GRP75-driven, cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles underlies distinct gene therapy effect in ovarian cancer.

Practice of tumor-targeted suicide gene therapy is hampered by unsafe and low efficient delivery of plasmid DNA (pDNA). Using HIV-Tat-derived peptide (Tat) to non-covalently form Tat/pDNA complexes advances the delivery performance. However, this innovative approach is still limited by intracellular delivery efficiency and cell-cycle status. In this study, Tat/pDNA complexes were further condensed into smaller, nontoxic nanoparticles by Ca2+ addition. Formulated Tat/pDNA-Ca2+ nanoparticles mainly use macropinocytosis for intercellular delivery, and their macropinocytic uptake was persisted in mitosis (M-) phase and highly activated in DNA synthesis (S-) phase of cell-cycle. Over-expression or phosphorylation of a mitochondrial chaperone, 75-kDa glucose-regulated protein (GRP75), promoted monopolar spindle kinase 1 (MPS1)-controlled centrosome duplication and cell-cycle progress, but also driven cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles. Further in vivo molecular imaging based on DF (Fluc-eGFP)-TF (RFP-Rluc-HSV-ttk) system showed that Tat/pDNA-Ca2+ nanoparticles exhibited highly suicide gene therapy efficiency in mouse model xenografted with human ovarian cancer. Furthermore, arresting cell-cycle at S-phase markedly enhanced delivery performance of Tat/pDNA-Ca2+ nanoparticles, whereas targeting GRP75 reduced their macropinocytic delivery. More importantly, in vivo targeting GRP75 combined with cell-cycle or macropinocytosis inhibitors exhibited distinct suicide gene therapy efficiency. In summary, our data highlight that mitochondrial chaperone GRP75 moonlights as a biphasic driver underlying cell-cycle-dependent macropinocytosis of Tat/pDNA-Ca2+ nanoparticles in ovarian cancer.

Development of a Prognostic Risk Model Based on Oxidative StressRelated Genes for Platinum-Resistant Ovarian Cancer Patients.

INTRODUCTION: Ovarian Cancer (OC) is a heterogeneous malignancy with poor outcomes. Oxidative stress plays a crucial role in developing drug resistance. However, the relationships between Oxidative Stress-related Genes (OSRGs) and the prognosis of platinum-resistant OC remain unclear. This study aimed to develop an OSRGs-based prognostic risk model for platinum-resistant OC patients. METHODS: Gene Set Enrichment Analysis (GSEA) was performed to determine the expression difference of OSRGs between platinum-resistant and -sensitive OC patients. Cox regression analyses were used to identify the prognostic OSRGs and establish a risk score model. The model was validated by using an external dataset. Machine learning was used to determine the prognostic OSRGs associated with platinum resistance. Finally, the biological functions of selected OSRG were determined via in vitro cellular experiments. RESULTS: Three gene sets associated with oxidative stress-related pathways were enriched (p < 0.05), and 105 OSRGs were found to be differentially expressed between platinum-resistant and - sensitive OC (p < 0.05). Twenty prognosis-associated OSRGs were identified (HR: 0:562-5.437; 95% CI: 0.319-20.148; p < 0.005), and seven independent OSRGs were used to construct a prognostic risk score model, which accurately predicted the survival of OC patients (1-, 3-, and 5-year AUC=0.69, 0.75, and 0.67, respectively). The prognostic potential of this model was confirmed in the validation cohort. Machine learning showed five prognostic OSRGs (SPHK1, PXDNL, C1QA, WRN, and SETX) to be strongly correlated with platinum resistance in OC patients. Cellular experiments showed that WRN significantly promoted the malignancy and platinum resistance of OC cells. CONCLUSION: The OSRGs-based risk score model can efficiently predict the prognosis and platinum resistance of OC patients. This model may improve the risk stratification of OC patients in the clinic.

Hypoxia-activated selectivity-improved anti-PKM2 antibody combined with prodrug TH-302 for potentiated targeting therapy in hepatocellular carcinoma.

