Hsc70 promotes anti-tumor immunity by targeting PD-L1 for lysosomal degradation.

Immune checkpoint inhibition targeting the PD-1/PD-L1 pathway has become a powerful clinical strategy for treating cancer, but its efficacy is complicated by various resistance mechanisms. One of the reasons for the resistance is the internalization and recycling of PD-L1 itself upon antibody binding. The inhibition of lysosome-mediated degradation of PD-L1 is critical for preserving the amount of PD-L1 recycling back to the cell membrane. In this study, we find that Hsc70 promotes PD-L1 degradation through the endosome-lysosome pathway and reduces PD-L1 recycling to the cell membrane. This effect is dependent on Hsc70-PD-L1 binding which inhibits the CMTM6-PD-L1 interaction. We further identify an Hsp90α/ß inhibitor, AUY-922, which induces Hsc70 expression and PD-L1 lysosomal degradation. Either Hsc70 overexpression or AUY-922 treatment can reduce PD-L1 expression, inhibit tumor growth and promote anti-tumor immunity in female mice; AUY-922 can further enhance the anti-tumor efficacy of anti-PD-L1 and anti-CTLA4 treatment. Our study elucidates a molecular mechanism of Hsc70-mediated PD-L1 lysosomal degradation and provides a target and therapeutic strategies for tumor immunotherapy.

ARIH1 activates STING-mediated T-cell activation and sensitizes tumors to immune checkpoint blockade.

Despite advances in cancer treatment, immune checkpoint blockade (ICB) only achieves complete response in some patients, illustrating the need to identify resistance mechanisms. Using an ICB-insensitive tumor model, here we discover cisplatin enhances the anti-tumor effect of PD-L1 blockade and upregulates the expression of Ariadne RBR E3 ubiquitin-protein ligase 1 (ARIH1) in tumors. Arih1 overexpression promotes cytotoxic T cell infiltration, inhibits tumor growth, and potentiates PD-L1 blockade. ARIH1 mediates ubiquitination and degradation of DNA-PKcs to trigger activation of the STING pathway, which is blocked by the phospho-mimetic mutant T68E/S213D of cGAS protein. Using a high-throughput drug screen, we further identify that ACY738, less cytotoxic than cisplatin, effectively upregulates ARIH1 and activates STING signaling, sensitizing tumors to PD-L1 blockade. Our findings delineate a mechanism that tumors mediate ICB resistance through the loss of ARIH1 and ARIH1-DNA-PKcs-STING signaling and indicate that activating ARIH1 is an effective strategy to improve the efficacy of cancer immunotherapy.

[Corrigendum] Lentivirus­mediated RNA interference of clusterin enhances the chemosensitivity of EJ bladder cancer cells to epirubicin in vitro.

Subsequently to the publication of the above paper, the authors have realized that Fig. 2A in this paper contained an error. The image selected to represent the experiment showing the invasion ability of EJ cells in the epirubicine/LV­NC group of Fig. 2A was chosen mistakenly during the figure compilation process. A corrected version of Fig. 2 is shown on the next page. Note that this error did not affect either the results or the conclusions reported in this paper, and all the authors agree to this Corrigendum. The authors are grateful to the Editor of Molecular Medicine Reports for allowing them the opportunity to publish this Corrigendum, and apologize to the readership for any inconvenience caused. [the original article was published in Molecular Medicine Reports 6: 1133­1139, 2012; DOI: 10.3892/mmr.2012.1017].

ARIH1 signaling promotes anti-tumor immunity by targeting PD-L1 for proteasomal degradation.

