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Chemotherapy induces tumor cell death and inhibits tumor progression, but the accompanying immune responses in the surrounding dying tissue cause significant inflammation. These responses, such as excessive neutrophil infiltration into tumor tissue, are the main causes of resistance to anticancer treatment. The development of drugs that reduce neutrophil infiltration into tumors is necessary to increase the anticancer effect of chemotherapy. Here, we show that the antitumor effect of the chemotherapy AC regimen (Adriamycin and cyclophosphamide) was increased by 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) cotreatment in the MDA-MB-231 triple-negative breast cancer xenograft mouse model. Tumor growth was inhibited up to 56% in mice treated with AC and inhibited up to 94% in mice cotreated with AC and PLAG. Side effects of chemotherapy, such as a reduction in body weight, were alleviated in mice cotreated with AC and PLAG. Excessive neutrophil infiltration caused by the AC regimen was successfully cleared in mice cotreated with AC and PLAG. We conclude that PLAG inhibits excessive neutrophil infiltration that aids tumor growth. Reduced neutrophils and increased lymphocytes in PLAG-treated mice can maximize the antitumor effect of the AC regimen and inhibit tumor growth.

Uterine serous carcinoma (USC) is an aggressive variant of endometrial cancer with a poor prognosis. Approximately 30% of USC overexpress HER2/neu, a recognized target for trastuzumab in advanced/recurrent HER2/neu-positive USC. We evaluated the efficacy of the pan-c-erb inhibitor neratinib and the poly (ADP-ribose)-polymerase (PARP) inhibitor olaparib as single agents and in combination against USC cell lines and xenografts.In-vitro cell-viability assays with olaparib, neratinib, and olaparib/neratinib were assessed using flow-cytometry based assays against a panel of USC cell lines with high and low HER2/neu expression. Homologous recombination deficiency (HRD) signatures were evaluated as described by Alexandrov et al. (Nature;2020;578:94-101) while downstream signaling affected by neratinib/olaparib exposure was assessed with immunoblotting. Efficacy of single- versus dual-agent inhibition was evaluated in-vivo using two USC-xenografts with 3+ HER2/neu expression.Neratinib was more potent than olaparib in suppression of in-vitro growth of HER2/neu 3+ cell lines (ARK1: p = 0.0047; ARK2: p = 0.0428) while no difference was noted against HER2/neu 1+ tumors (ARK4). Importantly, the combination of olaparib with neratinib synergistically improved tumor suppression compared to either single-agent in vitro. USC cells exposed to olaparib upregulated HER2/neu expression, while neratinib treatment increased PARP activity (ARK1: p0.0001; ARK2: p0.0001). Single-agent neratinib transiently inhibited in vivo growth of USC xenografts harboring HER2/neu gene amplification (ARK1: p0.05; ARK2: p0.05). In contrast, the combination of the two inhibitors caused a stronger and durable growth inhibition in both USC xenografts (ARK1: p0.05; ARK2: p0.05).The combination of olaparib and neratinib is active and synergistic against primary HER2/neu + USC. This combination may represent a novel therapeutic option for USC patients with HER2/neu+, homologous recombination-proficient tumors resistant to chemotherapy.

Bispecific T cell engagers (BiTEs) are bispecific antibodies that redirect T cells to target antigen-expressing tumors. We hypothesized that BiTE-secreting T cells could be a valuable therapy in solid tumors, with distinct properties in mono- or multi-valent strategies incorporating chimeric antigen receptor (CAR) T cells. Glioblastomas represent a good model for solid tumor heterogeneity, representing a significant therapeutic challenge. We detected expression of tumor-associated epidermal growth factor receptor (EGFR), EGFR variant III, and interleukin-13 receptor alpha 2 (IL13Rα2) on glioma tissues and cancer stem cells. These antigens formed the basis of a multivalent approach, using a conformation-specific tumor-related EGFR targeting antibody (806) and Hu08, an IL13Rα2-targeting antibody, as the single chain variable fragments to generate new BiTE molecules. Compared with CAR T cells, BiTE T cells demonstrated prominent activation, cytokine production, and cytotoxicity in response to target-positive gliomas. Superior response activity was also demonstrated in BiTE-secreting bivalent T cells compared with bivalent CAR T cells in a glioma mouse model at early phase, but not in the long term. In summary, BiTEs secreted by mono- or multi-valent T cells have potent anti-tumor activity in vitro and in vivo with significant sensitivity and specificity, demonstrating a promising strategy in solid tumor therapy.

Glioblastoma Multiforme (GBM) is one of the most aggressive and lethal types of all cancers, with an average 5-year survival rate of 5%. Since GBM tumors are highly vascularized tumors, and their growth is angiogenesis-dependent, antagonizing tumor angiogenesis by using angiogenesis inhibitors were considered as one of the promising approaches. In this context, intensive preclinical evaluation of a novel small molecule named F16 has exhibited potent anti-angiogenic and anti-tumor activities by selectively antagonizing Vascular Endothelial Growth Factor Receptor (VEGFR). Also, recent pharmacokinetic evaluation of F16 with tissue distribution analysis has shown that this molecule is transported across the blood-brain barrier (BBB) and accumulates in the brain regions with no signs of neurotoxicity. Therefore, further studies were conducted to determine the efficacy of F16 in delaying glioblastoma progression via inhibiting tumor angiogenesis. Our in vitro studies have clearly demonstrated the ability of F16 to inhibit migration and invasion of U87MG cells and also confirmed a potent cytotoxic effect against these cells in comparison to Temozolomide (TMZ). Our in vivo studies with the subcutaneously implanted (s.c.) xenograft tumor model and in vitro studies have clearly demonstrated the ability of F16 to delay tumor growth and inhibit migration and invasion.

ADAMTS8 expression has been identified to be low in many cancers including lung cancer. However, the specific functions and regulatory system of ADAMTS8 remain to be unveiled.To study the potential modulatory mechanism of ADAMTS8 in lung cancer in cell and xenograft mice models.Differential expression of ADAMTS8 in lung cancer was analyzed on online tools. So was the overall survival curve in association with ADAMTS8/VEGFA expression in lung cancer patients. RT-qPCR was applied to validate the ADAMTS8 expression in lung cancer cell lines H460 and A549, with the normal lung epithelial cell Beas-2b as a control. Thereafter, overexpressed and knockdown plasmids were constructed for transfection. Colony and flow cytometry methods were used for cell proliferation and apoptosis. RT-qPCR and Western blot methods validated the changes in VEGFA after ADAMTS8 regulation in cells. Tube formation and Transwell methods were applied to observe the changes in tube formation and migration in HUVECs induced by tumor conditioned medium (TCM). Stable-transfected cells were injected subcutaneously into nude mice. HE and Immunohistochemistry were applied to analyze the pathological differences and protein changes of ADAMTS8, VEGFA and CD31.High ADAMTS8 was correlated with high overall survival rate in lung cancer patients. ADAMTS8 was also abnormally downregulated in NSCLC cells. Upregulation of ADAMTS8 suppressed cell proliferation and enhanced apoptosis while downregulation of ADAMTS8 promoted cell proliferation and decreased apoptosis. VEGFA was negatively correlated with ADAMTS8 in lung cancer tissues. Upregulation of ADAMTS8 inhibited VEGFA in mRNA and protein levels. Further, knockdown of ADAMTS8 induced tube formation and migration of HUVECs and upregulation of ADAMTS8 inhibited this. In addition, upregulation of ADAMTS8 in nude mice inhibited tumor growth and also suppressed VEGFA and CD31 in tumors.ADAMTS8 inhibited lung cancer progression through suppressing VEGFA in lung cancer.

Prior to adoptive transfer, CAR T cells are activated, lentivirally infected with CAR transgenes, and expanded over 9 to 11 days. An unintended consequence of this process is the progressive differentiation of CAR T cells over time in culture. Differentiated T cells engraft poorly, which limits their ability to persist and provide sustained tumor control in hematologic as well as solid tumors. Solid tumors include other barriers to CAR T cell therapies, including immune and metabolic checkpoints that suppress effector function and durability. Sialic acids are ubiquitous surface molecules with known immune checkpoint functions. The enzyme C. perfringens neuraminidase (CpNA) removes sialic acid residues from target cells, with good activity at physiologic conditions. In combination with galactose oxidase (GO), NA has been found to stimulate T cell mitogenesis and cytotoxicity in vitro. Here we determine whether CpNA alone and in combination with GO promotes CAR T cell antitumor efficacy. We show that CpNA restrains CAR T cell differentiation during ex vivo culture, giving rise to progeny with enhanced therapeutic potential. CAR T cells expressing CpNA have superior effector function and cytotoxicity in vitro. In a Nalm-6 xenograft model of leukemia, CAR T cells expressing CpNA show enhanced antitumor efficacy. Arming CAR T cells with CpNA also enhanced tumor control in xenograft models of glioblastoma as well as a syngeneic model of melanoma. Given our findings, we hypothesize that charge repulsion via surface glycans is a regulatory parameter influencing differentiation. As T cells engage target cells within tumors and undergo constitutive activation through their CARs, critical thresholds of negative charge may impede cell-cell interactions underlying synapse formation and cytolysis. Removing the dense pool of negative cell-surface charge with CpNA is an effective approach to limit CAR T cell differentiation and enhance overall persistence and efficacy.

