Non-small cell lung carcinoma (NSCLC) is a major cause of cancer mortality. High expression of the epithelial-to-mesenchymal transition transcription factor (EMT-TF) Twist1 is strongly associated with metastatic cancers and with treatment resistance. Twist1 can also upregulate O-GlcNAcylation to suppress fail-safe programs such as KrasG12D oncogene-induced senescence (OIS) that accelerates NSCLC tumorigenesis. We wanted to decipher the critical domains and transcriptional targets required for Twist1 acceleration of lung tumorigenicity. We created a novel genetically-engineered mouse model for autochthonous lung cancer through lung epithelial expression of KrasG12D oncogene (CR) concomitantly with Twist1wt (CRT) or a Twist1F191G transactivation-deficient mutant (CRF191G). Compared to CR and CRF191G, CRT mice had shorter tumor-free survival and more aggressive tumors histologically. CRT lung tumors also showed higher proliferation and lower cell-cycle arrest suggesting that the Twist1 transactivation-domain is important for OIS suppression. Supporting these data, we observed in non-cancer human bronchial epithelial cells (HBECs) that the co-expression of human TWIST1wt enhanced tumorigenic/invasive programs and could suppress HRasG12V-induced senescence while co-expressing TWIST1F187G transactivation-deficient mutant could not. TWIST1wt co-expression with HRasG12V in HBECs differentially modulated MYC downstream transcriptional programs. Finally, OIS induction in HBECHRasG12V-TWIST1wt was rescued by O-GlcNAcylation inhibition or by treatment with a novel MYC inhibitor MYCi975 or by MYC knockdown. Altogether, these results indicate that the Twist1 transactivation domain is required for Twist1-dependent acceleration of lung tumorigenesis via MYC and nominate MYCi975 as a means to activate latent OIS programs. MYC targeting strategies could limit pro-tumorigenic programs and serve as a therapeutic for TWIST1-overexpressing NSCLCs.

The tissue microenvironment in prostate cancer is profoundly altered. While such alterations have been implicated in driving prostate cancer initiation and progression to aggressive disease, how prostate cancer cells and their precursors mediate those changes is unclear, in part due to the inability to longitudinally study the disease evolution in human tissues. To overcome this limitation, we performed extensive single-cell RNA-sequencing (scRNA-seq) and rigorous molecular pathology of the comparative biology between human prostate cancer and key time points in the disease evolution of a genetically engineered mouse model (GEMM) of prostate cancer. Our studies of human tissues, with validation in a large external data set, revealed that cancer cell-intrinsic activation of MYC signaling was the top up-regulated pathway in human cancers, representing a common denominator across the well-known molecular and pathological heterogeneity of human prostate cancer. Likewise, numerous non-malignant cell states in the tumor microenvironment (TME), including non-cancerous epithelial, immune, and fibroblast cell compartments, were conserved across individuals, raising the possibility that these cell types may be a sequelae of the convergent MYC activation in the cancer cells. To test this hypothesis, we employed a GEMM of prostate epithelial cell-specific MYC activation in two mouse strains. Cell communication network and pathway analyses suggested that MYC oncogene-expressing neoplastic cells, directly and indirectly, reprogrammed the TME during carcinogenesis, leading to the emergence of cascading cell state alterations in neighboring epithelial, immune, and fibroblast cell types that paralleled key findings in human prostate cancer. Importantly, among these changes, the progression from a precursor-enriched to invasive-cancer-enriched state was accompanied by a cell-intrinsic switch from pro-immunogenic to immunosuppressive transcriptional programs with coinciding enrichment of immunosuppressive myeloid and Treg cells in the immune microenvironment. These findings implicate activation of MYC signaling in reshaping convergent aspects of the TME of prostate cancer as a common denominator across the otherwise well-documented molecular heterogeneity of human prostate cancer.

