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Radiotherapy (RT) is one of the primary treatments of head and neck squamous cell carcinoma (HNSCC), which has a high-risk of locoregional failure (LRF). Presently, there is no reliable predictive biomarker of radioresistance in HNSCC. Here, we found that mutations in NFE2L2, which encodes Nrf2, are associated with a significantly higher rate of LRF in patients with oral cavity cancer treated with surgery and adjuvant (chemo)radiotherapy but not in those treated with surgery alone. Somatic mutation of NFE2L2 led to Nrf2 activation and radioresistance in HNSCC cells. Tumors harboring mutant Nrf2E79Q were substantially more radioresistant than tumors with wild-type Nrf2 in immunocompetent mice, whereas the difference was diminished in immunocompromised mice. Nrf2E79Q enhanced radioresistance through increased recruitment of intratumoral polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) and reduction of M1-polarized macrophages. Treatment with the glutaminase inhibitor CB-839 overcame the radioresistance induced by Nrf2E79Q or Nrf2E79K. RT increased expression of PMN-MDSC–attracting chemokines, including CXCL1, CXLC3, and CSF3, in Nrf2E79Q-expressing tumors via the TLR4, which could be reversed by CB-839. This study provides insights into the impact of NFE2L2 mutations on radioresistance and suggests that CB-839 can increase radiosensitivity by switching intratumoral myeloid cells to an antitumor phenotype, supporting clinical testing of CB-839 with RT in HNSCC with NFE2L2 mutations.Significance:NFE2L2 mutations are predictive biomarkers of radioresistance in head and neck cancer and confer sensitivity to glutaminase inhibitors to overcome radioresistance.

We have shown that decursin, a coumarin compound, induces cell cycle arrest and apoptosis in human prostate cancer cells (PCa); however, its molecular mechanisms are largely unexplored. We studied the mechanisms associated with its anticancer activity in advanced human prostate carcinoma cells. We found that decursin inhibited epidermal growth factor receptor (EGFR) signaling by inhibiting its activating phosphorylation at tyrosine 1068 residue in DU145 and 22Rv1 cells. This inhibition of EGFR was associated with the downregulation of ERK1/2 phosphorylation. Both EGFR and ERK1/2 are known to be deregulated/activated in many human malignancies. Consistent with our earlier study, decursin (25-100 µM) treatment for 24-72 h inhibited DU145 cell proliferation by 49%-87% (p 0.001) which was associated with strong G1 phase arrest and cell death. It also decreased (p 0.001) the number of surviving colonies. Decursin moderately increased the expression of Rb-related proteins p107 and p130 but decreased the levels of E2F family transcription factors including E2F-3, E2F-4 and E2F-5. Further, decursin strongly inhibited the growth of androgen-dependent prostate carcinoma 22Rv1 cells from 61% to 79% (p 0.001) and arrested these cells at G1 phase via induction of cyclin-dependent kinase inhibitor p27/Kip1 and downregulation of CDK2 and CDK4 protein expression. Additionally, EGFR inhibitor erlotinib- and EGF ligand-modulated EGFR activation validated EGFR signaling as a target of decursin-mediated cell growth inhibition and cytotoxicity. Decursin decreased EGF ligand-induced phosphorylation of EGFR (Y-1068) as well as activation of its downstream mediator, ERK1/2. Furthermore, inhibitory targeting of EGFR-ERK1/2 axis by combinatorial treatment of decursin and erlotinib further sensitized DU145 cells for the decursin-induced growth inhibition and cell death. Overall, these findings strongly suggest that anticancer efficacy of decursin against human PCa involves inhibitory targeting of EGFR-ERK1/2 signaling axis, a pathway constitutively active in advanced PCa.

Proportion of various immune cell type in TME of pancreatic cancer (TGCA).

Supplementary Figure S1: Proportion of total NK cell in the tumor microenvironment of 138 pancreatic cancer patients based on the CIBERSORTx analysis of TCGA. Supplementary Figure S2: Immunohistochemical staining of NK cells (Red circle) using NKp46. Supplementary Figure S3: Pan02 tumor in C57BL/6 mice as a syngeneic mouse model. Supplementary Figure S4: Representative flow cytometry gating strategy for NK cells (a) and percent of NK cells relative to immune cells in the C3aR antagonist-treated and vehicle-treated tumors (b). Supplementary Figure S5: Flow cytometry gating strategy for B cells (B220+), CD4+ T cells, CD8+ T cells, macrophages (Gr1-CD11b+F4/80+) and myeloid derived suppressor cells (MDSC) (Gr1+CD11b+) in the spleen, vehicle-treated (control) tumor and C3aR antagonist-treated tumor. Supplementary Figure S6: C3aR antagonism does not impact immune cell infiltration in the spleen of vehicle-treated and C3aR antagonist-treated mice. Supplementary Figure S7: Depletion of NK cells and CD8+ T cells and confirmatory analysis of depletion and survival of Pan02 tumor-bearing mice.

