The substance P (SP)/NK-1 receptor (NK1R) complex represents an intriguing anticancer target for a variety of tumors, including hepatoblastoma (HB). Therefore, NK1R antagonists, such as the clinical drug aprepitant, recently have been proposed as potent anticancer agents. However, very little is known regarding the molecular basis of NK1R inhibition in cancer. Using reverse phase protein array, Western blot, Super TOP/FOP, confocal microscopy, and sphere formation ability (SFA) assays, we identified the AKT and Wnt signaling pathways as the key targets of aprepitant in three human HB cell lines (HepT1, HepG2, and HuH6). Following NK1R blockage, we observed decreased phosphorylation of p70S6K and 4E-BP1/2 and inhibition of the canonical Wnt pathway with subsequent decrease of HB cell growth. This effect was dependent of high baseline Wnt activity either by mutational status of b-catenin or extrinsic Wnt activation. Wnt inhibition seemed to be strengthened by disruption of the FOXM1-b-catenin complex. Furthermore, treatment of HB cells with aprepitant led to reduced expression of (liver) stemness markers (AFP, CD13, SOX2, NANOG, and OCT4) and SFA when grown under cancer stem cell conditions. Taken together, we show for the first time that targeting the SP/NK1R signaling cascade inhibits canonical Wnt signaling in HB cells. These findings reveal important insight into the molecular mechanisms of the SP/NK1R complex as a critical component in a model of pediatric liver cancer and may support the development of novel therapeutic interventions for HB and other Wnt-activated cancers.

Therapeutic, V.

the delivery of M-Dox into cell nuclei, as compared to electroporation or M-Dox alone. Conclusion: Electroporation enhanced intracellular delivery of M-Dox in hepatocellular xenograft model. Such enhanced delivery could be utilized to improve the anti-tumor effect of irreversible electroporation.

Extracellular vesicles (EVs) are known effectors of cell signaling and exchange implicated in cancer development and progression, as well as remodeling and immunomodulation of the tumor microenvironment. EVs are detected in various biofluids and are considered to convey signature features of the cells from which they originate. Clinical assessment of plasma-derived EV molecular contents therefore offers promise of “liquid biopsies” for detection or subtyping of cancers as well as dynamic reporting of tumor status during and after treatment. To date, there have been no comprehensive analyses aimed at interrogating the diverse repertoire of EV protein cargoes in small cell lung carcinoma (SCLC). Using plasmas from a cohort of 18 SCLC cases, 16 lung adenocarcinoma (LUAD) cases and 34 controls matched based on smoking status, age, and sex, EVs were enriched via affinity capture using magnetic beads functionalized with phosphatidyl serine binding protein TIM4 in addition to single-step density flotation ultracentrifugation to deplete abundant protein background. In-depth proteomic profiling was performed on enriched EVs via nano liquid chromatography / high-resolution ion-mobility mass spectrometry. Profiles were intersected with proteomic profiles generated from intratumor EVs dissociated from 12 SCLC patient-derived xenograft (PDX) mouse models as well as EVs isolated from cell-conditioned medias of a panel of 17 SCLC cell lines. A total of 496 proteins were quantified in TIM4pos EVs. Unsupervised hierarchical clustering and principal component analyses showed that TIM4pos EV protein cargoes differentiate SCLC from LUAD and matched controls. Differential analyses identified 117 proteins that were elevated (AUC> 0.60) in TIM4pos SCLC EVs compared to controls. Of these 117 features, 101 (86%) were also elevated (AUC> 0.60) in TIM4pos SCLC EVs compared to TIM4pos LUAD EVs. Intersection of proteins elevated in TIM4pos SCLC EVs with proteomic profiles of intratumor EVs from SCLC PDX models and conditioned media-derived EVs from SCLC cell lines yielded 67 and 92 overlapping proteins, respectively. Overlapping proteins were characterized by YAP1-associated signatures of cytoskeletal re-arrangement and epithelial-to-mesenchymal transition and included a prevalence of neuronal tubulin and actin family members, YWHA family members, and annexins. We developed and applied an optimized EV isolation and proteomics workflow to profile EV protein cargoes and establish circulating EV-associated protein signatures of SCLC with biomarker potential. Our integrated analyses identified novel EV-associated proteins for detection of SCLC that are associated with oncogenic drivers. These studies demonstrate that EVs harbor cancer cell disseminated signatures and that continued efforts to query the EV proteome towards discovery of novel SCLC biomarkers are warranted. Citation Format: Jody Vykoukal, Taketo Kato, Hiroyuki Katayama, Allison Stewart, Ehsan Irajizad, Yining Cai, Fu-Chung Hsiao, Jennifer B. Dennison, Edwin J. Ostrin, Hai T. Tran, Carl M. Gay, Lauren A. Byers, Johannes F. Fahrmann, Samir M. Hanash. Cancer cell derived extracellular vesicles convey protein signatures of small cell lung carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P078.

