Targeting TUBB3 Suppresses Anoikis Resistance and Bone Metastasis in Prostate Cancer.

Bone metastases occur in more than 70% of advanced prostate cancer (PCa) patients, leading to a poor prognosis. Resistance to detachment-induced apoptosis, also known as anoikis, plays a crucial role in the onset of tumor metastasis. Targeting anoikis resistance is of immense therapeutic significance in repression of metastatic spread. In this study, based on an anoikis-related prognostic risk model of PCa, this study identifies TUBB3 as a key anoikis-related prognostic gene that is highly expressed in bone metastatic PCa. TUBB3 expression is increased in anoikis-resistant PCa cells, and TUBB3 depletion significantly reverses anoikis resistance during extracellular matrix (ECM) detachment and inhibits anoikis-resistance-induced PCa cell invasion and migration as well as epithelial-mesenchymal transition (EMT) process. TUBB3 knockdown significantly reduces αvß3/FAK/Src axis activation, blocking its downstream oncogenic signaling. In addition, this work develops bone-targeting lipid nanoparticles (BT-LNP) based on bisphosphonate-modified ionizable lipid for systemic delivery of siRNA targeting TUBB3 (siTUBB3). BT-LNP-delivered siTUBB3 therapy with localization in the bone microenvironment significantly attenuate PCa bone metastasis progression in vivo upon intravenous administration. These findings pinpoint that TUBB3, as a key regulator of anoikis resistance, is an effective therapeutic target in bone metastatic PCa and that BT-LNP-mediated systemic delivery of siTUBB3 can be developed as a novel therapeutic strategy for this disease.

T cells engineered with full-length NKG2D linked to signaling domains of 4-1BB and CD3ζ show enhanced antitumor activity.

Since NKG2D ligands (NKG2DLs) are primarily overexpressed on multiple types of solid tumors but absent on most normal tissues, NKG2DLs could be optimal antigens for CAR-T cells. To date, there have been two types of NKG2DL CARs: (i) the extracellular domain of NKG2D fused to the CD8a transmembrane domain, signaling domains of 4-1BB and CD3ζ (NKBz) and (ii) full-length NKG2D fused to the CD3ζ signaling domain (chNKz). Although NKBz- and chNKz-engineered T cells both showed antitumor activities, a comparison of their functions has not been reported. In addition, use of the 4-1BB signaling domain into the CAR construct could prolong the persistence and resistance to antitumor activities of CAR-T cells, we designed a new NKG2DL CAR, full-length NKG2D fused to the signaling domains of 4-1BB and CD3ζ (chNKBz). Among the two types of NKG2DL CAR-T cells reported in previous studies, we found that chNKz T cells had stronger antitumor ability than NKBz T cells in vitro, but their antitumor activity in vivo is similar. The chNKBz T cells showed antitumor activity superior to that of chNKz T cells and NKBz T cells in vitro and in vivo, providing a new option for the immunotherapy of NKG2DL-positive tumor patients.

Targeting Claudin 18.2 Using a Highly Specific Antibody Enables Cancer Diagnosis and Guided Surgery.

Claudin 18.2 (CLDN18.2) is a new potential target for cancer therapy, especially for advanced gastric cancer (AGC). A molecular targeting probe is of importance for patient stratification and therapeutic guidance. Here, we explored an antibody-dependent molecular imaging strategy for specific detection and surgery guidance based on a CLDN18.2-specific antibody, 5C9. Two imaging probes, 124I-5C9 and Cy5.5-5C9, were synthesized. The specificity to CLDN18.2 being evidenced in the cellular experiments with control, the diagnostic utility was assessed by immunopositron emission tomography (immuno-PET) and fluorescence imaging using xenograft models. A near-infrared fluorescent II imaging probe FD1080-5C9 was designed to facilitate the comprehensive surgical removal of lesions. 124I-5C9 immuno-PET imaging clearly delineated subcutaneous CLDN18.2-positive tumors, with a peak uptake (maximum standardized uptake value; SUVmax) of 2.25 ± 0.30, whereas the highest values for the 124I-IgG and blocking groups were 0.70 ± 0.13 and 0.66 ± 0.12, respectively. Cy5.5-5C9 fluorescence imaging showed similar results. As proof of the diagnosis and guided surgery (DGS) concept, 124I-5C9 and FD1080-5C9 were simultaneously administered in orthotopic CLDN18.2-positive tumor models, facilitating the comprehensive resection of tumor tissue. Combined, 124I-5C9 and FD1080-5C9 are both promising DGS tools: the former reveals CLDN18.2 in lesions as a PET probe, and the latter can guide surgery. These results provide a utility molecular imaging strategy for specific detection and surgery guidance based on a CLDN18.2-specific antibody both in AGC and other cancers.

