Nucleotides as new co-formers in co-amorphous systems: Enhanced dissolution rate, water solubility and physical stability.

Developing co-amorphous systems is an attractive strategy to improve the dissolution rate of poorly water-soluble drugs. Various co-formers have been investigated. However, previous studies revealed that it is a challenge to develop satisfied acidic co-formers, e.g., acidic amino acids showed much poorer co-former properties than neutral and basic amino acids. Only a few acidic co-formers have been reported, such as aspartic acid, glutamic acid, and some other organic acids. Thus, this study aims to explore the possibility of adenosine monophosphate and adenosine diphosphate used as acidic co-formers. Mebendazole, celecoxib and tadalafil were used as the model drugs. The drug-co-former co-amorphous systems were prepared via ball milling and confirmed using XRPD. The dissolution study suggested that the solubility and dissolution rate of the drug-co-formers systems were increased significantly compared to the corresponding crystalline and amorphous drugs. The stability study revealed that using the two nucleotides as co-formers enhanced the physical stability of pure amorphous drugs. Molecular interactions were observed in MEB-co-former and TAD-co-former systems and positively affected the pharmaceutical performance of the investigated co-amorphous systems. In conclusion, the two nucleotides could be promising potential acidic co-formers for co-amorphous systems.

Single-cell sequencing of tumour infiltrating T cells efficiently identifies tumour-specific T cell receptors based on the T cell activation score.

Adoptively transferred T cell receptor-engineered T cells are a promising cancer treatment strategy, and the identification of tumour-specific TCRs is essential. Previous studies reported that tumour-reactive T cells and TCRs could be isolated based on the expression of activation markers. However, since T cells with different cell states could not respond uniformly to activation but show a heterogeneous expression profile of activation and effector molecules, isolation of tumour-reactive T cells based on single activation or effector molecules could result in the absence of tumour-reactive T cells; thus, combinations of multiple activation and effector molecules could improve the efficiency of isolating tumour-specific TCRs. We enrolled two patients with lung adenocarcinoma and obtained their tumour infiltrating lymphocytes (TILs) and autologous tumour cells (ATCs). TILs were cocultured with the corresponding ATCs for 12 h and subjected to single-cell RNA sequencing. First, we identified three TCRs with the highest expression levels of IFNG and TNFRSF9 mRNA for each patient, yet only the top one or two recognized the corresponding ATCs in each patient. Next, we defined the activation score based on normalized expression levels of IFNG, IL2, TNF, IL2RA, CD69, TNFRSF9, GZMB, GZMA, GZMK, and PRF1 mRNA for each T cell and then identified three TCRs with the highest activation score for each patient. We found that all three TCRs in each patient could specifically identify corresponding ATCs. In conclusion, we established an efficient approach to isolate tumour-reactive TCRs based on combinations of multiple activation and effector molecules through single-cell RNA sequencing.

Single-cell RNA sequencing reveals aberrant sphingolipid metabolism in non-small cell lung cancer impacts tumor-associated macrophages and stimulates angiogenesis via macrophage inhibitory factor signaling.

BACKGROUND: Sphingolipids not only serve as structural components for maintaining cell membrane fluidity but also function as bioactive molecules involved in cell signaling and the regulation of various biological processes. Their pivotal role in cancer cell development, encompassing cancer cell proliferation, migration, angiogenesis, and metastasis, has been a focal point for decades. However, the contribution of sphingolipids to the complexity of tumor microenvironment promoting cancer progression has been rarely investigated. METHODS: Through the integration of publicly available bulk RNA-seq and single-cell RNA-seq data, we conducted a comprehensive analysis to compare the transcriptomic features between tumors and adjacent normal tissues, thus elucidating the intricacies of the tumor microenvironment (TME). RESULTS: Disparities in sphingolipid metabolism (SLM)-associated genes were observed between normal and cancerous tissues, with the TME characterized by the enrichment of sphingolipid signaling in macrophages. Cellular interaction analysis revealed robust communication between macrophages and cancer cells exhibiting low SLM, identifying the crucial ligand-receptor pair, macrophage inhibitory factor (MIF)-CD74. Pseudo-time analysis unveiled the involvement of SLM in modulating macrophage polarization towards either M1 or M2 phenotypes. Categorizing macrophages into six subclusters based on gene expression patterns and function, the SPP1+ cluster, RGS1+ cluster, and CXCL10+ cluster were likely implicated in sphingolipid-induced M2 macrophage polarization. Additionally, the CXCL10+, AGER+, and FABP4+ clusters were likely to be involved in angiogenesis through their interaction with endothelial cells. CONCLUSION: Based on multiple scRNA-seq datasets, we propose that a MIF-targeted strategy could potentially impede the polarization from M1 to M2 and impair tumor angiogenesis in low-SLM non-small cell lung cancer (NSCLC), demonstrating its potent antitumor efficacy.