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Activation of the nuclear factor erythroid 2-related factor 2 (NRF2) pathway, either through gain-of-function mutations in the NRF2 encoding gene NFE2L2 or through loss-of-function of its suppressor, Kelch-like ECH-associated protein 1 (KEAP1), is a frequent manifestation in various malignancies. NRF2 activation exerts pro-tumoral effects in part by altering cancer cell metabolism. We previously reported a novel mechanism of NRF2 tumoral immune suppression through selective upregulation of the tryptophan metabolizing enzyme kynureninase (KYNU) in lung adenocarcinoma. In the current study, we explored the Pan-Cancer relevance of NRF2-mediated KYNU upregulation. We analyzed the gene expression dataset for 9,801 tumors representing 32 cancer types in The Cancer Genome Atlas (TCGA). Elevated KYNU expression levels paralleled increased gene-based signatures of NRF2-activation and was strongly associated with an immunosuppressive tumor microenvironment, marked by high expression of gene-based signatures of Tregs as well as immune checkpoint blockade-related genes CD274 (PDL-1), PDCD1 (PD-1), and CTLA4, regardless of cancer type. Cox proportional hazard models further revealed that increased tumoral KYNU gene expression was prognostic for poor overall survival in several cancer types, including thymoma, acute myeloid leukemia, low grade glioma, kidney renal papillary cell carcinoma, gastric adenocarcinoma, and pancreatic ductal adenocarcinoma (PDAC). Using PDAC as a model system, we confirmed that siRNA-mediated knockdown of NRF2 reduces KYNU mRNA expression, whereas activation of NFE2L2 (the encoding gene for NRF2) through either small molecule agonists or siRNA-mediated knockdown of KEAP1 upregulated KYNU. Metabolomic analyses of conditioned media from PDAC cell lines revealed elevated levels of KYNU-derived anthranilate, confirming KYNU as enzymatically functional. Collectively, our findings highlight the multi-cancer relevance of the NRF2-KYNU axis and of tumoral KYNU as a prognostic marker of poor overall survival associated with immunosuppression. Citation Format: Ricardo A. Leon Letelier, Ali H. Abdel Sater, Yihui Chen, Ranran Wu, Jennifer B. Dennison, Soyoung Park, Ehsan Irajizad, Hiroyuki Katayama, Jody Vykoukal, Samir Hanash, Edwin J. Ostrin, Johannes F. Fahrmann. KYNU upregulation is a prominent feature of NRF2-activated cancers and is associated with tumor immunosuppression and poor prognosis [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 6031.

Purpose: Emerging evidence implicates the microbiome in the development of pancreatic cancer. We investigated whether increased levels of microbial-related metabolites in circulation are associated with pancreatic cancer risk. Methods: We applied metabolomics profiling to sera from the Prostate, Lung, Colorectal and Ovarian (PLCO) Cohort to quantify and build a model based on 14 microbial-related metabolites. The study involved samples collected from 172 subjects within five years prior to diagnosis and 863 matched controls. Data from five PLCO centers were used for training and from two centers for validation and model selection. The model was subsequently tested using samples from three independent centers. The contributions of non-microbial-associated metabolites as well as CA19-9 was also assessed. Results: A 3-marker microbial-related metabolite panel yielded in the PLCO testing set an AUC of 0.64 (95% CI: 0.53-0.76) for 5-year probability of pancreatic cancer. Five additional non-microbial metabolites were identified that when combined with the microbiome panel yielded an AUC of 0.79 (95% CI: 0.71-0.88) for 5-year probability of pancreatic cancer in the PLCO testing set. The combined metabolite panel and CA19-9 yielded an AUC of 0.86 (95% CI: 0.77-0.95) for 2-year probability of pancreatic cancer in the PLCO testing set, which was improved compared to CA19-9 alone (AUC: 0.70 (95% CI: 0.57-0.82), p< 0.001). Conclusion: We developed a metabolite panel derived in part from the microbiome for risk assessment of pancreatic cancer, which has relevance to prevention and early detection. Citation Format: Johannes F. Fahrmann, Ehsan Irajizad, Ana Kenney, Tiffany Tang, Jody Vykoukal, Ranran Wu, Jennifer B. Dennison, Marta Sans Escofet, James P. Long, Maureen Loftus, John A. Chabot, Michael D. Kluger, Fay Kastrinos, Lauren Brais, Ana Babic, Kunal Jajoo, Linda S. Lee, Thomas E. Clancy, Kimmie Ng, Andrea Bullock, Jeanine M. Genkinger, Anirban Maitra, Kim-Anh Do, Bin Yu, Brian M. Wolpin, Samir Hanash. Contribution of the microbiome to a metabolomic signature predictive of risk for pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr P076.