 7 to Group B, and 7 to Group C. The median overall survival was not reached in Groups A and B and was 335 days in Group C (p < 0.001). The 1-year survival rates were 100% in Group A, 60% in Group B, and 43% in Group C. Patients in Group C showed the shortest overall survival among the three groups. The vascular-poor pattern on late-phase CH-EUS had the highest sensitivity, specificity, and accuracy for aggressive PanNENs among the patterns analyzed on CH-EUS and CECT (84.6%, 91.7%, and 89.2%, respectively). CH-EUS is useful for the diagnosis of and predicting the prognosis of PanNENs.

Targeting L-type amino acid transporter 1 in urological malignancy: Current status and future perspective.

Amino acid transporters are responsible for the uptake of amino acids, critical for cell proliferation. L-type amino acid transporters play a major role in the uptake of essential amino acids. L-type amino acid transporter 1 (LAT1) exerts its functional properties by forming a dimer with 4F2hc. Utilizing this cancer-specificity, research on diagnostic imaging and therapeutic agents for malignant tumors targeting LAT1 progresses in various fields. In hormone-sensitive prostate cancer, the up-regulation of L-type amino acid transporter 3 (LAT3) through the androgen receptor (AR) has been identified. On the other hand, in castration-resistant prostate cancer, the negative regulation of LAT1 through AR has been determined. Furthermore, 4F2hc: a binding partner of LAT1, was identified as the specific downstream target of Androgen Receptor Splice Variant 7: AR-V7. LAT1 has been suggested to contribute to acquiring castration resistance in prostate cancer, making LAT1 a completely different therapeutic target from anti-androgens and taxanes. Increased expression of LAT1 has also been found in renal and bladder cancers, suggesting a contribution to acquiring malignancy and progression. In Japan, clinical trials of LAT1 inhibitors for solid tumors are in progress, and clinical applications are now underway. This article will summarize the relationship between LAT1 and urological malignancies.

Extracellular Vesicles in Bone Metastasis: Key Players in the Tumor Microenvironment and Promising Therapeutic Targets.

Extracellular vesicles (EVs) are lipid membranous vesicles that are released from every type of cell. It has become clear that EVs are involved in a variety of biological phenomena, including cancer progression, and play critical roles in intracellular communication through the horizontal transfer of cellular cargoes such as proteins, DNA fragments, RNAs including mRNA and non-coding RNAs (microRNA, piRNA, and long non-coding RNA) and lipids. The most common cause of death associated with cancer is metastasis. Recent investigations have revealed that EVs are deeply associated with metastasis. Bone is a preferred site of metastasis, and bone metastasis is generally incurable and dramatically affects patient quality of life. Bone metastasis can cause devastating complications, including hypercalcemia, pathological fractures, spinal compression, and bone pain, which result in a poor prognosis. Although the mechanisms underlying bone metastasis have yet to be fully elucidated, increasing evidence suggests that EVs in the bone microenvironment significantly contribute to cancer progression and cancer bone tropism. Emerging evidence on EV functions in bone metastasis will facilitate the discovery of novel treatments. In this review, we will discuss the remarkable effects of EVs, especially on the tumor microenvironment in bone.

TLP-mediated global transcriptional repression after double-strand DNA breaks slows down DNA repair and induces apoptosis.

Transcription and DNA damage repair act in a coordinated manner. Recent studies have shown that double-strand DNA breaks (DSBs) are repaired in a transcription-coupled manner. Active transcription results in a faster recruitment of DSB repair factors and expedites DNA repair. On the other hand, transcription is repressed by DNA damage through multiple mechanisms. We previously reported that TLP, a TATA box-binding protein (TBP) family member that functions as a transcriptional regulator, is also involved in DNA damage-induced apoptosis. However, the mechanism by which TLP affects DNA damage response was largely unknown. Here we show that TLP-mediated global transcriptional repression after DSBs is crucial for apoptosis induction by DNA-damaging agents such as etoposide and doxorubicin. Compared to control cells, TLP-knockdown cells were resistant to etoposide-induced apoptosis and exhibited an elevated level of global transcription after etoposide exposure. DSBs were efficiently removed in transcriptionally hyperactive TLP-knockdown cells. However, forced transcriptional shutdown using transcriptional inhibitors α-amanitin and 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole (DRB) slowed down DSB repair and resensitized TLP-knockdown cells to etoposide. Taken together, these results indicate that TLP is a critical determinant as to how cells respond to DSBs and triggers apoptosis to cells that have sustained DNA damage.

Duration of androgen deprivation therapy and nadir of testosterone at 20 ng/dL predict testosterone recovery to supracastrate level in prostate cancer patients who received external beam radiotherapy.