Pancreatic cancer is one of the deadliest cancers, against which current immunotherapy strategies are not effective. Herein, we analyzed the immune cell composition of the tumor microenvironment of pancreatic cancer samples in The Cancer Genome Atlas and found that the presence of intratumoral natural killer (NK) cells correlates with survival. Subsequent analysis also indicated that NK cell exclusion from the microenvironment is found in a high percentage of clinical pancreatic cancers and in preclinical models of pancreatic cancer. Mechanistically, NK cell exclusion is regulated in part by complement C3a and its receptor signaling. Inhibition of the C3a receptor enhances NK cell infiltration in syngeneic mouse models of pancreatic cancer resulting in tumor growth delay. However, tumor growth inhibition mediated by NK cells is not sufficient alone for complete tumor regression, but is potentiated when combined with radiotherapy. Our findings indicate that although C3a inhibition is a promising approach to enhance NK cell–based immunotherapy against pancreatic cancer, its combination with radiotherapy holds greater therapeutic benefit.Significance:Immunotherapeutic agents are not effective against pancreatic cancer. We show that the inhibition of complement C3a enhances NK cell infiltration in preclinical models of pancreatic cancer, resulting in tumor growth delay. This effect is further potentiated by radiotherapy, thereby leading to significant antitumor activity compared with either therapy alone.

Supplementary figure 1 shows the effect of silibinin and IR alone and in combination treatment for 48 h for the total cell number and number of colony formation in human prostate cancer 22RV1 cells. The combination treatment did not show any significant increase in the effect of IR for both the parameters. Supplementary figure 2 shows the representative histogram data for the analysis of cell cycle phase distribution. A prominent increase in G2/M phase peak can be noted in IR+silibinin combination treatment. Supplementary figure 3 shows that IR-induced expression of G2/M phase regulators are down-modulated in DU145 cells but not in HEK293 cells in the IR+silibinin combination treatment. This combination also decreased the expression of PCNA and survivin in DU145 and PC-3 cells. Supplementary figure 4 shows the measurement of ROS level which was higher in the IR+silibinin combination treatment and accompanied with down-modulation of antioxidant enzyme system in DU145 cells. Whereas as opposed to DU145 cells, ROS level was decreased in HEK-293 cells in the combination treatment which could provide protection to the cells. Supplementary figure 5 shows that IR induces nuclear translocation of EGFR in non-neoplastic JB6 cells which is not affected by the IR+silibinin combination treatment to the extent as observed in DU145 cells (figure 4). Supplementary figure 6 shows the parameters monitored for measuring the normal tissue toxicity in the tumor xenograft study in mice. The IR-induced adverse changes in the prostate and seminal vesicle, and decrease in neutrophil and monocytes counts were reversed in the combination treatment.

The table depicts the sequences of the gene primers used for the mRNA expression analysis.

The relationship between NFE2L2 mutation and radiation resistance in OC cancer. Kaplan-Meier analysis of PFS and OSin OC patients treated with surgery and postoperative (chemo)radiotherapy.

CB-839 overcomes the Nrf2E79Q-induced radiation resistance through regulating TLR4-mediated cytokine changes.

Mutational landscape and details of NFE2L2 gene mutation in a cohort of oral cavity cancer patients treated with surgery and postoperative (chemo)radiotherapy at Stanford University.

Nrf2 mutation leads to extrinsic radiation resistance through recruitment of PMN-MDSC in tumors.

Supplemental Table 1 shows patients distribution in OC cohorts from Stanford University. Supplemental Table 2 shows primers for mutagenesis. Supplemental Table 3 shows primers for real-time PCR. Supplemental Table 4 shows gene mutations identified by targeted next generation sequencing in a cohort of oral cavity cancer patients treated with surgery and postoperative (chemo)radiotherapy at Stanford University. Supplemental Table 5 shows tumor growth delay of indicated MOC1 xenografts using 1000mm3 tumor size as a proxy.