A cancer-inhibiting role of mesenchyme homeobox 2 (MEOX2) has been observed in several malignancies. However, the association between MEOX2 and breast carcinoma has not been addressed. This research focused on investigating the possible relevance of MEOX2 in breast carcinoma. Initial expression analysis by TCGA data uncovered low levels of MEOX2 in breast carcinoma. We then confirmed that MEOX2 was poorly expressed in clinical tumor specimens of breast carcinoma by real-time quantitative PCR and immunoblotting assays. Moreover, low levels of MEOX2 in breast carcinoma patients were found to be correlated with reduced overall survival. A series of cellular function assays showed that the forced expression of MEOX2 had anticancer effects, including the inhibition of cell proliferation, the induction of G0-G1 phase arrest, the restraint of metastatic potential, and the enhancement of chemosensitivity. Further analysis revealed that MEOX2 negatively modulated the phosphatidyl-inositol-3 kinase (PI3K)/AKT/mammalian target of the rapamycin (mTOR) and extracellular signal-regulated kinase (ERK1/2) pathways. Reactivation of AKT by a chemical activator reversed MEOX2-mediated anticancer effects. An in vivo xenograft assay validated the anticancer function of MEOX2 in breast carcinoma. Taken together, these data show that MEOX2 exerts a cancer-inhibiting role in breast carcinoma by affecting the PI3K/AKT/mTOR and ERK1/2 pathways. This work suggests MEOX2 as a new contributor for breast carcinoma progression, which may be a candidate target for anticancer therapy development.

Genistein (GEN) has been demonstrated to interfere with antitumor effects of cisplatin (CIS) in vitro. To analyze whether these findings are also relevant in vivo, we examined the effects of combined GEN and CIS treatment in an ovariectomized nude mouse breast cancer xenograft model. Tumor growth and markers for antitumor activity were determined after three weeks of treatment. Furthermore, the concentrations of GEN metabolites were measured in serum, liver, and xenograft tumor tissues using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Three weeks’ oral exposure to GEN at a dose of 5 mg kg-1·d-1 resulted in an average concentration of total GEN metabolite equivalent as high as 0.2729 nmol·g-1 wet weight in xenograft tumor tissues. At this dosage, GEN significantly antagonized the antitumor effects of CIS. Mechanistically, GEN blocked both the inhibition of cell proliferation and induction of apoptosis triggered by CIS. Moreover, GEN concentrations in xenograft tumor tissues were found to be significantly higher than in serum and liver. In conclusion, our findings suggested that oral GEN exposure at a level comparable to dietary exposure in humans could interfere with CIS chemotherapy.

Hepatocellular carcinoma (HCC), a heterogeneous cancer with high mortality, is resistant to single targeted therapy; thus, combination therapy based on synthetic lethality is a promising therapeutic strategy for HCC. Poly (adenosine diphosphate [ADP]-ribose) polymerase 1 (PARP1) is the most recognized target for synthetic lethality; however, the therapeutic effect of PARP1 inhibition on HCC is disappointing. Therefore, exploring new synthetic lethal partners for the efficient manipulation of HCC is urgently required. In this study, we identified Src and PARP1 as novel synthetic lethal partners, and the combination therapy produced significant anti-tumor effects without causing obvious side effects. Mechanistically, Src interacted with PARP1 and phosphorylated PARP1 at the Y992 residue, which further mediated resistance to PARP1 inhibition. Overall, this study revealed that Src-mediated PARP1 phosphorylation induced HCC resistance to PARP1 inhibitors and indicated a therapeutic window of the Y992 phosphorylation of PARP1 for HCC patients. Moreover, synthetic lethal therapy by co-targeting PARP1 and Src have the potential to broaden the strategies for HCC and might benefit HCC patients with high Src activation and resistance to PARP1 inhibitors alone.

ABL1 tyrosine kinase inhibitors (TKIs) dramatically improve the prognosis of chronic myeloid leukemia (CML), but 10-20% of patients achieve suboptimal responses with low TKIs sensitivity. Furthermore, residual leukemic stem cells (LSCs) are involved in the molecular relapse after TKIs discontinuation. Aberrant DNA hypermethylation contributes to low TKIs sensitivity and the persistence of LSCs in CML. DNMT1 is a key regulator of hematopoietic stem cells, suggesting that aberrant DNA hypermethylation targeting DNMT1 represents a potential therapeutic target for CML. We investigated the efficacy of OR-2100 (OR21), the first orally available single-compound prodrug of decitabine. OR21 exhibited anti-tumor effects as a monotherapy, and in combination therapy it increased TKI-induced apoptosis and induction of tumor suppressor genes including PTPN6 encoding SHP-1 in CML cells. OR21 in combination with imatinib significantly suppressed tumor growth in a xenotransplant model. OR21 and combination therapy decreased the abundance of LSCs and inhibited engraftment in a BCR-ABL1-transduced mouse model. These results demonstrate that targeting DNMT1 using OR21 exerts anti-tumor effects and impairs LSCs in CML. Therefore, combination treatment of TKIs and OR21 represents a promising treatment strategy in CML.

Bladder cancers, and specifically urothelial carcinoma, have few effective treatment options, and tumors typically develop resistance against standard of care chemotherapies leading to significant mortality. The development of alternative therapies with increased selectivity and improved tolerability would significantly impact this patient population. Here, we investigate a novel colchicine derivative, CR42-24, with increased selectivity for the βIII tubulin subtype as a treatment for urothelial carcinoma. βIII tubulin is a promising target due to its low expression in healthy tissues and its clinical association with poor prognosis. This study demonstrated that CR42-24 is selectively cytotoxic to several cancer cell lines at low nanomolar IC50, with high activity in bladder cancer cell lines both in vitro and in vivo. CR42-24 monotherapy in an aggressive urothelial carcinoma xenograft model results in effective control when treated early. We observed significant ablation of large tumors and patient-derived xenografts at low doses with excellent tolerability. CR42-24 was highly synergistic in combination with the standard of care chemotherapies gemcitabine and cisplatin, further increasing its therapeutic potential as a novel treatment for urothelial carcinoma.

Ubiquitin-specific protease 18 (USP18) is a deubiquitinating enzyme that reverses the post-translational modification of target proteins by ISG15 or ubiquitin, and is involved in a variety of cellular processes, including signal transduction, viral infection, and cancer development. Although high levels of USP18 mRNA have been observed in several types of cancer, its pathological significance in ovarian cancer (OV) is still elusive. Here, by integrating the Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and Genotypic Tissue Expression (GTEx) databases, we found that USP18 was abnormally up-regulated in OV tissues, and the increased expression of USP18 was associated with poor prognosis. We further showed that activated Jak-STAT3 signaling induced the expression of USP18, which in turn feedback maintained the activity of Jak-STAT3 signaling in OV. In addition, we found that USP18 played a cancer-promoting role in OV mainly through the transcriptional regulation of FBXO6. Silencing USP18 reduced the malignancy of OV, which can be largely reversed by overexpression of FBXO6. On the contrary, silencing FBXO6 significantly weaken the pro-proliferation function of USP18 in OV cells. In summary, our results indicate that USP18 is a downstream target gene of STAT3, and the USP18-FBXO6 axis might be a promising therapeutic target for OV.