Metabolic reprogramming induced by DCA enhances cisplatin sensitivity through increasing mitochondrial oxidative stress in cholangiocarcinoma.

Background: Cholangiocarcinoma has obvious primary multidrug resistance and is generally resistant to cisplatin and other chemotherapy drugs and high glycolytic levels may be associated with chemotherapy resistance of cholangiocarcinoma cells. Dichloroacetate (DCA) is a specific inhibitor of PDK, which can promote mitochondrial aerobic oxidation process by activating PDH. In the past few years, there have been an increasing number of studies supporting the action of DCA against cancer, which also provided evidence for targeting metabolism to enhance the efficacy of cholangiocarcinoma chemotherapy. Methods: Glucose uptake and lactic acid secretion were used to detect cell metabolism level. Cell apoptosis and cell cycle were detected to confirm cell fate induced by cisplatin combined with DCA. Mito-TEMPO was used to inhibit mtROS to explore the relationship between oxidative stress and cell cycle arrest induced by DCA under cisplatin stress. Finally, PCR array and autophagy inhibitor CQ were used to explore the potential protective mechanism under cell stress. Results: DCA changed the metabolic model from glycolysis to aerobic oxidation in cholangiocarcinoma cells under cisplatin stress. This metabolic reprogramming increased mitochondrial reactive oxygen species (mtROS) levels, which promoted cell cycle arrest, increased the expression of antioxidant genes and activated autophagy. Inhibition of autophagy further increased the synergistic effect of DCA and cisplatin. Conclusion: DCA increased cisplatin sensitivity in cholangiocarcinoma cells via increasing the mitochondria oxidative stress and cell growth inhibition. Synergistic effects of DCA and CQ were observed in cholangiocarcinoma cells, which further increased the cisplatin sensitivity via both metabolic reprogramming and inhibition of the stress response autophagy.

TCR extracellular domain genetically linked to CD28, 2B4/41BB and DAP10/CD3ζ -engineered NK cells mediates antitumor effects.

NK cells, especially FDA-approved NK-92 cells, could be used for TCR engineering owing to their specialized cytotoxicity against tumors, safety profile and potential use as an off-the-shelf cellular therapy. The TCR complex requires assembly of TCR- α/ ß chains with CD3 molecules (CD3δ, CD3γ, CD3ε, CD3ζ) to be correctly expressed at the cell membrane, and yet NK cells lack expression of these CD3 subunits besides CD3ζ. Since transmembrane regions of TCR α and ß chains are involved in TCR complex assembly, transmembrane regions of TCR replaced by CD28 transmembrane domain could result in the expression of TCR independent of its companion CD3 subunits. However, since the absence of CD3 signaling components can influence the transmission of TCR signals to NK cells, it is necessary to add the signaling molecules of NK cells followed by CD28 transmembrane domain. Both CD3ζ and DAP10 play an important role in the activation and cytotoxicity of NK cells; moreover, 2B4 and 4-1BB are the main costimulatory molecules in NK cells. Therefore, we designed a chimeric TCR that consisted of the extracellular domains of the TCR α and ß chains specific for NYESO-1 fused to the CD28 transmembrane domain followed by the 41BB and CD3ζ signaling domains as well as the 2B4 and DAP10 signaling domain, respectively. The chimeric TCR genetically engineered NK-92 cells exhibit antigen-specific recognition and lysis of tumor cells both in vitro and in vivo. In addition, TCR-28-2B10/BBζ can be feasibly expressed in primary NK cells and exhibit antigen-reactive recognition and effect function. The overall encouraging data highlight the value of NK-92 cells and primary NK cells engineered to express therapeutic chimeric TCR for adoptive immunotherapies.

Inhibition of TIGAR Increases Exogenous p53 and Cisplatin Combination Sensitivity in Lung Cancer Cells by Regulating Glycolytic Flux.