Background: Hypoxia induces hepatocellular carcinoma (HCC) malignancies; yet it also offers treatment opportunities, exemplified by developing hypoxia-activated prodrugs (HAPs). Although HAP TH-302 combined with therapeutic antibody (Ab) has synergistic effects, the clinical benefits are limited by the on-target off-tumor toxicity of Ab. Here, we sought to develop a hypoxia-activated anti-M2 splice isoform of pyruvate kinase (PKM2) Ab combined with TH-302 for potentiated targeting therapy. Methods: Codon-optimized and hypoxia-activation strategies were used to develop H103 Ab-azo-PEG5k (HAP103) Ab. Hypoxia-activated HAP103 Ab was characterized, and hypoxia-dependent antitumor and immune activities were evaluated. Selective imaging and targeting therapy with HAP103 Ab were assessed in HCC-xenografted mouse models. Targeting selectivity, systemic toxicity, and synergistic therapeutic efficacy of HAP103 Ab with TH-302 were evaluated. Results: Human full-length H103 Ab was produced in a large-scale bioreactor. Azobenzene (azo)-linked PEG5k conjugation endowed HAP103 Ab with hypoxia-activated targeting features. Conditional HAP103 Ab effectively inhibited HCC cell growth, enhanced apoptosis, and induced antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) functions. Analysis of HCC-xenografted mouse models showed that HAP103 Ab selectively targeted hypoxic HCC tissues and induced potent tumor-inhibitory activity either alone or in combination with TH-302. Besides the synergistic effects, HAP103 Ab had negligible side effects when compared to parent H103 Ab. Conclusion: The hypoxia-activated anti-PKM2 Ab safely confers a strong inhibitory effect on HCC with improved selectivity. This provides a promising strategy to overcome the on-target off-tumor toxicity of Ab therapeutics; and highlights an advanced approach to precisely kill HCC in combination with HAP TH-302.

Hypoxia-activated ADCC-enhanced humanized anti-CD147 antibody for liver cancer imaging and targeted therapy with improved selectivity.

Therapeutic antibodies (Abs) improve the clinical outcome of cancer patients. However, on-target off-tumor toxicity limits Ab-based therapeutics. Cluster of differentiation 147 (CD147) is a tumor-associated membrane antigen overexpressed in cancer cells. Ab-based drugs targeting CD147 have achieved inadequate clinical benefits for liver cancer due to side effects. Here, by using glycoengineering and hypoxia-activation strategies, we developed a conditional Ab-dependent cellular cytotoxicity (ADCC)-enhanced humanized anti-CD147 Ab, HcHAb18-azo-PEG5000 (HAP18). Afucosylated ADCC-enhanced HcHAb18 Ab was produced by a fed-batch cell culture system. Azobenzene (Azo)-linked PEG5000 conjugation endowed HAP18 Ab with features of hypoxia-responsive delivery and selective targeting. HAP18 Ab potently inhibits the migration, invasion, and matrix metalloproteinase secretion, triggers the cytotoxicity and apoptosis of cancer cells, and induces ADCC, complement-dependent cytotoxicity, and Ab-dependent cellular phagocytosis under hypoxia. In xenograft mouse models, HAP18 Ab selectively targets hypoxic liver cancer tissues but not normal organs or tissues, and has potent tumor-inhibiting effects. HAP18 Ab caused negligible side effects and exhibited superior pharmacokinetics compared to those of parent HcHAb18 Ab. The hypoxia-activated ADCC-enhanced humanized HAP18 Ab safely confers therapeutic efficacy against liver cancer with improved selectivity. This study highlights that hypoxia activation is a promising strategy for improving the tumor targeting potential of anti-CD147 Ab drugs.

GRP75-faciliated Mitochondria-associated ER Membrane (MAM) Integrity controls Cisplatin-resistance in Ovarian Cancer Patients.