OBJECTIVES: To determine the predictors of testosterone recovery after termination of androgen deprivation therapy in high/intermediate-risk prostate cancer patients receiving external beam radiation therapy with neoadjuvant and adjuvant androgen deprivation therapy. METHODS: A total of 82 patients who underwent external beam radiation therapy with androgen deprivation therapy for prostate cancer were retrospectively analyzed. Serum testosterone levels after androgen deprivation therapy terminations were studied. Cox proportional hazard models and the Kaplan-Meier method were used for statistical analysis. RESULTS: Median age, baseline testosterone, nadir testosterone and duration of androgen deprivation therapy were 73 years, 456 ng/dL, 16 ng/dL and 26 months, respectively. Androgen deprivation therapy duration of 33 months (hazard ratio 0.13; P = 0.0018), nadir testosterone of 20 ng/dL (hazard ratio 0.35; P = 0.0112) and testosterone >50 ng/dL at 6 months after androgen deprivation therapy termination (hazard ratio 0.21; P = 0.0075) were significantly associated with testosterone recovery to normal levels (200 ng/dL) on multivariate analysis. Androgen deprivation therapy duration of 33 months (hazard ratio 0.31; P = 0.0023) and nadir testosterone of 20 ng/dL (hazard ratio 0.38; P = 0.0012) were significantly associated with testosterone recovery to the supracastrate level (50 ng/dL) on multivariate analysis. After dividing patients into three risk groups, the rate of testosterone recovery to the normal level after 2 years of androgen deprivation therapy termination was 100% in the low-risk group versus 20.8% in the high-risk group (P < 0.0001); the rate of testosterone recovery to the supracastrate level was 100% in the low-risk group versus 51.5% in the high-risk group (P < 0.0001). CONCLUSIONS: Duration of androgen deprivation therapy and achievement of nadir testosterone 20 ng/dL both predict testosterone recovery to the supracastrate level in prostate cancer patients undergoing external beam radiation therapy with androgen deprivation therapy.

Testosterone Reduction of ≥ 480 ng/dL Predicts Favorable Prognosis of Japanese Men With Advanced Prostate Cancer Treated With Androgen-Deprivation Therapy.

INTRODUCTION: Reductions in testosterone concentration play a significant role in the treatment of prostate cancer. We studied the role of testosterone as a prognostic marker for advanced prostate cancer (stage C or higher) treated with primary androgen-deprivation therapy (ADT). PATIENTS AND METHODS: A total of 348 patients were treated using ADT as first-line therapy for prostate cancer at Chiba University Hospital between 1999 and 2016. Of these, 222 patients with advanced prostate cancer (stage C or higher) were enrolled onto this study. The prognostic values of serum testosterone level and other clinical factors were evaluated in association with prostate-specific antigen (PSA), progression-free survival during first-line therapy, and overall survival. RESULTS: Median age was 73 years. PSA at baseline was 86 ng/mL. Gleason scores of ≤ 6, 7, 8, and ≥ 9 were seen in 2.3%, 19.4%, 21.2%, and 41.9%, respectively. Mean follow-up was 60.5 months. Median testosterone at baseline was 482 ng/dL and nadir testosterone was 13 ng/dL. No variable associated with testosterone predicted progression-free survival. With regard to overall survival, multivariate analysis identified nadir testosterone ≤ 20 ng/dL (hazard ratio = 0.44, P = .026) and testosterone reduction ≥ 480 ng/dL (hazard ratio = 0.35, P = .030) as independent prognostic factors. With regard to progression-free survival, multivariate analysis identified nadir PSA ≤ 0.1 ng/mL (hazard ratio = 3.07, P < .001), presence of lymph node metastasis (hazard ratio = 1.67, P = .017), and time to nadir PSA (hazard ratio = 0.30, P < .001) as independent prognostic factors. CONCLUSION: Our data suggested both nadir testosterone (< 20 ng/dL; P = .026) and testosterone reduction (≥ 480 ng/dL; P = .030) to be key prognostic factors for primary ADT in advanced prostate cancer in Japanese men.

TBP-like Protein (TLP) Disrupts the p53-MDM2 Interaction and Induces Long-lasting p53 Activation.