The metabolism and apoptosis of tumor cells are important factors that increase their sensitivity to chemotherapeutic drugs. p53 and cisplatin not only induce tumor cell apoptosis, but also regulate the tumor cell metabolism. The TP53-induced glycolysis and apoptosis regulator (TIGAR) can inhibit glycolysis and promote more glucose metabolism in the pentose phosphate pathway. We speculate that the regulation of the TIGAR by the combination therapy of p53 and cisplatin plays an important role in increasing the sensitivity of tumor cells to cisplatin. In this study, we found that the combined treatment of p53 and cisplatin was able to inhibit the mitochondrial function, promote mitochondrial pathway-induced apoptosis, and increase the sensitivity. Furthermore, the expression of the TIGAR was inhibited after a combined p53 and cisplatin treatment, the features of the TIGAR that regulate the pentose phosphate pathway were inhibited, the glucose flux shifted towards glycolysis, and the localization of the complex of the TIGAR and Hexokinase 2 (HK2) on the mitochondria was also reduced. Therefore, the combined treatment of p53 and cisplatin may modulate a glycolytic flux through the TIGAR, altering the cellular metabolic patterns while increasing apoptosis. Taken together, our findings reveal that the TIGAR may serve as a potential therapeutic target to increase the sensitivity of lung cancer A549 cells to cisplatin.

Autophagic flux restoration enhances the antitumor efficacy of tumor infiltrating lymphocytes.

BACKGROUND: Although adoptive cell therapy with tumor infiltrating lymphocytes (TILs) has mediated effective antitumor responses in several cancers, dysfunction and exhaustion of TILs significantly impair the therapeutic effect of TILs. Thus, it is essential to elucidate the exhausted characteristics of TILs and improve the antitumor effect of TILs by reversing their exhaustion. Here, we focused on the influence of autophagy on TILs in terms of T-cell activation, proliferation, and differentiation in vitro and in vivo. METHODS: We first evaluated autophagy level of TILs and influence of spermidine treatment on autophagy levels of TILs. Furthermore, we assessed the proliferative potential, phenotypical characteristics, T cell receptor (TCR) repertoire and antitumor activity of TILs with and without spermidine treatment. RESULTS: We found that autophagic flux of TILs, especially exhausted TILs that express inhibitory immunoreceptors and have impaired proliferative capacity and decreased production of cytotoxic effector molecules, was significantly impaired. The restoration of autophagic flux via spermidine treatment resulted in increased diversity of the TCR repertoire, reduced expression of inhibitory immunoreceptors (PD1, TIM3, or LAG3), enhanced proliferation and effector functions, which subsequently demonstrated the superior in vitro and in vivo antitumor activity of TILs. Our findings unveil that spermidine, as an autophagy inducer, reverses dysfunction and exhaustion of TILs and subsequently improves the antitumor activity of TILs. CONCLUSIONS: These data suggest that spermidine treatment presents an opportunity to improve adoptive TIL therapy for the treatment of solid tumors.

Autophagic flux restoration of senescent T cells improves antitumor activity of TCR-engineered T cells.

Objectives: Although adoptive cell therapy with T-cell receptor-engineered T cells (TCR-Ts) has mediated effective antitumor responses in several cancers, senescence of T cells could impair the therapeutic effect of TCR-Ts. Thus, it is essential to elucidate the characteristics of senescent TCR-Ts and how to subsequently improve their antitumor effect. Here, we focused on the influence of autophagy on TCR-Ts, since autophagy is tightly associated with the regulation of T-cell activation, proliferation and differentiation. Methods: We first evaluated autophagy level of senescent TCR-Ts, and then the senescent TCR-Ts were expanded in vitro for 7 days with and without spermidine treatment, respectively. Furthermore, the proliferative potential, phenotypical characteristics and functionality of the propagated senescent TCR-Ts were analysed in vitro and in vivo after 7-day ex vivo expansion. Results: We found that autophagic flux of senescent TCR-T cells was significantly impaired. The restoration of autophagic flux via spermidine treatment reduced the expression of inhibitory immunoreceptors (PD-1, TIM-3 or LAG-3), enhanced proliferation and effector functions and subsequently demonstrated the superior in vitro and in vivo antitumor activity of TCR-Ts. Conclusion: These data suggest that spermidine treatment presents an opportunity to improve the antitumor effect of TCR-Ts for the treatment of solid tumors.