Pre-depletion of TRBC1+ T cells promotes the therapeutic efficacy of anti-TRBC1 CAR-T for T-cell malignancies.

Targeting T cell receptor ß-chain constant region 1 (TRBC1) CAR-T could specifically kill TRBC1+ T-cell malignancies. However, over-expressed CARs on anti-TRBC1 CAR transduced TRBC1+ T cells (CAR-C1) bound to autologous TRBC1, masking TRBC1 from identification by other anti-TRBC1 CAR-T, and moreover only the remaining unoccupied CARs recognized TRBC1+ cells, considerably reducing therapeutic potency of CAR-C1. In addition, co-culture of anti-TRBC1 CAR-T and TRBC1+ cells could promote exhaustion and terminal differentiation of CAR-T. These findings provide a rationale for pre-depleting TRBC1+ T cells before anti-TRBC1 CAR-T manufacturing.

Proteomic analysis revealed common, unique and systemic signatures in gender-dependent hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is the most common liver cancer and is highly malignant. Male prevalence and frequent activation of the Ras signaling pathway are distinct characteristics of HCC. However, the underlying mechanisms remain to be elucidated. By exploring Hras12V transgenic mice showing male-biased hepatocarcinogenesis, we performed a high-throughput comparative proteomic analysis based on tandem-mass-tag (TMT) labeling combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) on the tissue samples obtained from HCC (T) and their paired adjacent precancerous (P) of Hras12V transgenic male and female mice (Ras-Tg) and normal liver (W) of wild-type male and female mice (Non-Tg). The further validation and investigation were performed using quantitative real-time PCR and western blot. Totally, 5193 proteins were quantified, originating from 5733 identified proteins. Finally, 1344 differentially expressed proteins (DEPs) (quantified in all examined samples; |ratios| ≥ 1.5, p < 0.05) were selected for further analysis. Comparison within W, P, and T of males and females indicated that the number of DEPs in males was much higher than that in females. Bioinformatics analyses showed the common and unique cluster-enriched items between sexes, indicating the common and gender-disparate pathways towards HCC. Expression change pattern analysis revealed HCC positive/negative-correlated and ras oncogene positive/negative-correlated DEPs and pathways. In addition, it showed that the ras oncogene gradually and significantly reduced the responses to sex hormones from hepatocytes to hepatoma cells and therefore shrunk the gender disparity between males and females, which may contribute to the cause of the loss of HCC clinical responses to the therapeutic approaches targeting sex hormone pathways. Additionally, gender disparity in the expression levels of key enzymes involved in retinol metabolism and terpenoid backbone/steroid biosynthesis pathways may contribute to male prevalence in hepatocarcinogenesis. Further, the biomarkers, SAA2, Orm2, and Serpina1e, may be sex differences. In conclusion, common and unique DEPs and pathways toward HCC initiated by ras oncogene from sexually dimorphic hepatocytes provide valuable and novel insights into clinical investigation and practice.

Mycoplasma hyorhinis infection promotes gastric cancer cell motility via ß-catenin signaling.