Members of the PI3K signaling pathway, especially PIK3CA, the gene encoding the catalytic subunit of the PI3K complex, are highly mutated and amplified in various cancer types, including non-small cell lung cancer. Although PI3K inhibitors have been used in clinics for follicular lymphoma and chronic lymphocytic leukemia, no agents targeting PI3K aberrations in lung cancer have been approved by the FDA so far. In this study, we observed that PIK3CA-E545K, the most common mutation in lung cancer, harbored a modest induction of stem-like properties in lung epithelial cells, and drove development of adenocarcinoma autochthonously when paired with p53 loss in a murine mouse model. We also found that PIK3CA-mutant of amplified lung cancer cells were sensitive to EZH2 inhibition. EZH2 inhibition synergized with PI3K inhibition in human cancer cells in vitro and worked together efficiently in vivo. Mechanistically, EZH2 inhibition cooperated with PI3K inhibition to produce a more potent suppression of phospho-AKT downstream of PI3K. This study suggests a promising combination therapy to combat lung cancers with PIK3CA mutation or amplification. Both copanlisib, the PI3K inhibitor, and tazemetostat, the EZH2 inhibitor, are FDA-approved, which should enhance the clinical translation of this work.

Sorafenib and its derivative regorafenib are the first- and second-line targeted drugs for advanced HCC, respectively. Although both drugs improve overall survival, drug resistance remains the major barrier to their full efficacy. Thus, strategies to enhance sorafenib and regorafenib efficacy against HCC are solely needed. Interleukin-6 receptor alpha (IL-6Rα) is the receptor of IL-6, a multi-functional cytokine, which plays key roles in liver-regeneration, inflammation and development of hepatocellular carcinoma (HCC). Here we show the expression of IL-6Rα was induced in response to sorafenib. Depletion of IL-6Rα abolished IL-6 induced STAT3 phosphorylation at 705th tyrosine and tumor growth of HCC cells under sorafenib treatment. Mechanistically, activating transcription factor 3 (ATF3) was induced in response to sorafenib and subsequently bound to the promoter of IL-6Rα, leading to its transcriptional activation. Depletion of ATF3 or its upstream transcription factor, ATF4, attenuated IL-6Rα induction and IL-6 mediated sorafenib resistance. The ATF4-ATF3-IL-6Rα cascade is also activated by regorafenib. Furthermore, blockade of IL-6Rα with the FDA approved IL-6Rα antibody drug, Sarilumab, drastically attenuated both sorafenib and regorafenib resistance in patient-derived xenograft (PDX) tumors, where human IL-6 could be detected by a novel in situ hybridization technique, named RNAscope. Together, our data reveal that ATF3-mediated IL-6Rα up-regulation promotes both sorafenib and regorafenib resistance in HCC, and targeting IL-6Rα represents a novel therapeutic strategy to enhance sorafenib/regorafenib efficacy for advanced HCC treatment.

Colorectal cancer (CRC) is the third leading cause of cancer-related death worldwide. Dysregulation of circular RNAs (circRNAs) appears to be a critical factor in CRC progression. However, mechanistic studies delineating the role of circRNAs in CRC remain limited. In this study, qRT-PCR and western blot assays were used to measure the expression of genes and proteins. Migration, invasion, proliferation, and apoptosis were examined by wound-healing, transwell, CCK-8, colony formation, and flow cytometry assays, respectively. Molecular interactions were validated by a dual-luciferase report system. A xenograft animal model was established to examine in vivo tumor growth and lung metastasis. Our data indicated that circN4BP2L2 expression was increased in CRC tissues and cell lines. Notably, inhibition of circN4BP2L2 effectively inhibited proliferation, migration, and invasion of LoVo cells, and inhibited tumor growth and metastasis in vivo, whereas the forced expression of circN4BP2L2 facilitated the proliferation, migration, and invasion of HT-29 cells. Mechanistic studies revealed that circN4BP2L2 acted as a molecular sponge of miR-340-5p to competitively promote CXCR4 expression. Furthermore, inhibition of miR-340-5p reversed the anti-cancer effects of circN4BP2L2 or CXCR4 silencing. Our data indicated an oncogenic role of circN4BP2L2 in CRC via regulation of the miR-340-5p/CXCR4 axis, which may be a promising biomarker and target for CRC treatment. This study determined that circN4BP2L2 acts as an oncogene that promotes tumor growth and metastasis of colorectal cancer (CRC) by regulating the miR-340-5p/CXCR4 axis. The authors reveal a novel mechanism for the ceRNA regulatory network in CRC progression and identify a potential therapeutic target for CRC treatment.

Molecular biomarkers play an important guidance role in the diagnosis and treatment of glioma. It has been found that TRAM2 (translocation associated membrane protein 2) drives human cancers development. Here we report that TRAM2 activity is required for malignancy properties of glioma. In this study, we demonstrated that TRAM2 is over-expressed in glioma and cell lines, particularly in the mesenchymal subtype, and glioma patients with high expression of TRAM2 is associated with poorer survival. Silencing of TRAM2 significantly suppresses glioma cell proliferation, invasion, migration and EMT in vitro, and inhibits tumorigenicity of glioma cell in vivo. We further identify that TRAM2 is positively associated with activation of the PI3K/AKT/mTOR signaling in glioma. 740Y-P, a PI3K activator, reversed the effects of TRAM2 silencing on glioma cell proliferation, invasion, migration and EMT process. Taken together, these findings establish that TRAM2/PI3K/AKT/mTOR signaling drives malignancy properties of glioma and indicate that TRAM2 may act as a potential therapeutic target for glioma.

Central nervous system-penetrant therapies with intracranial efficacy against non-small cell lung cancer (NSCLC) brain metastases are urgently needed. We report preclinical studies investigating brain penetration and intracranial activity of the MET inhibitor tepotinib. After intravenous infusion of tepotinib in Wistar rats (n = 3), mean (±standard deviation) total tepotinib concentration was 2.87-fold higher in brain (505 ± 22 ng/g) than plasma (177 ± 20 ng/mL). In equilibrium dialysis experiments performed in triplicate, mean tepotinib unbound fraction was 0.35% at 0.3 and 3.0 µM tepotinib in rat brain tissue, and 4.0% at 0.3 and 1.0 µM tepotinib in rat plasma. The calculated unbound brain-to-plasma ratio was 0.25, indicating brain penetration sufficient for intracranial target inhibition. Of 20 screened subcutaneous patient-derived xenograft (PDX) models from lung cancer brain metastases (n = 1), two NSCLC brain metastases models (LU5349 and LU5406) were sensitive to the suboptimal dose of tepotinib of 30 mg/kg/qd (tumor volume change [%TV]: -12% and -88%, respectively). Molecular profiling (nCounter®; NanoString) revealed high-level MET amplification in both tumors (mean MET gene copy number: 11.2 and 24.2, respectively). Tepotinib sensitivity was confirmed for both subcutaneous models at a clinically relevant dose (125 mg/kg/qd; n = 5). LU5349 and LU5406 were orthotopically implanted into brains of mice and monitored by magnetic resonance imaging (MRI). Tepotinib 125 mg/kg/qd induced pronounced tumor regression, including complete or near-complete regressions, compared with vehicle in both orthotopic models (n = 10; median %TV: LU5349, -84%; LU5406, -63%). Intracranial antitumor activity of tepotinib did not appear to correlate with blood-brain barrier leakiness assessed in T1-weighted gadolinium contrast-enhanced MRI.

Anaplastic thyroid cancer is a rare but devastating malignancy. Anaplastic thyroid cancer cells exhibit the Warburg effect by preferentially undergoing glycolysis even in aerobic conditions, leading to high glucose use. Here we assess if targeted inhibition of glycolysis can diminish anaplastic thyroid cancer growth and improve outcomes.Human anaplastic thyroid cancer cell line 8505C was grown in medium containing high (25 mmol/L) or low (3 mmol/L) glucose concentration and hexokinase II inhibitor 3-bromopyruvate (200 μM). Cellular proliferation, migration, and invasion were measured. An orthotopic xenograft model of anaplastic thyroid cancer was generated in nude mice using 8505C cells. Animals were provided standard chow or a ketogenic diet and treated with 3-bromopyruvate (1.8 mg/kg). Overall survival time was monitored. Necropsies were performed to harvest tumors for analysis.Growth of 8505C in low-glucose medium with 3-bromopyruvate decreased cell proliferation by 89%, migration by 44%, and invasion by 73% (P.001 for all) compared with high glucose. Animals concomitantly receiving a ketogenic diet and 3-bromopyruvate exhibited smaller tumor volumes (P = .03), slower tumor growth rates (P = .01), and improved overall survival (P = .006) compared with standard-diet control subjects. Monotherapy with a ketogenic diet or 3-bromopyruvate alone did not reduce tumor size or increase survival over the standard-diet control group.Glycolytic inhibition with 3-bromopyruvate inhibits tumor growth and extends survival in a murine model of anaplastic thyroid cancer when combined with the ketogenic diet. Thus, targeted glycolytic inhibition of anaplastic thyroid cancer exhibits context-specific utility and may only be effective during ketosis induced by dietary restriction of glycolytic inputs.