Metabolic reprogramming by ex vivo glutamine inhibition endows CAR-T cells with less-differentiated phenotype and persistent antitumor activity.

The inadequate in vivo persistence of chimeric antigen receptor (CAR)-modified T cells has been shown to lead to poor therapeutic efficacy and disease recurrence. In vivo persistence is associated with the differentiation subsets infused, with less differentiated TN or TCM conferring superior renewal capacity and antitumor immunity compared to TEM or TEFF. However, ex vivo expanded CAR-T cells exhibit phenotypic heterogeneity with majority of TEM or TEFF subsets and very low populations of TN and TCM. The transition of differentiation subsets is closely correlated with T cell metabolism fitness. Effector T cell differentiation from TN or TCM requires glutamine uptake and metabolism. Using a CD19-specific CAR, we demonstrated that glutamine inhibition by adding the glutamine antagonist 6-Diazo-5-oxo-l-norleucine (DON) into the culture endows CAR-T cells with enhanced mitochondrial OXPHOS utilizing fatty acids and reduced glycolytic activity, and retains more TN or TCM subsets. DON- pretreated CAR-T cells exhibited stronger cytotoxic lysis in vitro and more robust elimination of tumor burdens in vivo. This study suggests that glutamine inhibition ex vivo would be a potential approach for modulating metabolism and differentiation state to improve the efficacy of CAR-T cell therapy.

p62 Promotes the Mitochondrial Localization of p53 through Its UBA Domain and Participates in Regulating the Sensitivity of Ovarian Cancer Cells to Cisplatin.

Chemotherapeutic drug-induced p53-dependent crosstalk among tumor cells affects the sensitivity of tumor cells to chemotherapeutic drugs, contributing to chemoresistance. Therefore, pharmacological targeting of p53 may contribute to overcoming drug resistance. The localization of p53 is closely related to its function. Thus, we assessed the effect of p62 on the coordination of p53 mitochondrial localization under chemotherapeutic drug treatment in ovarian cancer cells. We found that the combined use of the proteasome inhibitor epoxomicin and cisplatin led to the accumulation of p53 and sequestosome1(p62) in the mitochondria, downregulated mitochondrial DNA (mtDNA) transcription, inhibited mitochondrial functions, and ultimately promoted apoptosis by enhancing cisplatin sensitivity in ovarian cancer cells. Moreover, the ubiquitin-associated (UBA) domain of p62 was involved in regulating the mitochondrial localization of p53. Our findings suggest that the interaction between p62 and p53 may be a mechanism that determines the fate of tumor cells. In conclusion, p62 coordinated the mitochondrial localization of p53 through its UBA domain, inhibited mtDNA transcription, downregulated mitochondrial function, and promoted ovarian cancer cell death. Our study demonstrates the important role of p53 localization in tumor cell survival and apoptosis, and provides new insights into understanding the anti-tumor mechanism of targeting the ubiquitin-proteasome system in tumor cells.

The Mitochondrial PHB2/OMA1/DELE1 Pathway Cooperates with Endoplasmic Reticulum Stress to Facilitate the Response to Chemotherapeutics in Ovarian Cancer.

Interactions between the mitochondrial inner and outer membranes and between mitochondria and other organelles closely correlates with the sensitivity of ovarian cancer to cisplatin and other chemotherapeutic drugs. However, the underlying mechanism remains unclear. Recently, the mitochondrial protease OMA1, which regulates internal and external signals in mitochondria by cleaving mitochondrial proteins, was shown to be related to tumor progression. Therefore, we evaluated the effect of OMA1 on the response to chemotherapeutics in ovarian cancer cells and the mouse subcutaneous tumor model. We found that OMA1 activation increased ovarian cancer sensitivity to cisplatin in vivo and in vitro. Mechanistically, in ovarian cancer, OMA1 cleaved optic atrophy 1 (OPA1), leading to mitochondrial inner membrane cristae remodeling. Simultaneously, OMA1 induced DELE1 cleavage and its cytoplasmic interaction with EIF2AK1. We also demonstrated that EIF2AK1 cooperated with the ER stress sensor EIF2AK3 to amplify the EIF2S1/ATF4 signal, resulting in the rupture of the mitochondrial outer membrane. Knockdown of OMA1 attenuated these activities and reversed apoptosis. Additionally, we found that OMA1 protease activity was regulated by the prohibitin 2 (PHB2)/stomatin-like protein 2 (STOML2) complex. Collectively, OMA1 coordinates the mitochondrial inner and outer membranes to induce ovarian cancer cell death. Thus, activating OMA1 may be a novel treatment strategy for ovarian cancer.