Purpose/Objective(s) Non-small cell lung carcinoma (NSCLC) is a major cause of cancer mortality. High expression of the epithelial-to-mesenchymal transition transcription factor TWIST1 is strongly associated with metastatic cancers and treatment resistance. Additionally, TWIST1 can upregulate O-GlcNAcylation which (1) is required to suppress fail-safe programs such as oncogene (KRasG12D)-induced senescence (OIS) to accelerate tumorigenesis in primary NSCLC tumors, and (2) is a potential modulator of DNA repair/radiation response. We hypothesized that the transactivation function of TWIST1 and downstream target programs are critical in the promotion of tumorigenicity and radioresistance. Materials/Methods We created a novel genetically engineered mouse model (GEMM) allowing tetracycline-inducible expression in the lung epithelium (via lung specific CCSP-reverse tetracycline transactivator (C)) of KRasG12D (R) with Twist1wt (T) or with Twist1F191G transactivation-null mutant (F). We also created non-cancer Human Bronchial Epithelial Cell (HBEC) co-expressing HRasG12V oncogene with human TWIST1wt (HBEC-HRasG12V-TWIST1wt) or transactivation-null TWIST1F187G mutant (HBEC-HRasG12V-TWIST1F187G). Results CRT mice had shorter tumor-free survival and more aggressive tumors compared to CR/CRF mice indicating that the Twist1 transactivation domain is required for Twist1-dependent tumorigenesis acceleration. Also, Twist1wt expression promoted radioresistance in cell lines and GEMMs. Contrary to CRT, CRF showed a progressive loss of TWIST1F191G expression over time suggesting no functionality/no selective advantage. CRT lung tumors had higher proliferation (Ki67) and lower cell-cycle arrest (p16) compared to CR/CRF suggesting that the transactivation domain of Twist1 is important for OIS suppression. Supporting these data, we observed in HBEC that the co-expression of TWIST1wt could suppress HRasG12V-induced senescence while TWIST1F187G mutant could not. HBEC-HRasG12V-TWIST1wt also sustained tumorigenic/invasive programs. Interestingly, we observed that O-GlcNAcylation inhibition rescued OIS in HBEC-HRasG12V-TWIST1wt while O-GlcNAcylation stimulation in HBEC-HRasG12V-TWIST1F187G suppressed OIS. Importantly, TWIST1wt modulated MYC downstream targets, and MYC activity inhibition in HBEC-HRasG12V-TWIST1wt using the novel MYC inhibitor MYCi975 also rescued OIS induction. Conclusion Altogether, these results suggest that TWIST1 may suppress OIS via MYC signaling and nominate MYCi975 as a means to activate latent OIS programs. MYC inhibiting strategies could serve as a therapeutic sensitizer for TWIST1-expressing NSCLC. This work and our future studies on TWIST1 on the control of OIS, O-GlcNAcylation, and radioresistance mechanisms may help to identify new potential NSCLC therapeutic strategies.

alpha-particle emitters are emerging as a potent modality for disseminated cancer therapy because of their high linear energy transfer and localized absorbed dose profile. Despite great interest and pharmaceutical development, there is scant information on the distribution of these agents at the scale of the alpha-particle pathlength. We sought to determine the distribution of clinically approved [Ra-223]RaCl2 in bone metastatic castration-resistant prostate cancer at this resolution, for the first time to our knowledge, to inform activity distribution and dose at the near-cell scale. Methods: Biopsy specimens and blood were collected from 7 patients 24 h after administration. Ra-223 activity in each sample was recorded, and the microstructure of biopsy specimens was analyzed by micro-CT. Quantitative autoradiography and histopathology were segmented and registered with an automated procedure. Activity distributions by tissue compartment and dosimetry calculations based on the MIRD formalism were performed. Results: We revealed the activity distribution differences across and within patient samples at the macro- and microscopic scales. Microdistribution analysis confirmed localized high-activity regions in a background of low-activity tissue. We evaluated heterogeneous alpha-particle emission distribution concentrated at bone-tissue interfaces and calculated spatially nonuniform absorbed-dose profiles. Conclusion: Primary patient data of radiopharmaceutical therapy distribution at the small scale revealed that Ra-223 uptake is nonuniform. Dose estimates present both opportunities and challenges to enhance patient outcomes and are a first step toward personalized treatment approaches and improved understanding of alpha-particle radiopharmaceutical therapies.

ASPN expression is elevated in PCAFs.

Supplementary Figure S5. Mast cell quantification in ventral prostates of control versus PhIP-treated rats (n=2) at week 20 of the study (after 20 weeks of receiving dietary PhIP).

Supplementary Figure S4. IHC for E. coli in rat prostate tissues at 42 weeks post-inoculation.