Sunitinib is one of the first-line multi-tyrosine kinase inhibitors for metastatic renal cell carcinoma, and resistance to sunitinib continues to be a limiting factor for the successful treatment. As interleukin-6 (IL-6) is overexpressed in sunitinib-resistant cells, the purpose of this study was to explore the potential of IL-6 inhibition with tocilizumab, an IL-6 receptor inhibitor, to overcome resistance. In vitro, two sunitinib-resistant renal cell carcinoma cell lines (Caki-1 and SN12K1) were treated with tocilizumab. A mouse subcutaneous xenograft model was also used. Cell viability was studied by MTT assay, and apoptosis by morphology and ApopTag. Expression of IL-6, vascular endothelial growth factor (VEGF), and Bcl-2 was analyzed by qPCR. In vitro, tocilizumab induced significant cell death, and reduced the expression of IL-6, VEGF, and Bcl-2 in sunitinib-resistant cells. However, the in vitro findings could not be successfully translated in vivo, as tocilizumab did not decrease the growth of the tumors.

The immunosuppressive tumor microenvironment (TME) is a formidable barrier to the success of adoptive cell therapies for solid tumors. Oncolytic immunotherapy with engineered adenoviruses (OAd) may disrupt the TME by infecting tumor cells, as well as surrounding stroma, to improve the functionality of tumor-directed chimeric antigen receptor (CAR)-T cells, yet efficient delivery of OAds to solid tumors has been challenging. Here we describe how mesenchymal stromal cells (MSCs) can be used to systemically deliver a binary vector containing an OAd together with a helper-dependent Ad (HDAd; combinatorial Ad vector [CAd]) that expresses interleukin-12 (IL-12) and checkpoint PD-L1 (programmed death-ligand 1) blocker. CAd-infected MSCs deliver and produce functional virus to infect and lyse lung tumor cells while stimulating CAR-T cell anti-tumor activity by release of IL-12 and PD-L1 blocker. The combination of this approach with administration of HER.2-specific CAR-T cells eliminates 3D tumor spheroids in vitro and suppresses tumor growth in two orthotopic lung cancer models in vivo. Treatment with CAd MSCs increases the overall numbers of human T cells in vivo compared to CAR-T cell only treatment and enhances their polyfunctional cytokine secretion. These studies combine the predictable targeting of CAR-T cells with the advantages of cancer cell lysis and TME disruption by systemic MSC delivery of oncolytic virotherapy: incorporation of immunostimulation by cytokine and checkpoint inhibitor production through the HDAd further enhances anti-tumor activity.

Mechanistic target of rapamycin (mTOR) forms two distinct complexes, mTOR complex 1 (mTORC1) and mTORC2. Here we investigated the antitumor effect of dual mTORC1/2 inhibitor AZD2014 on epithelial ovarian cancer (EOC) and its potential effect on immunosuppressive myeloid-derived suppressor cells (MDSCs). Immunohistochemical analysis of mTORC1 and mTORC2 was performed on a human ovarian cancer tissue microarray. High mTORC2 expression level was associated with shorter survival in EOC, whereas mTORC1 was not correlate with patients’ prognosis. AZD2014 suppressed mTOR signaling pathway in ovarian cancer cells, inhibited proliferation and induced G1-phase cell cycle arrest and apoptosis. In tumor-bearing mice, AZD2014 treatment limited tumor growth, reduced peritoneal ascites, and prolonged survival. AZD2014 specifically reduced MDSCs migration and accumulation in EOC peritoneal fluid but not in the spleen. Moreover, subsequent AZD2014 treatment after cisplatin chemotherapy delayed EOC recurrence. Collectively, we observed that high mTORC2 expression level in EOC indicated a poor prognosis. Remarkably, in tumor-bearing mice, AZD2014 diminished MDSC accumulation and delayed tumor growth and recurrence.

Graphical abstract, Introduction Previously, we have reported a withanolide-type steroid, named tubocapsenolide A (TA), which shows potent anti-proliferative activity in several cancer cell lines. However, its inhibitory effect on the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway and therapeutic potential on osteosarcoma have not been reported. Objectives In the present study, we aimed to investigate the effect and molecular mechanism of TA in osteosarcoma. Methods The biological functions of TA in U2OS cells were investigated using colony formation, 5-ethynyl-20-deoxyuridine (EDU) staining, and cell cycle/apoptosis assays. The interaction between TA and Src homology 2 phosphatase 2 (SHP-2) was detected by enzyme activity and validated by target-identification methods such as drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), and biolayer interferometry (BLI). The in vivo anti-tumor efficacy of TA was analyzed in the xenograft tumor model. Western blotting analysis was performed to detect the protein expression levels. Results TA exhibited antitumor activity against osteosarcoma both in vitro and in vivo by regulating the JAK/STAT3 signaling pathway. Mechanically, TA interacted with SHP-2 directly and activated its phosphatase activity. Importantly, protein tyrosine phosphatase (PTP) inhibitor, SHP-2 inhibitor, and SHP-2 siRNA could reverse the inhibitory effect of TA on the JAK/STAT3 signaling pathway and restored the TA-induced cell death. Conclusion TA activated the phosphatase activity of SHP-2, which resulted in the inhibition of the JAK/STAT3 pathway and contributed to the antitumor efficacy of TA. Collectively, these findings suggested that TA could serve as a novel therapeutic agent for the treatment of osteosarcoma.

The purpose of this study was to determine whether statins can enhance anticancer effects in head and neck squamous cell carcinoma (HNSCC) when used with cisplatin and act as immunogenic cell death (ICD) inducers that can be used in cancer immunotherapy. Statins alone showed both in vitro and in vivo inhibitory effects against HNSCC, and synergistic antitumor effects were observed when combined with cisplatin in a syngeneic murine HNSCC model. Statins increased calreticulin exposure and endoplasmic reticulum stress-related signals in HNSCC cells. In addition, it was confirmed that statins could activate antigen-presenting cells and tumor-specific CD8+ T cells with an increase in their numbers in the tumor tissues and draining lymph nodes, with this effect showing significant improvement following the combination therapy with cisplatin. Moreover, in triple combination with both cisplatin and anti-programmed cell death 1 receptor (anti-PD-1) antibody, statins dramatically induced further tumor eradication and improved the survival of tumor-bearing mice. Taken together, these results demonstrate that statins, administered in combination with anti-PD-1 antibody, could enhance the anticancer effect of cisplatin and potentiate the efficacy of immunotherapy for HNSCC and present a rationale for repurposing statins as an adjuvant immunotherapeutic option for HNSCC.

A major challenge to the treatment of advanced prostate cancer (PCa) is the development of resistance to androgen-deprivation therapy (ADT) and chemotherapy. It is imperative to discover effective therapies to overcome drug resistance and improve clinical outcomes. We have developed a novel class of silicon-containing compounds and evaluated the anticancer activities and mechanism of action using cellular and animal models of drug-resistant PCa. Five organosilicon compounds were evaluated for their anticancer activities in the NCI-60 panel and established drug-resistant PCa cell lines. GH1504 exhibited potent in vitro cytotoxicity in a broad spectrum of human cancer cells, including PCa cells refractory to ADT and chemotherapy. Molecular studies identified several potential targets of GH1504, most notably androgen receptor (AR), AR variant 7 (AR-v7) and survivin. Mechanistically, GH1504 may promote the protein turnover of AR, AR-v7 and survivin, thereby inducing apoptosis in ADT-resistant and chemoresistant PCa cells. Animal studies demonstrated that GH1504 effectively inhibited the in vivo growth of ADT-resistant CWR22Rv1 and chemoresistant C4-2B-TaxR xenografts in subcutaneous and intraosseous models. These preclinical results indicated that GH1504 is a promising lead that can be further developed as a novel therapy for drug-resistant PCa.