Background: Control of ER-mitochondrial Ca2+ fluxes is a critical checkpoint to determine cell fate under stress. The 75-kDa glucose-regulated protein (GRP75) is a key tether protein facilitating mitochondria-associated ER membrane (MAM) formation through the IP3R-GRP75-VDAC1 complex. Although GRP75 contributes to cisplatin (CP)-resistance of ovarian cancer (OC), the underlying mechanisms are not clear. Methods: CP-resistant and -sensitive OC cell lines with GRP75 stable modulation were established. Confocal, PLA, co-IP, and TEM analysis were utilized to detect MAM integrity. Live cell Ca2+ imaging, intracellular ATP, ROS, and NAD+ assays were utilized to investigate ER-to-mitochondrial Ca2+ transfer and mitochondrial bioenergetics. Western blot, flow cytometry, CCK-8, Δψm, and mPTP assays were utilized to examine apoptotic cell death. Bioinformatics, patient's specimens, and immunohistochemistry were conducted to obtain the clinical relevance for GRP75-facilitated MAM formation. Results: GRP75-faciliated MAM formation was enriched in CP-resistant OC cells. CP-exposure only increased MAM formation in CP-sensitive OC cells, and enrichment of GRP75 and VDAC1 at MAMs is indispensable to CP-resistance. Diminishing MAM integrity by GRP75-deficiency reduced ER-to-mitochondria Ca2+ transfer, accelerated CP-induced mitochondrial dysfunction, provoked catastrophic ROS, and enhanced CP-triggered apoptotic cell death in OC cells. Clinical investigations confirmed the enrichment of GRP75-faciliated MAM formation in relapsed OC patients, and such enrichment was associated with the CP-resistance phenotype. Conclusion: GRP75-overexpression confers CP-resistance by distinctively managing MAM-facilitated Ca2+ fluxes and the pro-survival ROS signal, whereas GRP75-deficiency induces cell death via bioenergetic crisis and apoptotic ROS accumulation in OC cells. Our results show that GRP75-faciliated MAM formation is a potential target to overcome CP-resistance of OC.

Arf6-mediated macropinocytosis-enhanced suicide gene therapy of C16TAB-condensed Tat/pDNA nanoparticles in ovarian cancer.

The use of cell-penetrating peptides (CPPs), typically HIV-Tat, to deliver therapeutic genes for cancer treatment is hampered by the inefficient delivery and complicated uptake route of plasmid DNA (pDNA). On the one hand, surface charges, particle size and shape essentially contribute to the endocytosis pathway of Tat/pDNA nanocomplexes, and on the other hand, endogenous cellular factors dominantly determine their intracellular trafficking fate and biological outcome. Recent advances in surfactant-modified nanomaterial and dual molecular imaging technology have offered new opportunities for suicide gene therapy. In this study, we employed the cationic surfactant C16TAB to further condense Tat/pDNA nanocomplexes for improving their delivery efficiency and tested the therapeutic effect of Tat/pDNA/C16TAB (T-P-C) nanoparticles carrying the GCV-converted HSV-ttk suicide gene for ovarian cancer. The cellular endocytosis pathway and underlying signal mechanism of T-P-C nanoparticles were further determined. The obtained T-P-C nanoparticles exhibited a small size, positive surface charge, irregular granular shape and high pDNA encapsulation efficiency. The in vitro experiments showed that T-P-C nanoparticles mainly used the macropinocytosis pathway for uptake in ovarian cancer cells. Their internalization and payload gene expression were controlled by the Arf6 GTPase-dependent, Rab GTPase-activated signal axis. Further in vivo molecular imaging based on DF (Fluc-eGFP)-TF (RFP-Rluc-HSV-ttk) system showed that T-P-C nanoparticles significantly increased the targeted delivery and suicide gene therapy in a mouse model xenografted with human ovarian cancer. More importantly, Arf6-mediated macropinocytosis remarkably enhanced the delivery efficiency and suicide gene therapy effect of T-P-C nanoparticles. Therefore, these C16TAB-condensed Tat/pDNA nanoparticles combined with the dual molecular imaging strategy provides a novel intracellular delivery platform for high-efficient, precise suicide gene therapy of ovarian cancer.