Radiotherapy (RT) is one of the primary treatments of head and neck squamous cell carcinoma (HNSCC), which has a high-risk of locoregional failure (LRF). Presently, there is no reliable predictive biomarker of radioresistance in HNSCC. Here, we found that mutations in NFE2L2, which encodes Nrf2, are associated with a significantly higher rate of LRF in patients with oral cavity cancer treated with surgery and adjuvant (chemo)radiotherapy but not in those treated with surgery alone. Somatic mutation of NFE2L2 led to Nrf2 activation and radioresistance in HNSCC cells. Tumors harboring mutant Nrf2E79Q were substantially more radioresistant than tumors with wild-type Nrf2 in immunocompetent mice, whereas the difference was diminished in immunocompromised mice. Nrf2E79Q enhanced radioresistance through increased recruitment of intratumoral polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC) and reduction of M1-polarized macrophages. Treatment with the glutaminase inhibitor CB-839 overcame the radioresistance induced by Nrf2E79Q or Nrf2E79K. RT increased expression of PMN-MDSC–attracting chemokines, including CXCL1, CXLC3, and CSF3, in Nrf2E79Q-expressing tumors via the TLR4, which could be reversed by CB-839. This study provides insights into the impact of NFE2L2 mutations on radioresistance and suggests that CB-839 can increase radiosensitivity by switching intratumoral myeloid cells to an antitumor phenotype, supporting clinical testing of CB-839 with RT in HNSCC with NFE2L2 mutations.Significance:NFE2L2 mutations are predictive biomarkers of radioresistance in head and neck cancer and confer sensitivity to glutaminase inhibitors to overcome radioresistance.

CB-839 overcomes RT resistance in Nrf2E79Q MOC1 cells through regulating the tumor microenvironment.

Somatic mutation of NFE2L2 leads to Nrf2 activation and promotes intrinsic radiation resistance in human head and neck squamous cell carcinoma (HNSCC).

NFE2L2 mutation leads to extrinsic radiation resistance.

An immunosuppressive microenvironment causes poor tumour T-cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumours is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identify complement receptor C5aR1 as a druggable target which when inhibited improves radiotherapy even in tumours displaying immunosuppressive features and poor CD8+ T-cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we find that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumour-cell specific functions. C5aR1 targeting results in increased NF-κB-dependent apoptosis specifically in tumours and not normal tissues; indicating that in malignant cells, C5aR1 primarily regulates cell fate. Collectively, these data reveal that increased complement gene expression is part of the stress response mounted by irradiated tumours and that targeting C5aR1 can improve radiotherapy even in tumours displaying immunosuppressive features.

Pancreatic cancer is one of the deadliest cancers, against which current immunotherapy strategies are not effective. Herein, we analyzed the immune cell composition of the tumor microenvironment of pancreatic cancer samples in The Cancer Genome Atlas and found that the presence of intratumoral natural killer (NK) cells correlates with survival. Subsequent analysis also indicated that NK cell exclusion from the microenvironment is found in a high percentage of clinical pancreatic cancers and in preclinical models of pancreatic cancer. Mechanistically, NK cell exclusion is regulated in part by complement C3a and its receptor signaling. Inhibition of the C3a receptor enhances NK cell infiltration in syngeneic mouse models of pancreatic cancer resulting in tumor growth delay. However, tumor growth inhibition mediated by NK cells is not sufficient alone for complete tumor regression, but is potentiated when combined with radiotherapy. Our findings indicate that although C3a inhibition is a promising approach to enhance NK cell–based immunotherapy against pancreatic cancer, its combination with radiotherapy holds greater therapeutic benefit. Significance: Immunotherapeutic agents are not effective against pancreatic cancer. We show that the inhibition of complement C3a enhances NK cell infiltration in preclinical models of pancreatic cancer, resulting in tumor growth delay. This effect is further potentiated by radiotherapy, thereby leading to significant antitumor activity compared with either therapy alone.