Introduction We hypothesised that the combined use of radiation therapy and a phosphoinositide 3-kinaseγδ inhibitor to reduce immune suppression would enhance the efficacy of an immune checkpoint inhibitor. Methods Murine breast cancer cells (4T1) were grown in both immune-competent and -deficient BALB/c mice, and tumours were irradiated by 3 fractions of 24 Gy. A PD-1 blockade and a phosphoinositide 3-kinase (PI3K)γδ inhibitor were then administered every other day for 2 weeks. The same experiments were performed in humanised patient-derived breast cancer xenograft model and its tumour was sequenced to identify immune-related pathways and profile infiltrated immune cells. Transcriptomic and clinical data were acquired from The Cancer Genome Atlas pan-cancer cohort, and the deconvolution algorithm was used to profile immune cell repertoire. Results Using a PI3Kγδ inhibitor, radiation therapy (RT) and PD-1 blockade significantly delayed primary tumour growth, boosted the abscopal effect and improved animal survival. RT significantly increased CD8+cytotoxic T-cell fractions, immune-suppressive regulatory T cells (Tregs), myeloid-derived suppressor cells and M2 tumour-associated macrophages (TAMs). However, the PI3Kγδ inhibitor significantly lowered the proportions of Tregs, myeloid-derived suppressor cells and M2 TAMs, achieving dramatic gains in splenic, nodal, and tumour CD8+ T-cell populations after triple combination therapy. In a humanised patient-derived breast cancer xenograft model, triple combination therapy significantly delayed tumour growth and decreased immune-suppressive pathways. In The Cancer Genome Atlas cohort, high Treg/CD8+ T cell and M2/M1 TAM ratios were associated with poor overall patient survival. Conclusion These findings indicate PI3Kγ and PI3Kδ are clinically relevant targets in an immunosuppressive TME, and combining PI3Kγδ inhibitor, RT and PD-1 blockade may overcome the therapeutic resistance of immunologically cold tumours. Synopsis Combining PI3Kγδ inhibitor, RT, and PD-1 blockade may be a viable clinical approach, helping to overcome the therapeutic resistance of immunologically cold tumours such as breast cancer.

Hepatocellular carcinoma (HCC) lacks effective treatment, and the patients rapidly develop the acquired resistance to sorafenib with less defined mechanisms. Here, we demonstrate that transcriptional factor myocyte enhancer factor 2D (MEF2D) overexpression is detected in sorafenib-resistant HCC specimens and HCC cell lines and predicts poor prognosis of sorafenib-treated HCC patients. Mechanistically, MEF2D in complex with histone deacetylase HDAC4 directly binds to the SPRY4 promoter regions and suppresses the transcriptional expression of SPRY4, which is a negative regulator of MAPK/ERK signaling pathway. Inhibition of HDAC4 with its clinically used inhibitor induces SPRY4 expression and inhibition of ERK activity, resulting in sensitization of HCC cells to sorafenib-induced apoptosis and greatly improved inhibition of liver tumor growth in mice with sorafenib treatment. These findings highlight the critical role of coupling HDAC4 with MEF2D in activation of ERK by suppressing SPRY4 and underscore the great potential to improve HCC treatment by combined administration of sorafenib with HDAC4 inhibitors.

BACKGROUND & AIMS: SIRT5 plays pleiotropic roles via post-translational modifications, serving as a tumor suppressor, or an oncogene, in different tumors. However, the role SIRT5 plays in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC) remains unknown. METHODS: Published datasets and tissue arrays with SIRT5 staining were used to investigate the clinical relevance of SIRT5 in PDAC. Furthermore, to define the role of SIRT5 in the carcinogenesis of PDAC, we generated autochthonous mouse models with conditional Sirt5 knockout. Moreover, to examine the mechanistic role of SIRT5 in PDAC carcinogenesis, SIRT5 was knocked down in PDAC cell lines and organoids, followed by metabolomics and proteomics studies. A novel SIRT5 activator was utilized for therapeutic studies in organoids and patient-derived xenografts. RESULTS: SIRT5 expression negatively regulated tumor cell proliferation and correlated with a favorable prognosis in PDAC patients. Genetic ablation of Sirt5 in PDAC mouse models promoted acinar-to-ductal metaplasia, precursor lesions, and pancreatic tumorigenesis, resulting in poor survival. Mechanistically, SIRT5 loss enhanced glutamine and glutathione metabolism via acetylation-mediated activation of GOT1. A selective SIRT5 activator, MC3138, phenocopied the effects of SIRT5 overexpression and exhibited anti-tumor effects on human PDAC cells. MC3138 also diminished nucleotide pools, sensitizing human PDAC cell lines, organoids, and patient-derived xenografts to gemcitabine. CONCLUSIONS: Collectively, we identify SIRT5 as a key tumor suppressor in PDAC, whose loss promotes tumorigenesis through increased non-canonical utilization of glutamine via GOT1, and that SIRT5 activation is a novel therapeutic strategy to target PDAC.

Adoptive immunotherapy has emerged as a powerful approach to cure cancer and chronic infections. Currently, the generation of a massive number of T cells that provide long-lasting immunity is challenged by exhaustion and differentiation-associated senescence, which inevitably arise during in vitro cloning and expansion. To circumvent these problems, several studies have proposed an induced pluripotent stem cell (iPSC)-mediated rejuvenation strategy to revitalize the exhausted/senescent T cell clones. Because iPSC-derived cytotoxic T lymphocytes (iPSC-CTLs) generated via commonly used monolayer systems have unfavorable, innate-like features such as aberrant natural killer (NK) activity and limited replication potential, we modified the redifferentiation culture to generate CD8αβ(+)CD5(+)CCR7(+)CD45RA(+)CD56(−)-adaptive iPSC-CTLs. The modified iPSC-CTLs exhibited early memory phenotype, including high replicative capacity and the ability to give rise to potent effector cells. In expansion culture with an optimized cytokine cocktail, iPSC-CTLs proliferated more than 10(15)-fold in a feeder-free condition. Our redifferentiation and expansion package of early memory iPSC-CTLs could supply memory and effector T cells for both autologous and allogeneic immunotherapies.

Anti-tumor immunity through checkpoint inhibitors, specifically anti-programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) interaction, is a promising approach for cancer therapy. However, as early clinical trials indicate that colorectal cancers (CRCs) do not respond well to immune-checkpoint therapies, new effective immunotherapy approaches to CRC warrant further study. Simvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (CoA) reductase (HMGCR), the rate-limiting enzyme of the mevalonate (MVA) pathway for the cholesterol biosynthesis. However, little is known about the functions of simvastatin in the regulation of immune checkpoints or long noncoding RNA (lncRNA)-mediated immunoregulation in cancer. Here, we found that simvastatin inhibited PD-L1 expression and promoted anti-tumor immunity via suppressing the expression of lncRNA SNHG29. Interestingly, SNHG29 interacted with YAP and inhibited phosphorylation and ubiquitination-mediated protein degradation of YAP, thereby facilitating downregulation of PD-L1 transcriptionally. Patient-derived tumor xenograft (PDX) models and the clinicopathological analysis in samples from CRC patients further supported the role of the lncRNA SNHG29-mediated PD-L1 signaling axis in tumor microenvironment reprogramming. Collectively, our study uncovers simvastatin as a potential therapeutic drug for immunotherapy in CRC, which suppresses lncRNA SNHG29-mediated YAP activation and promotes anti-tumor immunity by inhibiting PD-L1 expression.

Selenium nanoparticles (SeNPs), a potential cancer therapeutic agent, have attracted widespread attention owing to their high bioavailability and remarkable anticancer activity. Nevertheless, the poor water solubility and dispersibility of SeNPs seriously limit their applications. In the present study, we synthesized stable and individual spherical selenium nanoparticles (CL90-Tw-SeNP2) with an average diameter of approximately 79 nm using a polysaccharide extracted from Citrus limon (CL90) and Tween-80 as the decorator and stabilizers. The proportion of selenium in CL90-Tw-SeNP2 was 10.6%. CL90-Tw-SeNP2 possessed high stability and good dispersion in water for more than three months. The subsequent biological assay revealed that CL90-Tw-SeNP2 showed remarkable antitumor effects against HepG2 cells, with an IC50 value of 49.13 μg/mL, by inducing cell apoptosis. Furthermore, an in vivo zebrafish assay to explore possible applications indicated that CL90-Tw-SeNP2 could inhibit the proliferation and migration of tumors and the zebrafish angiogenesis. These results indicated that CL90-Tw-SeNP2 could be a potential agent for cancer treatment, especially against human liver hepatoma cancer.

In today's world, cancer is still the leading cause of human death. Among them, the incidence and mortality of lung cancer remain high, and have become the focus of research in the world. BEAP, a protein with anti-lung cancer activity, was isolated and purified from the edible mushroom Boletus edulis. Previous studies have shown that BEAP can inhibit the proliferation of non-small cell lung cancer A549 cells by inducing apoptosis and cell cycle arrest in vitro and in vivo. However, there are many ways in which antitumor proteins from edible and medicinal mushroom play their roles. It is worth exploring whether there are other antitumor mechanisms of BEAP, which can provide reference value for the development of new drugs targeting non-small cell lung cancer and the repurposing of existing drugs.