Supplementary Table 3 from Cancer-Related Epigenome Changes Associated with Reprogramming to Induced Pluripotent Stem Cells

Prostate cancer (PCa) is the second most common cancer and constitutes about 14.7% of total cancer cases. PCa is highly prevalent and more aggressive in African-American (AA) men than in European-American (EA) men. PCa tends to be highly heterogeneous, and its complex biology is not fully understood. We use metabolomics to better understand the mechanisms behind PCa progression and disparities in its clinical outcome. Adenosine deaminase (ADA) is a key enzyme in the purine metabolic pathway; it was found to be upregulated in PCa and is associated with higher-grade PCa and poor disease-free survival. The inosine-to-adenosine ratio, which is a surrogate for ADA activity was high in PCa patient urine and higher in AA PCa compared to EA PCa. To understand the significance of high ADA in PCa, we established ADA overexpression models and performed various in vitro and in vivo studies. Our studies have revealed that an acute increase in ADA expression during later stages of tumor development enhances in vivo growth in multiple pre-clinical models. Further analysis revealed that mTOR signaling activation could be associated with this tumor growth. Chronic ADA overexpression shows alterations in the cells' adhesion machinery and a decrease in cells' ability to adhere to the extracellular matrix in vitro. Losing cell-matrix interaction is critical for metastatic dissemination which suggests that ADA could potentially be involved in promoting metastasis. This is supported by the association of higher ADA expression with higher-grade tumors and poor patient survival. Overall, our findings suggest that increased ADA expression may promote PCa progression, specifically tumor growth and metastatic dissemination.

This file consists of the Supplementary Materials and Methods and the Supplementary Figure Legends.

ABSTRACTEvaluating the complex interplay of cell types in the tissue microenvironment is critical to understanding the origin and progression of diseases in the prostate and potential opportunities for intervention. Mouse models are an essential tool to investigate the molecular and cell-type-specific contributions of prostate disease at an organismal level. While there are well-documented differences in the extent, timing, and nature of disease development in various genetically engineered mouse models in different mouse strains and prostate lobes within each mouse strain, yet, the underlying molecular phenotypic differences in cell types across mouse strains and prostate lobes are incompletely understood. To address this, we examined the single-cell transcriptomes of individual mouse prostate lobes from two commonly used mouse strains, FVB/NJ and C57BL/6J. Data dimensionality reduction and clustering analysis revealed that basal and luminal cells possessed strain-specific transcriptomic differences, with luminal cells also displaying marked lobe-specific differences. Additionally, three rare populations of epithelial cells clustered independently of strain and lobe: one population of luminal cells expressing Foxi1 and components of the vacuolar ATPase proton pump (Atp6v0d2andAtp6v1g3), another population expressing Psca and other stem cell-associated genes (Ly6a/Sca-1, Tacstd2/Trop-2), and a neuroendocrine population expressingChga, Chgb, andSyp. In contrast, stromal cell clusters, including fibroblasts, smooth muscle cells, endothelial cells, pericytes, and immune cell types, were conserved across strain and lobe, clustering largely by cell type and not by strain or lobe. One notable exception to this was the identification of two distinct fibroblast populations that we term subglandular fibroblasts and interstitial fibroblasts based on their strikingly distinct spatial distribution in the mouse prostate. Altogether, these data provide a practical reference of the transcriptional profiles of mouse prostate from two commonly used mouse strains and across all four prostate lobes.

Frequency of germline ASPN D-repeat-length in JHH prostate cancer cases and controls.

The biological influence of physicochemical parameters of "targeted" nanoparticles on their delivery to cancer tumors remains poorly understood. A comparative analysis of nanoparticle distributions in tumors following systemic delivery across several models can provide valuable insights. Methods: Bionized nanoferrite nanoparticles (iron oxide core coated with starch), either conjugated with a targeted anti-HER2 antibody (BH), or unconjugated (BP), were intravenously injected into athymic nude or NOD-scid gamma (NSG) female mice bearing one of five human breast cancer tumor xenografts growing in a mammary fat pad. Tumors were harvested 24 hours after nanoparticle injection, fixed, mounted, and stained. We performed detailed histopathology analysis by comparing spatial distributions of nanoparticles (Prussian blue) with various stromal cells (CD31, SMA, F4/80, CD11c, etc.) and the target antigen-expressing (HER2) tumor cells. Results: Only BH nanoparticles were retained in tumors and generally concentrated in the tumor periphery, with nanoparticle content diminishing towards the tumor interior. Nanoparticle distribution correlated strongly with specific stromal cells within each tumor type, which varied among tumor types and between mouse strains. Weak or no correlation between nanoparticle distribution and HER2 positive cells, or CD31 cells was observed. Conclusion: Antibody-labeled nanoparticles were retained across all tumors, irrespective of presence of the "target" antigen. Though presence of antibody on nanoparticles correlated with retention, non-cancerous host stromal cells were responsible for their retention in the tumor microenvironment. This study highlights gaps in our understanding of the complex biological interplay between disease and host immune biology, and the need to account for the influence of underlying aberrant tumor biology as factors determining nanoparticle fate in vivo.