HDAC9 Contributes to Serous Ovarian Cancer Progression through Regulating Epithelial-Mesenchymal Transition.

Epithelial ovarian cancer has the highest mortality rate of all gynecological malignant tumors. Metastasis is the main cause of poor prognosis in patients with ovarian cancer. Epigenetic and protein post-translational modifications play important roles in tumor metastasis. As a member of class IIa histone deacetylases, histone deacetylase 9 (HDAC9) is involved in many biological processes by deacetylating histone and nonhistone proteins. However, its roles in ovarian cancer remain unclear. In this study, we found that patients with serous ovarian cancer with high expression of HDAC9 had poor prognoses. On the contrary, patients with non-serous ovarian cancer with high expression of HDAC9 had higher survival rates. In serous ovarian cancer, overexpressed HDAC9 may promote cell migration through the forkhead box protein O1 (FOXO1)/transforming growth factor-beta (TGF-ß) axis. In non-serous ovarian cancer, overexpressed HDAC9 exerts antitumor effects that might be caused by the suppression of ß-catenin signaling. Therefore, HDAC9 may be a potential target for individualized treatment of patients with different histological subtypes of ovarian cancer.

Remodeling metabolic fitness: Strategies for improving the efficacy of chimeric antigen receptor T cell therapy.

The dramatic success of adoptive transfer of engineered T cells expressing chimeric antigen receptor (CAR-T) has been achieved with effective responses in some relapsed or refractory hematologic malignancies, which is not yet met in solid tumors. The efficacy of CAR-T therapy is associated with its fate determination and their interaction with cancer cells in tumor microenvironment (TME), which is closely correlated with T cell metabolism fitness. Indeed, modulating T cell metabolism reprogramming has been proven crucial for their survival and reinvigorating antitumor immunity, and thus is considered as a promising strategy to improve the clinical performance of CAR-T cell therapy in difficult-to-treat cancers. This review briefly summarizes the T cell metabolic profiles and key metabolic challenges it faces in TME such as nutrient depletion, hypoxia, and toxic metabolites, then emphatically discusses the potential strategies to modulate metabolic properties of CAR-T cells including improving CARs construct design, optimizing manufacture process via addition of exogenous cytokines or targeting specific signaling pathway, manipulating ROS levels balance or relieving the unfavorable metabolic TME including adaptation to hypoxia and blocking inhibitory effect of toxic metabolites, eventually strengthening the anti-tumor response.

Induction of EBV latent membrane protein-2A (LMP2A)-specific T cells and construction of individualized TCR-engineered T cells for EBV-associated malignancies.

BACKGROUND: Latent membrane protein-2A (LMP2A)-specific TCR-engineered T cells could be a promising treatment approach to Epstein-Barr virus-associated malignancies. However, previous studies mainly reported LMP2A-reactive TCRs only focusing on specific HLA subtypes and corresponding epitopes, and thus, they were only suitable for patients with specific HLA. METHODS: Due to hugely varied HLA subtypes and presented LMP2A epitopes in different individuals, our study attempted to develop an individualized approach, based on the weekly in vitro stimulation of peripheral T cells for 2 weeks with autologous dendritic cells (DCs) pulsed with a pool of LMP2A peptides covering LMP2A whole protein and combination analysis of high throughput TCRß sequencing of prestimulated and poststimulated T cells and single-cell TCR sequencing of poststimulated T cells, and to identify LMP2A-specific TCRs of which poststimulated frequencies significantly increased than corresponding prestimulated frequencies. RESULTS: Following this approach, multiple LMP2A-reactive TCRs were identified, optimized and cloned into lentiviral vector, and then transduced into peripheral T cells. These engineerd T cells were demonstrated to specifically recognize the LMP2A presented by autologous DCs and lymphoblastoid cell lines in vitro and in vivo. CONCLUSIONS: This approach provides an efficient procedure to isolate individualized LMP2A-specific TCRs for basic and translational research, as well as for clinical applications.