In Asia, prostate cancer is becoming a growing concern, impacting both socially and economically, compared with what is seen in western countries. Hence, it is essential to know the mechanisms associated with the development and tumorigenesis of PCa for primary diagnosis, risk management, and development of therapy strategies against PCa. Kinesin family member 15 (KIF15), a kinesin family member, is a plus-end-directed kinesin that functions to form bipolar spindles. There is emerging evidence indicating that KIF15 plays a significant role in several malignancies, such as pancreatic cancer, hepatocellular carcinoma, lung adenocarcinoma, and breast cancer. Still, the function of KIF15 remains unclear in prostate cancer. Here, we study the functional importance of KIF15 in the tumorigenesis of PCa. The bioinformatic analysis from PCa patients revealed high KIF15 expression compared to normal prostate tissues. High expression hinting at a possible functional role of KIF15 in regulating cell proliferation of PCa, which was demonstrated by both in vitro and in vivo assays. Downregulation of KIF15 silenced the expression of CDK2, p-RB, and Cyclin D1 and likewise blocked the cells at the G1 stage of the cell cycle. In addition, KIF15 downregulation inhibited MEK-ERK signaling by significantly silencing p-ERK and p-MEK levels. In conclusion, this study confirmed the functional significance of KIF15 in the growth and development of prostate cancer and could be a novel therapeutic target for the treatment of PCa.

Temozolomide is a first-line therapeutic drug for glioblastoma (GBM), and it has a low solubility, short biological half-life, and resistance to drug limits in clinical applications. Therefore, it is necessary to find more effective anti-tumor drugs to overcome drug resistance and enhance its anti-glioma activity. We therefore used n-butanol, n-hexanol, n-octanol, 1-dodecanol and 1-hexadecanol to synthesize a series of temozolomide ester compounds (TMZEs) and then investigated their physicochemical properties and anti-glioma efficacy. Our results showed that TMZEs had a higher lipophilicity compared to TMZ and could stably exist in plasma and brain homogenates. TMZEs had significantly increased cytotoxicity and cellular uptake in C6 glioma cells as chain lengths increased. Additionally, the IC50 of TMZ-16E towards TMZ-resistant cells (T98G) was 85.9-fold lower than that of TMZ (p

The Hippo and mTOR signaling cascades are major regulators of cell growth and division. Aberrant regulation of these pathways has been demonstrated to contribute to gliomagenesis and result in enhanced glioblastoma proliferation and invasive characteristics. Several crosstalk mechanisms have been described between these two pathways, although a complete picture of these signaling interactions is lacking and is required for effective therapeutic targeting. Here we report the ability of mTORC2 to directly phosphorylate YAP at serine 436 (Ser436) positively regulating YAP activity. We show that mTORC2 activity enhances YAP transcriptional activity and the induction of YAP-dependent target gene expression while its ablation via genetic or pharmacological means has the opposite affects on YAP function. mTORC2 interacts with YAP via Sin1 and mutational analysis of serine 436 demonstrates that this phosphorylation event affects several properties of YAP leading to enhanced transactivation potential. Moreover, YAP serine 436 mutants display altered glioblastoma growth, migratory capacity and invasiveness both in vitro and in xenograft experiments. We further demonstrate that mTORC2 is able to regulate a Hippo pathway resistant allele of YAP suggesting that mTORC2 can regulate YAP independent of Hippo signaling. Correlative associations between the expression of these components in GBM patient samples also supported the presence of this signaling relationship. These results advance a direct mTORC2/YAP signaling axis driving GBM growth, motility and invasiveness.

Ovarian cancer (OC) is one of the most lethal type of cancer in women due to a lack of effective targeted therapies and high rates of treatment resistance and disease recurrence. Recently Poly (ADP-ribose) polymerase inhibitors (PARPi) have shown promise as chemotherapeutic agents; however, their efficacy is limited to a small fraction of patients with BRCA mutations. Here we show a novel function for the Hedgehog (Hh) transcription factor Glioma associated protein 1 (GLI1) in regulation of key Fanconi anemia (FA) gene, FANCD2 in OC cells. GLI1 inhibition in HR-proficient OC cells induces HR deficiency (BRCAness), replication stress and synergistic lethality when combined with PARP inhibition. Treatment of OC cells with combination of GLI1 and PARP inhibitors shows enhanced DNA damage, synergy in cytotoxicity, and strong in vivo anticancer responses.

Background Circular RNA (circRNA) has been confirmed to play a vital role in melanoma progression. Objective The regulatory function of circ_0062270, a novel circRNA, in melanoma progression is unclear. Methods Relative expression levels of circ_0062270 and microRNA (miR)-331-3p were determined using qRT-PCR. Cell counting kit 8 assay, EdU staining and flow cytometry were used to measure cell proliferation, cell cycle distribution and apoptosis. The protein levels of proliferation, apoptosis and metastasis-related markers, as well as EPH receptor A2 (EPHA2), were tested using western blot analysis. Besides, cell migration and invasion were evaluated using transwell assay. Meanwhile, the interaction between miR-331-3p and circ_0062270 or EPHA2 was confirmed by dual-luciferase reporter assay or RIP assay. Additionally, tumor xenograft models were constructed to investigate the function of circ_0062270 on melanoma tumor growth in vivo. Results The expression of circ_0062270 was increased in melanoma tissues and cells. Knockdown of circ_0062270 inhibited proliferation, promoted apoptosis, and repressed metastasis in melanoma. Moreover, circ_0062270 could serve as miR-331-3p sponge, and miR-331-3p could target EPHA2. Furthermore, miR-331-3p inhibitor and EPHA2 overexpression reversed the inhibitory effect of circ_0062270 silencing on melanoma progression. In addition, silenced circ_0062270 also could inhibit melanoma tumor growth in vivo. Conclusion Circ_0062270 accelerated the progression of melanoma through regulating the miR-331-3p/EPHA2 axis, suggesting that circ_0062270 might be a novel potential therapeutic target for melanoma.

In recent years, increasing evidence indicates the significant roles of circRNAs in carcinogenesis. However, their roles in lung cancer remain largely unclear. We profiled the circRNA expression in 10 paired non-small cell lung cancer (NSCLC) and adjacent non-cancer tissues using high-throughput sequencing. A total of 183 up-regulated and 428 down-regulated circRNAs were identified in the NSCLC tissues (fold change ≥ 2, P < 0.05). Circ_0089823, an up-regulated circRNA (5.4-fold, P = 0.0017), was further investigated through loss-of-function and gain-of-function. The circ_0089823 level in NSCLC samples was related to the gender, tumor size, pathological type, TNM stage and smoking history. Knockdown of circ_0089823 suppressed cell proliferation, induced cell cycle arrest and apoptosis of NSCLC cells in vitro. Additionally, circ_0089823-silenced xenografts grew much slowly. On the contrary, its over-expression promoted the malignant behaviors of NSCLC cells. Furthermore, SOX4, a tumor-promoting transcription factor, was highly expressed in NSCLC tissues and positively regulated by circ_0089823. Bioinformatic analysis revealed several potential binding sites for miR-507, miR-557, miR-579-3p and miR-1287-5p in circ_0089823 and SOX4 3′-untranslated region, which was later confirmed by luciferase reporter assay. Interestingly, silencing SOX4 countervailed the effects of circ_0089823 over-expression on NSCLC cells. Here, we revealed that circ_0089823 might act as a sponge of microRNAs targeting SOX4, thus increasing the expression of SOX4, thereby reinforcing the malignant behaviors of NSCLC cells. This study indicates that circ_0089823 has the potential to become a candidate target for NSCLC treatment.

T cell malignancies are a group of hematologic cancers with high recurrence and mortality rates. CD5 is highly expressed in ∼85% of T cell malignancies, although normal expression of CD5 is restricted to thymocytes, T cells, and B1 cells. However, CD5 expression on chimeric antigen receptor (CAR)-T cells leads to CAR-T cell fratricide. Once this limitation is overcome, CD5-targeting CAR-T therapy could be an attractive strategy to treat T cell malignancies. Here, we report the selection of novel CD5-targeting fully human heavy-chain variable (FHVH) domains for the development of a biepitopic CAR, termed FHVH3/VH1, containing FHVH1 and FHVH3, which were validated to bind different epitopes of the CD5 antigen. To prevent fratricide in CD5 CAR-T cells, we optimized the manufacturing procedures of a CRISPR-Cas9-based CD5 knockout (CD5KO) and lentiviral transduction of anti-CD5 CAR. In vitro and in vivo functional comparisons demonstrated that biepitopic CD5KO FHVH3/VH1 CAR-T cells exhibited enhanced and longer lasting efficacy; produced moderate levels of cytokine secretion; showed similar specificity profiles as either FHVH1, FHVH3, or the clinically tested H65; and is therefore suitable for further development.