Purpose- BIN1 is in deleted in ~5% of all prostate cancer (PC) patients and interestingly is highly enriched in the Speckle-type POZ protein (SPOP) mutant subclass (15%) of PC. BIN1 was originally identified as a repressor of cMYC activity and our lab has shown that patients with SPOPmut;MYChigh PC have worse clinical outcomes. Our observation of BIN1 deletion in the SPOP mutant subclass leads us to hypothesize that BIN1 deletion leads to enhanced AR signaling and more aggressive PC. Experimental Procedures- We analyzed baseline protein and mRNA levels of BIN1 in 13 PC cell lines via quantitative polymerase chain reaction (qPCR) and immunoblot. To understand the role of BIN1 in PC cells, we overexpressed BIN1 and evaluated changes in cell proliferation, migration, and AR target gene expression. To elucidate the role of BIN1 in vivo prostate development, we also generated a prostate-specific knockout of BIN1 and investigated changes in prostate development and AR signaling axis over time in this mouse model. Results- Our results show that none of the 13 PC cell lines analyzed had strong protein expression at baseline, but all had detectable mRNA levels. Interestingly, the three cell lines with the highest mRNA expression were AR negative (DU-145), AR independent (LN95) or castration resistant (MDVR). We also saw a decrease in downstream AR target genes (KLK3 and NKX3.1) measured using qPCR after BIN1 overexpression in 22Rv1 cells. We further corroborated this finding by measuring KLK3 expression using a KLK3-luc reporter assay after BIN1 overexpression. BIN1 overexpression also reduced invasive activity of 22Rv1 measured using a Matrigel invasion assay system. Since BIN1 deletion was enriched in SPOP mutant PC clinical data, we evaluated changes in cell proliferation in the wildtype and mutant SPOP cell lines with and without BIN1OE and found that BIN1OEhindered growth of SPOP mutant PC cells. Analysis of the TCGA primary PC dataset illustrate that PC patients harboring BIN1 deletion had higher AR activity compared to PC patients expressing wildtype BIN1. Importantly, mice with prostate specific BIN1 deletion had larger prostate mass at 2 months of age compared to age matched controls and several mice at the 6&9 month had severely enlarged prostates. Conclusion- Our findings show for the first time that BIN1 functions as an inhibitor of the AR signaling axis and deletion of BIN1 leads to higher levels of AR signaling. We also show that BIN1 deletion in mice affects prostate development and leads to enlarged prostates. These novel findings illustrate the clinical significance of BIN1 deletion in PC patients and highlights the AR-signaling axis as a potential target for PC patients with BIN1 deletion Citation Format: Collin McColl, Darlene Skapura, Elisa Echartea, Jenny Deng, Cristian Coarfa, Salma Kaochar, Brian Simons, Aleksandra Rusin. Elucidating the effects of the Alzheimer's disease associated gene BIN1 on cancer tumorigenesis [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A008.

This document contains experimental details for microarray data acquisition and analysis, immunohistochemistry, immunofluorescence, western blotting, migration assay, invasion assay, anoikis assay, soft agar colony formation assay, RNA-immunoprecipitation, protein co-immunoprecipitation, Chromatin immunoprecipitation (ChIP), ChIP-Re-ChIP.

Figure S1 shows cell growth and lipid distribution data. Figure S2 shows lipid distribution and invasion data. Figure S3 shows additional transcriptomic data. Figure S4 and S5 shows additional orthotopic data.

Supplementary Table S2 shows bivariate and multivariable analyses of germline ASPN D13/14 compared to ASPN D13/13 for lymph node involvement and metastatic recurrence. Supplementary Table S3 show bivariate analyses of ASPN D genotypes/alleles for biochemical recurrence. Supplementary Table S4 shows multivariable analyses of ASPN D13/14 for biochemical recurrence.

Supplementary Figure Legends