BACKGROUND: We previously identified that Mycoplasma hyorhinis infection promotes gastric cancer cell motility. The ß-catenin signaling pathway is critical to determining malignant cancer cell phenotypes; however, the association between M hyorhinis and the ß-catenin signaling pathway is unclear. METHODS: We performed subcellular fractionation and immunofluorescence staining to observe ß-catenin accumulation in the nucleus. The expression of downstream ß-catenin genes was detected by quantitative RT-PCR. Gastric cancer cell motility was examined by transwell chamber migration and wound healing assays, and a co-immunoprecipitation assay was used to detect the proteins associated with the membrane protein p37 of M hyorhinis. RESULTS: We found that M hyorhinis infection promoted nuclear ß-catenin accumulation and enhanced the expression of downstream ß-catenin genes. M hyorhinis-promoted gastric cancer cell motility was counteracted by treatment with the ß-catenin inhibitor XAV939 or ß-catenin knockdown. We further detected a protein complex containing LRP6, GSK3ß, and p37 in M hyorhinis-infected cells. M hyorhinis also induced LRP6 phosphorylation in a GSK3ß-dependent fashion. Knockdown of LRP6 or GSK3ß abolished M hyorhinis-induced cell motility. CONCLUSION: Our results reveal that the ß-catenin signaling pathway could be activated by M hyorhinis infection, thereby contributing to M hyorhinis-induced gastric cancer cell motility.

mTOR and ERK regulate VKORC1 expression in both hepatoma cells and hepatocytes which influence blood coagulation.

Deficiency of γ-glutamyl carboxylation of coagulation factors, as evidenced by the elevated level of Des-γ-carboxyl prothrombin (DCP), is a common feature in hepatocellular carcinoma patients. Additionally, treatment of cancer patients with mTOR inhibitors significantly increases hemorrhagic events. However, the underlying mechanisms remain unknown. In the present study, Vitamin K epoxide reductase complex subunit 1 (VKORC1) was found to be significantly down-regulated in clinical hepatoma tissues and most tested hepatoma cell lines. In vitro investigations showed that VKORC1 expression was promoted by p-mTOR at the translational level and repressed by p-ERK at the transcriptional level. By exploring Hras12V transgenic mice, a hepatic tumor model, VKROC1 was significantly down-regulated in hepatic tumors and showed prolonged activated partial prothrombin time (APTT). In vivo investigations further showed that VKORC1 expression was promoted by p-mTOR and repressed by p-ERK in both hepatoma and hepatocytes. Consistently, APTT and prothrombin time were significantly prolonged under the mTOR inhibitor treatment and significantly shortened under the ERK inhibitor treatment. Conclusively, these findings indicate that mTOR and ERK play crucial roles in controlling VKORC1 expression in both hepatoma and hepatocytes, which provides a valuable molecular basis for preventing hemorrhage in clinical therapies.

Metabolomic and transcriptomic profiling of hepatocellular carcinomas in Hras12V transgenic mice.

Activation of the Ras/MAPK pathway is prevalently involved in the occurrence and development of hepatocellular carcinoma (HCC). However, its effects on the deregulated cellular metabolic processes involved in HCC in vivo remain unknown. In this study, a mouse model of HCC induced by hepatocyte-specific expression of the Hras12V oncogene was investigated using an integrative analysis of metabolomics and transcriptomics data. Consistent with the phenotype of abundant lipid droplets in HCC, the lipid biosynthesis in HCC was significantly enhanced by (1) a sufficient supply of acetyl-CoA from enhanced glycolysis and citrate shuttle activity; (2) a sufficient supply of NADPH from enhanced pentose phosphate pathway (PPP) activity; (3) upregulation of key enzymes associated with lipid biosynthesis; and (4) downregulation of key enzymes associated with bile acid biosynthesis. In addition, glutathione (GSH) was significantly elevated, which may result from a sufficient supply of 5-oxoproline and L-glutamate as well as an enhanced reduction in the process of GSSG being turned into GSH by NADPH. The high level of GSH along with elevated Bcl2 and Ucp2 expression may contribute to a normal level of reactive oxygen species (ROS) in HCC. In conclusion, our results suggest that the lipid metabolism, glycolysis, PPP, tricarboxylic acid (TCA) cycle, citrate shuttle activity, bile acid synthesis, and redox homeostasis in the HCC induced by ras oncogene are significantly perturbed, and these altered metabolic processes may play crucial roles in the carcinogenesis, development, and pathological characteristics of HCC.