Mitochondrial integration and ovarian cancer chemotherapy resistance.

Ovarian cancer has been nicknamed the "silent killer". Most patients with ovarian cancer are diagnosed at an advanced stage of the disease for the first time because of its insignificant early clinical symptoms. In addition to the difficulty of early screening and delay in diagnosis, the high recurrence rate and relapsed refractory status of patients with ovarian cancer are also important factors for their high mortality. Patients with recurrent ovarian cancer often use neoadjuvant chemotherapy followed by surgery as the first choice. However, this is often accompanied by chemotherapy resistance, leading to treatment failure and a mortality rate of more than 90%. In the past, it was believed that the anti-tumor effect of chemotherapeutics represented by cisplatin was entirely attributable to its irreversible damage to DNA, but current research has found that it can inhibit cell growth and cytotoxicity via nuclear and cytoplasmic coordinated integration. As an important hub and integration platform for intracellular signal communication, mitochondria are responsible for multiple key factors during tumor occurrence and development, such as metabolic reprogramming, acquisition of metastatic ability, and chemotherapy drug response. The role of mitochondria in ovarian cancer chemotherapy resistance is becoming increasingly recognized. In this review, we discuss the cellular interactive regulatory network surrounding mitochondria, elucidate the mechanisms of tumor cell survival under chemotherapy, and discuss potential means of interfering with mitochondrial function as a novel anti-cancer therapy.

PGC1α regulates mitochondrial oxidative phosphorylation involved in cisplatin resistance in ovarian cancer cells via nucleo-mitochondrial transcriptional feedback.

Mitochondria play an important role in effective cell energy production and cell survival under stress conditions, such as treatment with chemotherapeutic drugs. Mitochondrial biogenesis is increased in ovarian cancer tissues, which is accompanied by alteration of mitochondrial energy metabolism, structure, and dynamics. These factors are involved in tumorigenesis and apoptosis resistance, highlighting the role of mitochondria in resisting cisplatin toxicity. Cisplatin-resistant ovarian cancer cells are dependent on mitochondrial OXPHOS for energy supply, and intracellular PGC1α-mediated mitochondrial biogenesis levels are increased in this cell line, indicating the important role of mitochondrial oxidative phosphorylation in cisplatin resistance. As PGC1α is a key molecule for integrating and coordinating nuclear DNA and mitochondrial DNA transcriptional machinery, an investigation into the regulatory mechanism PGC1α in mitochondrial energy metabolism via transcription may provide new clues for solving chemotherapy resistance. In the present study, it was demonstrated that inhibiting the expression of PGC1α decreased nuclear and mitochondrial DNA transcription factor expression, leading to increased lactic acid production and decreased cellular oxygen consumption and mitochondrial oxidative phosphorylation. Furthermore, mitochondrial stress-induced ROS production, as a feedback signal from mitochondria to the cell nucleus, increased PGC1α expression in SKOV3/DDP cells, which was involved in mitochondrial oxidative phosphorylation regulation. Collectively, the present study provides evidence that PGC1α-mediated nuclear and mitochondrial transcription feedback regulates energy metabolism and is involved in ovarian cancer cells escaping apoptosis during cisplatin treatment.

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.

HOXB7 mediates cisplatin resistance in esophageal squamous cell carcinoma through involvement of DNA damage repair.

BACKGROUND: DNA damage repair is an important mechanism of platinum resistance. HOXB7 is one member of HOX family genes, which are essential developmental regulators and frequently dysregulated in cancer. Recently, its relevance in chemotherapy resistance and DNA damage repair has also been addressed. However, little is known regarding the association between HOXB7 and chemotherapy resistance in esophageal squamous cell carcinoma (ESCC). METHODS: The association between HOXB7 expression detected by immunohistochemisty and tumor regression grade (TRG) and long-term survival was analyzed in 143 ESCC patients who underwent neoadjuvant chemotherapy. CCK8 assay was used to examine the effect of cisplatin in a panel of four ESCC cell lines. A stable cell strain with HOXB7 knockdown of KYSE150 and KYSE450 was established to explore the effect on cisplatin sensitivity. The interaction of HOXB7 with Ku70, Ku80 and DNA-PKcs was determined by GST-pull down, coimmunoprecipitation and immunofluorescent colocalization. Finally, we investigated whether disrupting HOXB7 function by a synthetic peptide HXR9 blocking the formation of HOXB7/PBX could enhance cisplatin sensitivity in vitro and in vivo. RESULTS: High expression of HOXB7 was associated with cisplatin resistance and worse chemotherapy efficacy. HOXB7 knockdown reinforced cisplatin sensitivity. It was identified that HOXB7 interacts with Ku70, Ku80 and DNA-PKcs. HOXB7 knockdown was related to the downregulation of Ku70, Ku80 and DNA-PKcs as well as arrested cell cycle in S phase. HOXB7 inhibition by HXR9 had a synergistic effect to improve cisplatin sensitivity. CONCLUSION: HOXB7 may be a biomarker for the prediction of chemoresistance of ESCC and serves as a promising therapeutic target.