Cancer expression of PD-L1 suppresses anti-tumor immunity. PD-L1 has emerged as a remarkable therapeutic target. However, the regulation of PD-L1 degradation is not understood. Here, we identify several compounds as inducers of PD-L1 degradation using a high-throughput drug screen. We find EGFR inhibitors promote PD-L1 ubiquitination and proteasomal degradation following GSK3α-mediated phosphorylation of Ser279/Ser283. We identify ARIH1 as the E3 ubiquitin ligase responsible for targeting PD-L1 to degradation. Overexpression of ARIH1 suppresses tumor growth and promotes cytotoxic T cell activation in wild-type, but not in immunocompromised mice, highlighting the role of ARIH1 in anti-tumor immunity. Moreover, combining EGFR inhibitor ES-072 with anti-CTLA4 immunotherapy results in an additive effect on both tumor growth and cytotoxic T cell activation. Our results delineate a mechanism of PD-L1 degradation and cancer escape from immunity via EGFR-GSK3α-ARIH1 signaling and suggest GSK3α and ARIH1 might be potential drug targets to boost anti-tumor immunity and enhance immunotherapies.

Pharmacological targeting of MCL-1 promotes mitophagy and improves disease pathologies in an Alzheimer's disease mouse model.

There is increasing evidence that inducing neuronal mitophagy can be used as a therapeutic intervention for Alzheimer's disease. Here, we screen a library of 2024 FDA-approved drugs or drug candidates, revealing UMI-77 as an unexpected mitophagy activator. UMI-77 is an established BH3-mimetic for MCL-1 and was developed to induce apoptosis in cancer cells. We found that at sub-lethal doses, UMI-77 potently induces mitophagy, independent of apoptosis. Our mechanistic studies discovered that MCL-1 is a mitophagy receptor and directly binds to LC3A. Finally, we found that UMI-77 can induce mitophagy in vivo and that it effectively reverses molecular and behavioral phenotypes in the APP/PS1 mouse model of Alzheimer's disease. Our findings shed light on the mechanisms of mitophagy, reveal that MCL-1 is a mitophagy receptor that can be targeted to induce mitophagy, and identify MCL-1 as a drug target for therapeutic intervention in Alzheimer's disease.

Hepatoid adenocarcinoma of the renal pelvis in a 59-year-old male with nephrolithiasis: Case report and review of the literature.

BACKGROUND: Hepatoid adenocarcinoma arising from urological system is extremely rare, and the pathogenesis and therapeutic regimen have been poorly understood. CASE REPORT: we report a unique case of ɑ-fetaprotein (AFP)-producing neoplasm of renal pelvis associated with nephrolithiasis. A 59-year-old male patient was diagnosed with right renal tumor and nephrolithiasis with no evidence of lesions in his digestive or reproductive system. He was successfully treated with right laparoscopic radical nephroureterectomy and lymph node dissection. Pathology analysis showed moderately or poorly hepatocellular differentiation and adenocarcinoma differentiation with lymph node reactive hyperplasia. Immunohistochemical analysis demonstrated that the cancer cells were positive for AFP, HepPar-1, GPC3, CK7, and PLAP. The patient's recovery was on schedule and no sign of recurrence was observed for 3 months. We recently reviewed AFP-producing nongerm cell tumors in upper urinary tract and discussed the clinical aspect, morphology features, pathogenesis, and therapeutic regimen for a better understanding of this rare entity. CONCLUSION: The present case is the first documented of hepatoid adenocarcinoma of renal pelvis complicated with nephrolithiasis, which was treated with laparoscopic approach. The prognosis of the hepatoid adenocarcinomas arising from renal pelvis and ureter seems good.

Characterization and Analysis of Strain Heterogeneity at Grain-Scale of Titanium Alloy with Tri-Modal Microstructure during Tensile Deformation.