Olaparib, a potent PARP inhibitor, has been shown to have great anti-tumor effects in some tumor types. Although biliary tract cancer (BTC) is a good candidate for DNA damage response (DDR)-targeted agents, targeted DDR inhibitors, including olaparib, are currently rarely evaluated in BTC. In our project, a total of ten BTC cell lines were used to assess the efficacy of olaparib. Olaparib alone showed moderate anti-proliferative effects in BTC cells and increased p-ATR and PD-L1 expression levels. In combination with an ATR inhibitor (AZD6738, ceralasertib) showed synergistic anti-proliferative effects and increased DNA strand breaks in vitro. PD-L1 induced by olaparib was also downregulated by ceralasertib through p-STAT-3 and YAP reduction with or without human primary peripheral blood mononuclear cells. In SNU478-xenograft models, the combination treatment significantly suppressed tumor growth. PD-L1 and YAP were strongly downregulated, similar to in vitro conditions, and expression of CXCR2 and CXCR4 was further reduced. In the current ongoing clinical trial (NCT04298021), BTC patients treated with olaparib and ceralasertib combination have shown tumor response. In conclusion, co-targeting of PARP and ATR might be a potential therapeutic approach for patients with BTC.

Highlights • Deletion of fibronectin in cancer cells prolongs survival • Pharmacologic inhibition of fibronectin deposition slows down cancer progression in two models • Decreasing fibronectin in the matrix diminishes proliferation in tumors and can serve as an adjuvant therapy, Understanding how the extracellular matrix affects cancer development constitutes an emerging research field. Fibronectin and collagen are two intriguing matrix components found in cancer. Large concentrations of fibronectin or collagen type I have been implicated in poor prognosis in patients. In a mouse model, we had shown that genetically decreasing circulating fibronectin resulted in smaller tumors. We therefore aimed to manipulate fibronectin pharmacologically and determine how cancer development is affected. Deletion of fibronectin in human breast cancer cells (MDA-MB-231) using shRNA (knockdown: Kd) improved survival and diminished tumor burden in a model of metastatic lesions and in a model of local growth. Based on these findings, it seemed reasonable to attempt to prevent fibronectin accumulation using a bacterial derived peptide called pUR4. Treatment with this peptide for 10 days in the breast cancer local growth model or for 5 days in a melanoma skin cancer model (B16) was associated with a significant suppression of cancer growth. Treatment aimed at inhibiting collagen type I accumulation without interfering with fibronectin could not affect any changes in vivo. In the absence of fibronectin, diminished cancer progression was due to inhibition of proliferation, even though changes in blood vessels were also detected. Decreased proliferation could be attributed to decreased ERK phosphorylation and diminished YAP expression. In summary, manipulating fibronectin diminishes cancer progression, mostly by suppressing cell proliferation. This suggests that matrix modulation could be used as an adjuvant to conventional therapy as long as a decrease in fibronectin is obtained., Graphical abstract Image, graphical abstract

Adoptively transferred CD19 chimeric antigen receptor (CAR) T cells have led to impressive clinical outcomes in B cell malignancies. Beyond induction of remission, the persistence of CAR-T cells is required to prevent relapse and provide long-term disease control. To improve CAR-T cell function and persistence, we developed a composite co-stimulatory domain of a B cell signaling moiety, CD79A/CD40, to induce a nuclear translocating signal, NF-κB, to synergize with other T cell signals and improve CAR-T cell function. CD79A/CD40 incorporating CD19CAR-T cells (CD19.79a.40z) exhibited higher NF-κB and p38 activity upon CD19 antigen exposure compared with the CD28 or 4-1BB incorporating CD19CAR-T cells (CD19.28z and CD19.BBz). Notably, we found that CD19.79a.40z CAR-T cells continued to suppress CD19(+) target cells throughout the co-culture assay, whereas a tendency for tumor growth was observed with CD19.28z CAR-T cells. Moreover, CD19.79a.40z CAR-T cells exhibited robust T cell proliferation after culturing with CD19(+) target cells, regardless of exogenous interleukin-2. In terms of in vivo efficiency, CD19.79a.40z demonstrated superior anti-tumor activity and in vivo CAR-T cell proliferation compared with CD19.28z and CD19.BBz CD19CAR-T cells in Raji-inoculated mice. Our data demonstrate that the CD79A/CD40 co-stimulatory domain endows CAR-T cells with enhanced proliferative capacity and improved anti-tumor efficacy in a murine model.

PURPOSE: Recent preclinical studies suggest combining the HSP90 inhibitor AT13387 (Onalespib) with radiation (IR) against colon cancer and head and neck squamous cell carcinoma (HNSCC). These studies emphasized that AT13387 downregulates HSP90 client proteins involved in oncogenic signaling and DNA repair mechanisms as major drivers of enhanced radiosensitivity. Given the large array of client proteins HSP90 directs, we hypothesized that other key proteins or signaling pathways may be inhibited by AT13387 and contribute to enhanced radiosensitivity. Metabolomic analysis of HSP90 inhibition by AT13387 was conducted to identify metabolic biomarkers of radiosensitization and whether modulations of key proteins were involved in IR-induced tumor vasculogenesis, a process involved in tumor recurrence. METHODS AND MATERIALS: HNSCC and non-small cell lung cancer cell lines were used to evaluate the AT13387 radiosensitization effect in vitro and in vivo. Flow cytometry, immunofluorescence, and immunoblot analysis were used to evaluate cell cycle changes and HSP90 client protein’s role in DNA damage repair. Metabolic analysis was performed using liquid chromatography−Mass spectrometry. Immunohistochemical examination of resected tumors post-AT13387 and IR treatment were conducted to identify biomarkers of IR-induced tumor vasculogenesis. RESULTS: In agreement with recent studies, AT13387 treatment combined with IR resulted in a G2/M cell cycle arrest and inhibited DNA repair. Metabolomic profiling indicated a decrease in key metabolites in glycolysis and tricarboxylic acid cycle by AT13387, a reduction in Adenosine 5’-triphosphate levels, and rate-limiting metabolites in nucleotide metabolism, namely phosphoribosyl diphosphate and aspartate. HNSCC xenografts treated with the combination exhibited increased tumor regrowth delay, decreased tumor infiltration of CD45 and CD11b+ bone marrow−derived cells, and inhibition of HIF-1 and SDF-1 expression, thereby inhibiting IR-induced vasculogenesis. CONCLUSIONS: AT13387 treatment resulted in pharmacologic inhibition of cancer cell metabolism that was linked to DNA damage repair. AT13387 combined with IR inhibited IR-induced vasculogenesis, a process involved in tumor recurrence post-radiotherapy. Combining AT13387 with IR warrants consideration of clinical trial assessment. © 2021 Elsevier Inc. All rights reserved.

ATP hydrolysis and downstream signaling pathways in the extracellular space have a major impact upon tumor progression and metastasis. The complexity and interdependence of various cell types in the extracellular space have been increasingly appreciated in recent years. With increased awareness of the importance of this signaling pathway in the pathogenic development and progression of malignancies, there has been attention to therapeutic strategies targeting extracellular adenosine metabolism and signaling. This review summarizes the molecular and physiologic roles of extracellular ATP and adenosine in normal and disease states, and potential therapeutic applications.

Fucoidan is a sulfated polysaccharide, derived from various marine brown seaweeds, that has immunomodulatory effects. In this study, we analyzed the effects of five different fucoidans, which were extracted from Ascophyllum nodosum, Undaria pinnatifida, Macrocystis pyrifera, Fucus vesiculosus, and Ecklonia cava, on natural killer (NK) cell activation in mice. Among these, E. cava fucoidan (ECF) promoted an increase in the number of NK cells in the spleen and had the strongest effect on the activation of NK cells. Additionally, we observed that DC stimulation was required for NK cell activation and that ECF had the most potent effect on splenic dendritic cells (DC). Finally, ECF treatment effectively prevented infiltration of CT-26 carcinoma cells in the lungs of BALB/c mice in an NK cell dependent manner. Collectively, these results suggest that ECF could be a suitable candidate for enhancing NK cell-mediated anti-cancer immunity.