Inhibition of PI3K/mTOR increased the sensitivity of hepatocellular carcinoma cells to cisplatin via interference with mitochondrial-lysosomal crosstalk.

OBJECTIVES: The genotoxicity of cisplatin towards nuclear DNA is not sufficient to explain the cisplatin resistance of hepatocellular carcinoma (HCC) cells; cisplatin interacts with many organelles, which can influence the sensitivity. Here, we explored the role of mitochondrial-lysosomal crosstalk in the cisplatin resistance of HCC cells. MATERIALS AND METHODS: Huh7 and HepG2 cells were subjected to different treatments. Flow cytometry was conducted to detect mitochondrial reactive oxygen species, mitochondrial mass, lysosomal function, mitochondrial membrane potential and apoptosis. Western blotting was performed to evaluate protein levels. The oxygen consumption rate was measured to evaluate mitochondrial function. RESULTS: Cisplatin activated mitophagy and lysosomal biogenesis, resulting in crosstalk between mitochondria and lysosomes and cisplatin resistance in HCC cells. Furthermore, a combination of cisplatin with the phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) inhibitor PKI-402 induced lysosomal membrane permeabilization. This effect changed the role of the lysosome from a protective one to that of a cell death promoter, completely destroying the mitochondrial-lysosomal crosstalk and significantly enhancing the sensitivity of HCC cells to cisplatin. CONCLUSIONS: This is the first evidence of the importance of mitochondrial-lysosomal crosstalk in the cisplatin resistance of HCC cells and of the destruction of this crosstalk by a PI3K/mTOR inhibitor to increase the sensitivity of HCC cells to cisplatin. This mechanism could be developed as a novel target for treatment of HCC in the future.

Targeting HOX/PBX dimer formation as a potential therapeutic option in esophageal squamous cell carcinoma.

Homeobox genes are known to be classic examples of the intimate relationship between embryogenesis and tumorigenesis, which are a family of transcriptional factors involved in determining cell identity during early development, and also dysregulated in many malignancies. Previously, HOXB7, HOXC6 and HOXC8 were found abnormally upregulated in esophageal squamous cell carcinoma (ESCC) tissues compared with normal mucosa and seen as poor prognostic predictors for ESCC patients, and were shown to promote cell proliferation and anti-apoptosis in ESCC cells. These three HOX members have a high level of functional redundancy, making it difficult to target a single HOX gene. The aim of the present study was to explore whether ESCC cells are sensitive to HXR9 disrupting the interaction between multiple HOX proteins and their cofactor PBX, which is required for HOX functions. ESCC cell lines (KYSE70, KYSE150, KYSE450) were treated with HXR9 or CXR9, and coimmunoprecipitation and immunofluorescent colocalization were carried out to observe HOX/PBX dimer formation. To further investigate whether HXR9 disrupts the HOX pro-oncogenic function, CCK-8 assay and colony formation assay were carried out. Apoptosis was assessed by flow cytometry, and tumor growth in vivo was investigated in a xenograft model. RNA-seq was used to study the transcriptome of HXR9-treated cells. Results showed that HXR9 blocked HOX/PBX interaction, leading to subsequent transcription alteration of their potential target genes, which are involved in JAK-signal transducer and activator of transcription (STAT) activation and apoptosis inducement. Meanwhile, HXR9 showed an antitumor phenotype, such as inhibiting cell proliferation, inducing cell apoptosis and significantly retarding tumor growth. Therefore, it is suggested that targeting HOX/PBX may be a novel effective treatment for ESCC.