Tumor cells alter the immune microenvironment to facilitate metastatic progression. Galectin-1 (Gal1) is a known modulator of tumor growth and progression in multiple cancer types including head and neck cancer (HNC). However, its contribution to the regulation of metastasis is poorly understood. Through this study we report a previously unknown role for Gal1 in modulating the STING pathway to regulate metastasis. The cGAS-STING pathway has paradoxical roles in cancer depending on the kinetics and strength of its induction, with chronic STING activation being linked to immune suppression and tumor progression while acute activation being associated with immune activation. However, little is known about the pathways that promote chronic STING activation. Using HNC and lung cancer models, we show that Gal-1 enhances STING protein stability, leading to sustained NF-κΒ activation and heightened chemokine-driven recruitment of myeloid derived suppressor cells (MDSCs). We show that Gal1-STING connection fosters the establishment of pre-metastatic niche through polymorphonuclear MDSCs (PMN-MDSCs), which modify the local lung microenvironment to support metastatic spread. Notably, RNA sequencing of MDSCs isolated from pre-metastatic lungs indicate the role of PMN-MDSCs in remodeling collagen and the extracellular matrix in the pre-metastatic compartment. Our findings reveal an unexpected role of STING activation in metastatic progression of HNC and lung cancer models and establish Gal1 as an endogenous positive regulator of STING. Citation Format: Dhanya K. Nambiar, Vignesh Vignesh Viswanathan, Hongbin Cao, Weiruo Zhang, Li Guan, Manish Chamoli, Brittany Holmes, Christina Kong, Rachel Hildebrand, Amanda Koong, Rie Eyben, Amato Giaccia, Lingyin Li, Edgar Engleman, Quynh Thu Le. Galectin-1 mediated chronic tumoral-STING activation promotes metastasisthrough MDSC recruitment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 66.

Neoangiogenesis constitutes one of the first steps of tumor progression beyond a critical size of tumor growth, which supplies a dormant mass of cancerous cells with the required nutrient supply and gaseous exchange through blood vessels essentially needed for their sustained and aggressive growth. In order to understand any biological process, it becomes imperative that we use models, which could mimic the actual biological system as closely as possible. Hence, finding the most appropriate model is always a vital part of any experimental design. Angiogenesis research has also been much affected due to lack of simple, reliable, and relevant models which could be easily quantitated. The angiogenesis models have been used extensively for studying the action of various molecules for agonist or antagonistic behaviour and associated mechanisms. Here, we have described two protocols or models which have been popularly utilized for studying angiogenic parameters. Rat aortic ring assay tends to bridge the gap between in vitro and in vivo models. The chorioallantoic membrane (CAM) assay is one of the most utilized in vivo model system for angiogenesis-related studies. The CAM is highly vascularized tissue of the avian embryo and serves as a good model to study the effects of various test compounds on neoangiogenesis.

Hypoxia (low oxygen conditions) in prostate tumors is associated with aggressive phenotype and poor prognosis. Also, hypoxia is considered the first major challenge faced by growing mass of neoplastic cells, as cells in hypoxic areas are surrounded by metabolic waste, acidic pH and necrotic cells. To overcome these hostile conditions, tumor cells activate transcriptional machinery leading to neo-angiogenesis, altered metabolism and selection of resistant clones. This suggests that prostate cancer (PCa) growth and progression could be prevented via targeting hypoxia-induced signaling. Here, we analyzed silibinin effect on hypoxia-induced proliferation, angiogenesis and metabolic changes in human PCa cells both in vitro and in vivo. Silibinin inhibited LNCaP cells clone formation (exposed to 1% O2 for 48 hrs and then returned to normoxic culture conditions) by 90-100% at 25-50 µM doses. Under similar hypoxic conditions in 22Rv1 cells, silibinin inhibited the clone formation by 63-77% at 25-50 µM doses. Importantly, conditioned media collected from PCa cells under hypoxic conditions (1% O2 for 24 hrs) induced tube formation by human umbilical vein endothelial cells (HUVEC) on matrigel, whereas conditioned media from silibinin-treated hypoxic PCa cells or silibinin addition in hypoxic-conditioned media abrogated HUVEC tube formation. Interestingly, we observed higher lipid accumulation in LNCaP cells under hypoxia, which was decreased (∼70%) by silibinin. Molecular analyses showed that silibinin decreases hypoxia-induced HIF-1α expression without affecting HIF-1β in PCa cells. Furthermore, silibinin increased the level of FIH (factor inhibiting HIF) and prolyl-hydroxylase 2, involved in HIF-1α degradation. Also, silibinin decreased the level of phosphorylated mTOR and ERK1/2 in PCa cells under hypoxia, and strongly inhibited lipid synthesis via activating AMP-activated protein kinase and decreasing acetyl Co A carboxylase, fatty acid synthase, acetyl Co A synthetase, fatty-acyl Co A synthetase and lipin expression. In vivo, silibinin effect on hypoxia-induced proliferation, angiogenesis and metabolism was analyzed in 22Rv1 xenograft model. Mice with established 22Rv1 tumors were imaged at base-line as well as 6 and 15 days following silibinin administration (200 mg/kg body weight) by diffusion weighted (DWI), dynamic contrast enhanced (DCE)-MRI and 18FDG-PET. At the study end, endogenous tumor metabolites were assed by NMR and various biomarkers by IHC. In vivo results showed that silibinin feeding reduced the tumor volume (by 47%), inhibited HIF-1α signaling, decreased proliferation and angiogenesis (DCE-MRI), induced apoptotic death and modulated tumor metabolism (PET and NMR). Together, these findings further support silibinin usefulness in PCa prevention through inhibiting hypoxia-induced proliferation, angiogenesis and lipogenesis. Citation Format: Gagan Deep, Anand M. Ramteke, Dhanya K. Nambiar, Anil K. Jain, Natalie J. Serkova, Chapla Agarwal, Rajesh Agarwal. Silibinin inhibits hypoxia-induced proliferation, angiogenesis and lipogenesis in prostate cancer cells both in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4114. doi:10.1158/1538-7445.AM2014-4114