Biguanides are a class of anti-diabetic drugs that includes phenformin and metformin, however, the former was withdrawn from approval in many countries due to its toxicity. Findings from retrospective epidemiological studies in diabetic populations and pre-clinical laboratory models have demonstrated that biguanides possess anti-tumor activities that suggest their repurposing for cancer prevention and treatment. However, a better understanding of how these biguanides behave as anti-tumor agents is needed to guide their improved applications in cancer therapy, spurring increased interest in their pharmacology. Here, we present evidence for proposed mechanisms of action related to their anti-tumor activity, including their effects on central carbon metabolism in cancer cells and immune-modulating activity, and then review progress on biguanide repurposing in cancer therapeutics and the possible re-evaluation of phenformin as a cancer therapeutic agent.

Background It is difficult to distinguish between a tumor and its liver segment with traditional use of indocyanine green (ICG) alone. In the present study, a method was used to limit ICG to the liver segment adjacent to a tumor. A spectrally-distinct fluorescently-labeled tumor-specific antibody against human carcinoembryonic antigen-related cell-adhesion molecules was used to label the metastatic tumor in a patient-derived orthotopic xenograft mouse model to enable color-coded visualization and distinction of a colon-cancer liver metastases and its adjacent liver segment. Materials and Methods Nude mice received surgical orthotopic implantation in the liver of colon-cancer liver metastases derived from two patients. An anti- carcinoembryonic antigen-related cell-adhesion molecules monoclonal antibody (mAb 6G5j) was conjugated to a near-infrared dye IR700DX (6G5j-IR700DX). After three weeks, mice received 6G5j-IR700DX via tail-vein injection 48 hours before surgery. ICG was intravenously injected after ligation of the left or left lateral Glissonean pedicle resulting in labeling of the segment with preserved blood-flow in the liver. Imaging was performed with the Pearl Trilogy and FLARE Imaging Systems. Results The metastatic liver tumor had a clear fluorescence signal due to selective tumor targeting by 6G5j-IR700DX, which was imaged on the 700 nm channel. The adjacent liver segment, with preserved blood-flow in the liver, had a clear fluorescence ICG 800 nm signal, while the left or left lateral segment had no fluorescence signal. Overlay of the images showed clear color-coded differentiation between the tumor fluorescing at 700 nm and the adjacent liver segment fluorescing at 800 nm. Conclusions Color-coding of a liver tumor and uninvolved liver segment has the potential for improved liver resection.

Gemcitabine and nab-paclitaxel (Abraxane®) is a standard of care chemotherapy combination used in the treatment of patients with advanced pancreatic cancer. While the combination has shown a survival benefit when compared to gemcitabine monotherapy, it is associated with significant off-target toxicity. Ultrasound targeted microbubble destruction (UTMD) has emerged as an effective strategy for the site-specific deposition of drug-payloads. However, loading a single microbubble formulation with two drug payloads can be challenging and often involves several manipulations post-microbubble preparation that can be cumbersome and generally results in low / inconsistent drug loadings. In this manuscript, we report the one-pot synthesis of a gemcitabine functionalised phospholipid and use it to successfully generate stable microbubble formulations loaded with gemcitabine (Lipid-Gem MB) or a combination of gemcitabine and paclitaxel (Lipid-Gem-PTX MB). Efficacy of the Lipid-Gem MB and Lipid-Gem-PTX MB formulations, following ultrasound (US) stimulation, was evaluated in a three-dimensional (3D) PANC-1 spheroid model of pancreatic cancer and a mouse model bearing ectopic BxPC-3 tumours. The results demonstrated a significant reduction in the cell viability in spheroids for both formulations reducing from 90 ± 10% to 62 ± 5% for Lipid-Gem MB and 84 ± 10% to 30 ± 6% Lipid-Gem-PTX MB following US irradiation. When compared with a clinically relevant dose of free gemcitabine and paclitaxel (i.e. non-particle bound) in a BxPC-3 murine pancreatic tumour model, both formulations also improved tumour growth delay with tumours 40 ± 20% and 40 ± 30% smaller than the respective free drug formulation when treated with Lipid-Gem MB and Lipid-Gem-PTX MB respectively, at the conclusion of the experiment. These results highlight the potential of UTMD mediated Gem / PTX as a treatment for pancreatic cancer and the facile preparation of Lipid-Gem-PTX MBs using a gemcitabine functionalised lipid should expedite clinical translation of this technology.

A novel nitrogen mustard CBISC has been synthesized and evaluated as an anticancer agent. CBISC has been shown to exhibit enhanced cell proliferation inhibition properties against mutant p53 cell lines colorectal cancer WiDr, pancreatic cancer (MIAPaCa-2 and PANC-1), and triple negative breast cancer (MDA-MB-231 and MDA-MB-468). In vitro mechanism of action studies revealed perturbations in the p53 pathway and increased cell death as evidenced by western blotting, immunofluorescent microscopy and MTT assay. Further, in vivo studies revealed that CBISC is well tolerated in healthy mice and exhibited significant in vivo tumor growth inhibition properties in WiDr and MIAPaCa-2 xenograft models. These studies illustrate the potential utility of CBISC as an anticancer agent.

Background Lung cancer is the greatest cause of cancer mortality in the United States, necessitating ongoing improvements in current treatment techniques. Photodynamic therapy (PDT) involves the interaction between a photosensitizer, light, and oxygen. The resulting release of reactive oxygen species causes tumor necrosis. It has been used as an endoscopic technique for the palliation of lung cancer. Porfimer sodium (Photofrin) is the only Food and Drug Administration–approved photosensitizer for PDT but has limited depth of penetration and produces prolonged skin phototoxicity. Multiple newer photosensitizers are in development, including PS785. The effectiveness of PS785 was compared with porfimer sodium in the treatment of human lung cancer xenografts in mice. Methods Human non–small cell lung cancer (NSCLC) xenografts were established in severe combined immunodeficient mice and grouped into small (3-5 mm) and large tumors (6-10 mm). PS785 or porfimer sodium was administered intravenously, and PDT was executed at 24, 48, or 72 h after injection. The primary endpoint was the delay of tumor regrowth after PDT. Results Porfimer sodium and PS785 produced statistically similar delays of tumor regrowth after PDT when small tumors were treated at 24 and 48 h. At 72 h, PS785 performed better in small tumors. However, for large tumors, PS785 produced no delay in tumor regrowth at any time point. Conclusions PS785 and porfimer sodium were able to effectively treat NSCLC to a depth of ≤5 mm. However, porfimer sodium was more effective in treating NSCLC tumors to a depth of 6-10 mm. Further efforts are required to produce photosensitizers that will facilitate PDT of larger tumors.

Purpose The poor response of breast cancer to immune checkpoint blockade might result from low immunogenicity and the immune-suppressive tumor microenvironment. We hypothesized that in situ tumor vaccination via radiation therapy (RT) and suppression of immune tolerance via phosphoinositide 3-kinase δ (PI3Kδ) inhibition would enhance the efficacy of immune checkpoint blockade. Methods and Materials 4T1 murine breast cancer cells were grown in both immune-competent and -deficient BALB/c mice, and tumors were irradiated with 24 Gy in 3 fractions. A PD-1 blockade and a PI3Kαδ inhibitor were then administered every other day for 2 weeks. Fluorescence-activated cell sorting and immunohistochemistry served to monitor subsequent changes in immune cell repertoire. Results The triple combination of RT, PD-1 blockade, and PI3Kαδ inhibitor significantly delayed tumor growth. The immune-deficient syngeneic 4T1 murine tumor model failed to show this tumor growth delay. Use of RT and PI3Kαδ inhibitor increased the proportions of CD8+ T cells; PI3Kαδ inhibitor led to a decrease in regulatory T cells and polymorphonuclear myeloid-derived suppressor cells. The triple combination resulted in a remarkable increase in cytotoxic CD8+ T cells, suggesting a prominent immune-modulatory effect. The abscopal effect was most prominent in the triple-combination therapy group, and it correlated with splenic CD8+ T cell accumulation. Conclusions These findings collectively indicate that combining RT, PI3Kαδ inhibitor, and PD-1 blockade could be a viable approach, helping to overcome the therapeutic resistance of immunologically cold tumors, such as breast cancer, with an immunosuppressive tumor microenvironment.