B lymphocytes repress hepatic tumorigenesis but not development in Hras12V transgenic mice.

Increasing reports show noninflammation underlying HCC, challenging our understanding of the roles of the immune system in hepatocarcinogenesis. By exploring a mouse model of hepatic tumor induced by hepatocyte-specific expression of the Hras12V oncogene without obvious inflammation, we found that the proportion of B cells, but not T cells, progressively and significantly decreased in 3, 5-month-old transgenic mice (Tg) compared with non-transgenic mice. Notably, the proportions of total and activated B and T cells all significantly decreased in 9-month-old Tg with multiple massive hepatic tumors. Together with the decreased B cell proportion, serum IgG1/2 also significantly decreased in 5, 9-month-old Tg. Interestingly, homozygous Tg showed significantly higher B cell proportion and IgG2 levels, accompanied by significantly lower incidences of liver nodules but not adenomas and carcinomas compared with heterozygous Tg. Treatment of Tg with PCI-32765, a potent Bruton's tyrosine kinase (BTK) inhibitor for suppressing B cell proliferation and activation, significantly decreased the B cell proportion and IgG2 levels, accompanied by a significantly higher incidence of liver nodules, but had no effects on adenoma and carcinoma. Treatment of Tg with insulin-like growth factor 1 (IGF-1) significantly increased the B cell proportion and IgG2 levels, accompanied by a significantly lower incidence of liver nodules and carcinoma, but had no effects on adenoma. Conclusively, B cells and IgG2 may play important roles in suppressing hepatic tumorigenesis, but not development. In addition, hepatocyte-specific expression of the ras oncogene may play roles in suppressing B cells, while developed hepatic tumors suppress both B and T cells.

Differential Proteomic Analysis of Gender-dependent Hepatic Tumorigenesis in Hras12V Transgenic Mice.

Male prevalence is an outstanding characteristic of hepatocellular carcinoma (HCC), and the underlying mechanisms for this have remained largely unknown. In the present study, Hras12V transgenic mice, in which hepatocyte-specific expression of the ras oncogene induces male-biased hepatic tumorigenesis, were studied, and altered proteins were detected by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). Protein samples from hepatic tumor tissues (T) and peritumor tissues (P) of transgenic males and females and the corresponding normal liver tissues (Wt) of nontransgenic males and females were subjected to pairwise comparisons based on proteomic analysis. Among 2381 autodetected protein spots, more than 1600 were differentially expressed based on a pairwise comparison (|ratio| > = 1.5, p < = 0.05). Of these, 180 spots were randomly selected for matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF MS) identification; finally, 89 distinct proteins were obtained. Among these 89 proteins, 7 and 50 proteins were further validated by Western blotting and literature investigation, respectively. Intriguingly, compared with Wt, the altered proteins were relatively concentrated in T in transgenic females but in P in transgenic males. Consistently, the levels of p-ERK and p-mTOR were significantly higher in the T of females compared with that of males. The pathway enrichment assay showed that 5 pathways in males but only 1 in females were significantly altered in terms of the upregulated proteins in T compared with Wt. These data indicate that female hepatocytes are disturbed by oncogenes with great difficulty, whereas male hepatocytes readily do so. In addition, 33 proteins were gender-dependently altered in hepatic tumorigenesis. Moreover, 4% DNA packaging and 4% homeostasis-related functional proteins were found in females but not in males, and more nucleus proteins were found in females (8%) than in males (3%). In conclusion, the proteomic data and comparative analysis presented here offer crucial clues for elucidating the mechanisms that underlie the male prevalence in HCC.