Aberrant lipid metabolism is a major factor in growth, progression and invasion/metastasis of prostate tumors. Prostate cancer (PCa) cells are unique in terms of their low glycolytic rate and weak expression of glucose transporters. Concomitantly, PCa cells overexpress fatty acid synthase (FASN) which is considered a metabolic oncogene. As glucose consumption is not elevated in PCa, a metabolic switch to lipogenesis and fatty acid oxidation provides energy and cellular building blocks for abnormal cell growth and proliferation. In the present study, we evaluated the effect and related molecular mechanisms of a natural flavonoid silibinin in inhibiting lipid metabolism in PCa cells. Results showed that silibinin (30-90 µM) inhibited lipid and cholesterol accumulation specifically in human prostate carcinoma LNCaP and DU145 cells but not in non-neoplastic prostate epithelial PWR-1E cells. This inhibition by silibinin was mediated via a decrease in the expression of key enzymes regulating fatty acid and cholesterol synthesis including FASN, acetyl CoA carboxylase, acetyl CoA synthetase and HMG CoA reductase. Since most of these lipogenic enzymes are transcriptionally controlled by SREBP, we next analyzed the effect of silibinin on this master transcriptional regulator of lipogenesis. Silibinin inhibited SREBP1 (as well as SREBP2) protein expression without affecting its mRNA level in PCa cells suggesting a post-translational mode of regulation. SREBP1 activation involves proteolytic cleavage of precursor protein (125 kDa), generating a 68 kDa fragment that translocates to the nucleus and binds to sterol response element on target genes. Confocal and Immunoblot analyses confirmed that silibinin inhibited the proteolytic cleavage and subsequent nuclear translocation of SREBP1. Importantly, silibinin activated AMPK that phosphorylates SREBP1 (at Ser-372 site) inhibiting its nuclear translocation and transcriptional activity. Moreover, AMPK inhibition, through Compound C, reversed silibinin-mediated inhibition of nuclear SREBP1 and lipid accumulation. The central role of SREBP1 in silibinin-mediated effects on lipid accumulation and cell growth inhibition was also established by using a specific synthetic SREBP inhibitor, fatostatin. Parallely, we also observed that silibinin attenuates fatty acid uptake through down regulating LDL receptor expression. Furthermore, silibinin also significantly reduced AMACR (α-methylacyl-CoA racemase) expression in PCa cells, suggesting that it could also negatively regulate fatty acid oxidation. Overall, we found that silibinin targets SREBP1 and metabolically starves PCa cells by reducing de novo lipogenesis and lipolysis leading to a strong growth inhibition. Considering the critical role of lipid metabolism in prostate carcinogenesis, non-toxic silibinin could be useful in PCa prevention and treatment. Citation Format: Dhanya K. Nambiar, Gagan Deep, Rana P. Singh, Chapla Agarwal, Rajesh Agarwal. Silibinin inhibits lipid metabolism by primarily targeting the master regulator sterol response element binding protein 1 (SREBP1) in prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2452. doi:10.1158/1538-7445.AM2014-2452