Grain-scale strain heterogeneity characteristics play a critical role in the ductile damage behavior and mechanical properties of two-phase titanium alloys. In this work, the grain-scale strain distribution, strain heterogeneity, and strain localization of titanium alloy with tri-modal microstructure (consisting of equiaxed α (αp), lamellar α (αl), and ß transformed matrix (ßt)) during tensile deformation were experimentally investigated. The results show that the strain probability distribution of the whole microstructure obeys normal distribution during deformation. Significant strain heterogeneities exist in each constituent (αp, αl, and ßt) and the whole microstructure. At lower macro-strain, αp and αl exhibit higher average strain than those of ßt and the whole of the microstructure. Meanwhile, strain heterogeneity of each constituent is small and has a negligible change. The strain heterogeneity of the whole microstructure is mainly determined by αp. At larger macro-strain, some highly deformed regions produce and their positions do not change during further deformation. As a result, the strain heterogeneity of each constituent increases fast, and the strain heterogeneity of whole microstructure is mainly related to αl in this deformation stage. On the other hand, two types of strain localization may be generated within αp and αl and at the αp/ßt and αl/ßt boundaries, respectively. The former type is caused by transgranular intense slip deformation and presents crystal orientation dependence. The latter type is related to the boundary sliding and presents spatial distribution dependence for αl. These strain localizations greatly determine the micro-damages, thus forming the corresponding micro-voids within αp and αl and the micro-cracks at αp/ßt and αl/ßt boundaries in tri-modal microstructure at larger deformation.

Pelvic mass: Schwannoma of the left seminal vesicle.

Schwannomas rarely occur in seminal vesicles. Here, we report a schwannoma of the left seminal vesicle. A 55-year-old man presented no clinical symptoms, and a mass in the left region of the seminal vesicle was found incidentally in a medical examination. A computed tomography and magnetic resonance imaging of pelvic were obtained and revealed a 5.17 × 2.59 × 3.5 cm mass on the left seminal vesicle. Transrectal ultrasound-guided seminal biopsy revealed a diagnosis of seminal vesical schwannoma. Laparoscopic resection of the tumour was performed. Postoperative pathology and immunohistochemical analysis revealed schwannoma arising from seminal vesical.

CasHRA (Cas9-facilitated Homologous Recombination Assembly) method of constructing megabase-sized DNA.

Current DNA assembly methods for preparing highly purified linear subassemblies require complex and time-consuming in vitro manipulations that hinder their ability to construct megabase-sized DNAs (e.g. synthetic genomes). We have developed a new method designated 'CasHRA (Cas9-facilitated Homologous Recombination Assembly)' that directly uses large circular DNAs in a one-step in vivo assembly process. The large circular DNAs are co-introduced into Saccharomyces cerevisiae by protoplast fusion, and they are cleaved by RNA-guided Cas9 nuclease to release the linear DNA segments for subsequent assembly by the endogenous homologous recombination system. The CasHRA method allows efficient assembly of multiple large DNA segments in vivo; thus, this approach should be useful in the last stage of genome construction. As a proof of concept, we combined CasHRA with an upstream assembly method (Gibson procedure of genome assembly) and successfully constructed a 1.03 Mb MGE-syn1.0 (Minimal Genome of Escherichia coli) that contained 449 essential genes and 267 important growth genes. We expect that CasHRA will be widely used in megabase-sized genome constructions.

MEGA (Multiple Essential Genes Assembling) deletion and replacement method for genome reduction in Escherichia coli.

Top-down reduction of the bacterial genome to construct desired chassis cells is important for synthetic biology. However, the current progress in the field of genome reduction is greatly hindered by indispensable life-essential genes that are interspersed throughout the chromosomal loci. Here, we described a new method designated as "MEGA (Multiple Essential Genes Assembling) deletion and replacement" that functions by assembling multiple essential genes in an E. coli-S. cerevisiae shuttle vector, removing targeted chromosomal regions containing essential and nonessential genes using a one-round deletion, and then integrating the cloned essential genes into the in situ chromosomal loci via I-SceI endonuclease cleavage. As a proof of concept, we separately generated three large deletions (80-205 kbp) in the E. coli MDS42 chromosome. We believe that the MEGA deletion and replacement method has potential to become widely used in large-scale genome reductions in other sequenced organisms in addition to E. coli.