Stress-induced activation of p53 is an essential cellular response to prevent aberrant cell proliferation and cancer development. The ubiquitin ligase MDM2 promotes p53 degradation and limits the duration of p53 activation. It remains unclear, however, how p53 persistently escapes MDM2-mediated negative control for making appropriate cell fate decisions. Here we report that TBP-like protein (TLP), a member of the TBP family, is a new regulatory factor for the p53-MDM2 interplay and thus for p53 activation. We found that TLP acts to stabilize p53 protein to ensure long-lasting p53 activation, leading to potentiation of p53-induced apoptosis and senescence after genotoxic stress. Mechanistically, TLP interferes with MDM2 binding and ubiquitination of p53. Moreover, single cell imaging analysis shows that TLP depletion accelerates MDM2-mediated nuclear export of p53. We further show that a cervical cancer-derived TLP mutant has less p53 binding ability and lacks a proliferation-repressive function. Our findings uncover a role of TLP as a competitive MDM2 blocker, proposing a novel mechanism by which p53 escapes the p53-MDM2 negative feedback loop to modulate cell fate decisions.

Ubiquitin-proteasome-dependent degradation of TBP-like protein is prevented by direct binding of TFIIA.

Although the majority of gene expression is driven by TATA-binding protein (TBP)-based transcription machinery, it has been reported that TBP-related factors (TRFs) are also involved in the regulation of gene expression. TBP-like protein (TLP), which is one of the TRFs and exhibits the highest affinity to TFIIA among known proteins, has recently been showed to have significant roles in gene regulation. However, how the level of TLP is maintained in vivo has remained unknown. In this study, we explored the mechanism by which TLP protein is turned over in vivo and the factor that maintains the amount of TLP. We showed that TLP is rapidly degraded by the ubiquitin-proteasome system and that tight interaction with TFIIA results in protection of TLP from ubiquitin-proteasome-dependent degradation. The half-life of TLP was shown to be less than a few hours, and the proteasome inhibitor MG132 specifically suppressed TLP degradation. Moreover, knockdown and over-expression experiments showed that TFIIA is engaged in stabilization of TLPin vivo. Thus, we showed a novel characteristic of TLP, that is, interaction with TFIIA is essential to suppress proteasome-dependent turnover of TLP, providing a further insight into TLP-governed gene regulation.

TBP-like protein (TLP) represses myogenesis via inhibition of the myogenin promoter.

TBP-like protein (TLP) is one of the metazoan-restricted transcription factors participating in development and differentiation, though the molecular mechanism by which TLP regulates these processes remains unclear. In this study, we investigated the relationship between TLP and myogenesis of mouse C2C12 myoblasts. We found that TLP gene expression decreases during myogenic differentiation. Overexpression and knockdown of TLP revealed that the levels of muscle-specific myosin heavy chain and the myogenic transcription factor myogenin are downregulated by TLP. TLP inhibits the progression of morphological change from myoblasts to myotubes, thereby suppressing myogenesis. We further show that TLP represses the promoter activity of myogenin. The proximal AT-rich sequence of the myogenin promoter is responsible for TLP-mediated transcriptional repression. The results of this study suggest that TLP inhibits myogenesis through downregulation of the myogenin gene.

Novel Methodology for Rapid Detection of KRAS Mutation Using PNA-LNA Mediated Loop-Mediated Isothermal Amplification.

Detecting point mutation of human cancer cells quickly and accurately is gaining in importance for pathological diagnosis and choice of therapeutic approach. In the present study, we present novel methodology, peptide nucleic acid-locked nucleic acid mediated loop-mediated isothermal amplification (PNA-LNA mediated LAMP), for rapid detection of KRAS mutation using advantages of both artificial DNA and LAMP. PNA-LNA mediated LAMP reactions occurred under isothermal temperature conditions of with 4 primary primers set for the target regions on the KRAS gene, clamping PNA probe that was complimentary to the wild type sequence and LNA primers complementary to the mutated sequences. PNA-LNA mediated LAMP was applied for cDNA from 4 kinds of pancreatic carcinoma cell lines with or without KRAS point mutation. The amplified DNA products were verified by naked-eye as well as a real-time PCR equipment. By PNA-LNA mediated LAMP, amplification of wild type KRAS DNA was blocked by clamping PNA probe, whereas, mutant type KRAS DNA was significantly amplified within 50 min. Mutant alleles could be detected in samples which diluted until 0.1% of mutant-to-wild type ratio. On the other hand, mutant alleles could be reproducibly with a mutant-to-wild type ratio of 30% by direct sequencing and of 1% by PNA-clamping PCR. The limit of detection (LOD) of PNA-LNA mediated LAMP was much lower than the other conventional methods. Competition of LNA clamping primers complementary to two different subtypes (G12D and G12V) of mutant KRAS gene indicated different amplification time depend on subtypes of mutant cDNA. PNA-LNA mediated LAMP is a simple, rapid, specific and sensitive methodology for the detection of KRAS mutation.