Lentivirus-mediated RNA interference of clusterin enhances the chemosensitivity of EJ bladder cancer cells to epirubicin in vitro.

Clusterin (CLU) is a glycoprotein that is over-expressed in a number of malignant tumors and has been proven to correlate closely with the chemoresistance of several cancer cells to chemotherapeutic agents. However, the effect of CLU expression on the chemoresistance of bladder cancer to epirubicin remains unknown. In the present study, we aimed to elucidate the role of CLU in the chemoresistance of bladder cancer cells to epirubicin. Lentivirus-mediated RNA interference was applied to knock down CLU in EJ bladder cancer cells. The efficiency was examined by RT-PCR and western blot analysis. After stable CLU silencing, an EJ cell line was established and cells were treated with or without epirubicin. Cell viability, migration, invasiveness, clone formation and cell cycle progression were assessed by MTT assay, wound healing assay, Matrigel invasion assay, plate clone formation assay and flow cytometry, respectively. The results indicated that lentivirus-mediated RNA interference effectively silenced CLU at the RNA and protein levels. CLU knockdown increased the cytotoxicity of epirubicin to EJ bladder cancer cells. Combined treatment with lentivirus-mediated shRNA targeting CLU and epirubicin had maximum effects in bladder cancer cells on cell viability, migration, invasiveness and clone-forming ability. Furthermore, cell cycle analysis indicated that CLU knockdown reinforced the efficacy of epirubicin on G0/G1 cell cycle arrest. Taken together, our results suggest that CLU silencing enhances chemosensitivity of EJ bladder cancer cells to epirubicin. Lentivirus-mediated shRNA targeting CLU may be an alternative approach in the treatment of bladder cancer.

[Thermal coagulation of human benign prostatic hyperplasia tissues induced changes in the absorption and scattering properties in spectral range from 590 to 1 064 nm in vitro].

The optical properties and their differences of native and coagulated human benign prostatic hyperplasia (BPH) tissues were studied in the spectral range from 590 to 1 064 nm in vitro. The measurements were performed using a spectrophotometer with an integrating sphere attachment, and the absorption and scattering properties were assessed from these measurements using the inverse adding-doubling method. The results of measurement showed that the thermal coagulation of BPH tissues induced obviously the decrease in the absorption coefficients in the spectral range from 590 to 1 064 nm. The peaks in the absorption coefficients for native and coagulated BPH tissues were respectively 0.438 and 0.416 mm(-1) corresponding to the same wavelength of 990 nm, the maximum difference in the absorption coefficients of native and coagulated BPH tissues is 86.79% at 1 064 nm, and the minimum difference is 4.74% at 920 nm. The thermal coagulation of BPH tissues induced an increase in the reduced scattering coefficients in the spectral range from 600 to 1 064 nm obviously, and induced a decrease in the reduced scattering coefficients at 590 nm obviously. The peaks in the reduced scattering coefficients for native and coagulated BPH tissues were respectively 1.090 and 1. 449 mm(-1) corresponding to the same wavelength of 970 nm, and other peaks in the reduced scattering coefficients for native and coagulated BPH tissues were respectively 1.116 and 1.627 mm(-1) corresponding to the same wavelength of 1 050 nm, the maximum difference in the reduced scattering coefficients of native and coagulated BPH tissues is 47.73% at 1 064 nm, and the minimum difference is 4.86% at 600 nm.

[Absorption and scattering characteristics of human benign prostatic hyperplasia tissue with Ti: sapphire laser irradiation in vitro].

The optical properties and their differences of human benign prostatic hyperplasia (BPH) tissues removed using transurethral plasma kinetic resection of the prostate (PKRP) and transurethral vaporization of the prostate (TUVP) at 640, 660, 680, 700, 720, 740, 760, 780, 800, 820, 840, 860 and 880 nm of Ti: Sapphire laser were studied in vitro. The measurements were performed using a double-integrating-sphere setup, and the absorption and scattering properties were assessed using the inverse adding-doubling method. The results of measurement showed that the absorption coefficients and reduced scattering coefficients of BPH tissues removed using PKRP and TURP obviously decreased with the increase in the wavelength for thirteen different laser wavelengths. The absorption coefficient and reduced scattering coefficient of BPH tissues removed using PKRP at a certain laser wavelength were obviously smaller than that of BPH tissues removed using TUVP at the same laser wavelength. The maximum absorption coefficient and maximum reduced scattering coefficient of BPH tissues removed using PKRP and TURP were respectively (0. 885 +/- 0. 022) and (0.955 +/- 0.024)mm(-1), and (1.564 +/- 0.039) and (1.658 +/- 0.042)mm(-1) at 640 nm, their differences were respectively 7.91% and 6.01%, and the minimum absorption coefficient and minimum reduced scattering coefficient of BPH tissues removed using PKRP and TURP were respectively (0.443 +/- 0.011) and (0.455 +/- 0.011) mm(-1), and (1.117 +/- 0.028) and (1.197 +/- 0.030)mm(-1) at 640 nm, their differences were respectively 2.71% and 9.13%. The maximum difference in the absorption coefficients of BPH tissues removed using PKRP and TURP is 8.95% at 660 nm, and the minimum difference is 1.75% at 860 nm. The maximum difference in the reduced scattering coefficients of BPH tissues removed using PKRP and TURP is 9.13% at 800 nm, and the minimum difference is 6.01% at 640 nm.

[Superficial bladder cancer detection using diffuse reflectance spectral ratio R540/R575 of oxygenated hemoglobin bands].

A low-cost, fast, and noninvasive method for early diagnosis of malignant lesions of mucosa tissue based on diffuse reflectance spectra was applied in the study of the optical biopsy of superficial human bladder cancer. In the present paper, differential diagnosis of superficial human bladder cancer was studied using the diffuse reflectance spectral ratio (R540/R575) of the oxygenated hemoglobin absorption bands at 540 and 575 nm in vitro. Diffuse reflectance spectra for mucosa/submucosa tissues of normal bladder and superficial bladder cancer were measured using a spectrophotometer with an integrating sphere attachment. The results of measurement showed that there were three the diffuse reflectance spectral dips at 415, 542 and 577 nm respectively for mucosa/submucosa tissues of normal bladder and superficial bladder cancer in the spectral range from 400 to 600 nm. The mean diffuse reflectance spectral ratio (R540/R575) of normal bladder mucosa/submucosa tissue decreased slowly with time increase after surgical excision, and the mean diffuse reflectance spectral ratio (R540/R575) of superficial bladder cancer mucosa/ submucosa tissue also decreased slowly with time increasing after surgical excision. The mean diffuse reflectance spectral ratios (R540/R575) of normal bladder mucosa/submucosa tissue were 111%, 107%, 104% and 102% after 2, 3, 4 and 5 h after surgical excision respectively, and those of superficial bladder cancer mucosa/submucosa tissue were 98.4%, 95.5%, 93.1% and 91.6% after 2, 3, 4 and 5 h after surgical excision respectively. There were significant differences in mean diffuse reflectance spectral ratio (R540/R575) for mucosa/submucosa tissues between normal bladder and superficial bladder cancer after 2, 3, 4 and 5 h after surgical excision respectively (p < 0.05). Differences in mean diffuse reflectance spectral ratio (R540/R575) for mucosa/ submucosa tissues between normal bladder and superficial bladder cancer were 12.6%, 11.5%, 10.9% and 10.4% after 2, 3, 4 and 5 h after surgical excision respectively. It is obvious that pathological changes in bladder mucosa/submucosa tissues induced changes in the component and structure of the tissues, and especially quantitative changes in oxyhemoglobin and de-oxyhemoglobin of tissues obviously. Conclusion of the study provides a new method that can be applied to rapid, low-cost and noninvasive optical biopsy of superficial bladder cancer.