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As epithelial tumors grow from single cells to a malignant mass of invasive tissue, they must exploit the innate inflammatory response, while evading the adaptive immune system. Prognosis of solid tumors has historically focused on macroscopic features such as size, grade, and mitotic index. It is now clear that prognosis assessment must also consider the stromal and immune cells that surround and infiltrate the tumor. Tumors promote growth, angiogenesis, and tissue remodeling by subverting the normal functions of macrophages and other cells of the innate immune system that inhabit their microenvironment. Simultaneously, tumor cells escape from and inactivate the adaptive immune system by exploiting the mechanisms preventing damaging auto-immune responses in cytotoxic T cells. The presence of CD8+ T cells within epithelial tumors is now a well-supported marker of better prognosis in many tumor types. However, this benefit is counterbalanced by immune regulatory cell populations that promote tumor escape from immune surveillance and metastasis. Therapeutic approaches that kill tumor cells selectively by re-activating immune checkpoints are becoming an established therapeutic option, but it is not yet clear how to identify which patients will benefit from this treatment modality. Evidence is accumulating that identifying the presence of T cell-activating neoantigens, produced by mutated proteins in tumors, will play an important role in checkpoint inhibitor prognosis. This review provides an overview of the evidence that lymphocytic infiltration of tumors has prognostic value in many epithelial tumor types and is linked to the success of chemical and immune checkpoint therapeutic strategies. •The presence of tumor-infiltrating lymphocytes is a positive prognostic marker in many solid tumors, including breast, colon, and melanoma.•Both pathological review of tissue sections and gene transcription analysis effectively quantify lymphocytic infiltrate.•The role of tumor-infiltrating lymphocytes in breast cancer is mediated by the tumor's molecular subtype.•Patients whose tumors bear a higher somatic mutational burden are more likely to benefit from immune checkpoint inhibitors.

> Circulating tumour DNA (ctDNA) in blood plasma is an emerging tool for clinical cancer genotyping and longitudinal disease monitoring 1 . However, owing to past emphasis on targeted and low-resolution profiling approaches, our understanding of the distinct populations that comprise bulk ctDNA is incomplete 2 – 12 . Here we perform deep whole-genome sequencing of serial plasma and synchronous metastases in patients with aggressive prostate cancer. We comprehensively assess all classes of genomic alterations and show that ctDNA contains multiple dominant populations, the evolutionary histories of which frequently indicate whole-genome doubling and shifts in mutational processes. Although tissue and ctDNA showed concordant clonally expanded cancer driver alterations, most individual metastases contributed only a minor share of total ctDNA. By comparing serial ctDNA before and after clinical progression on potent inhibitors of the androgen receptor (AR) pathway, we reveal population restructuring converging solely on AR augmentation as the dominant genomic driver of acquired treatment resistance. Finally, we leverage nucleosome footprints in ctDNA to infer mRNA expression in synchronously biopsied metastases, including treatment-induced changes in AR transcription factor signalling activity. Our results provide insights into cancer biology and show that liquid biopsy can be used as a tool for comprehensive multi-omic discovery. Deep whole-genome sequencing of serial blood samples and matched metastatic tissue reveals that circulating tumour DNA profiling enables detailed study of treatment-driven subclone dynamics, epigenomics and genome-wide somatic evolution in metastatic human cancers.

Calcium carbonate is an abundant substance that can be created in several mineral forms by the reaction of dissolved carbon dioxide in water with calcium ions. Through biomineralization, organisms can harness and control this process to form various functional materials that can act as anything from shells through to lenses. The early stages of calcium carbonate formation have recently attracted attention as stable prenucleation clusters have been observed, contrary to classical models. Here we show, using computer simulations combined with the analysis of experimental data, that these mineral clusters are made of an ionic polymer, composed of alternating calcium and carbonate ions, with a dynamic topology consisting of chains, branches and rings. The existence of a disordered, flexible and strongly hydrated precursor provides a basis for explaining the formation of other liquid-like amorphous states of calcium carbonate, in addition to the non-classical behaviour during growth of amorphous calcium carbonate. Prenucleation clusters have been observed during the early stages of calcium carbonate formation, contrary to classical models. Here, computer simulations indicate that the clusters are composed of an ionic polymer with alternating calcium and carbonate ions, and a dynamic topology of chains, branches and rings.

Whole-exome sequencing is used to compare the mutational landscape of adenomas from three mouse models of non-small-cell lung cancer, induced either by exposure to carcinogens or by genetic mutation of Kras ; the results reveal that the two types of tumour have different mutational profiles and adopt different routes to tumour development. Adenoma gene profiles compared Allan Balmain and colleagues use whole-exome sequencing to compare the mutational landscape of adenomas from three mouse models of non-small cell lung cancer, induced by exposure to the carcinogens methyl-nitrosourea (MNU) and urethane, or by genetic activation of Kras ( Kras LA2 ). Although the MNU-induced and Kras LA2 tumours carried the same initiating Kras mutation, MNU tumours also carry numerous non-synonymous point mutations. At the same time, Kras LA2 tumours carried numerous copy number alterations. This suggests that carcinogen-induced and genetically engineered models adopt different routes to tumour development. The results argue for a major role of germline Kras status in mutation selection during initiation. Collectively, these data demonstrate the utility of carcinogen models for understanding the complex mutation spectra seen in human cancers. Next-generation sequencing of human tumours has refined our understanding of the mutational processes operative in cancer initiation and progression, yet major questions remain regarding the factors that induce driver mutations and the processes that shape mutation selection during tumorigenesis. Here we performed whole-exome sequencing on adenomas from three mouse models of non-small-cell lung cancer, which were induced either by exposure to carcinogens (methyl-nitrosourea (MNU) and urethane) or by genetic activation of Kras ( Kras LA2 ). Although the MNU-induced tumours carried exactly the same initiating mutation in Kras as seen in the Kras LA2 model (G12D), MNU tumours had an average of 192 non-synonymous, somatic single-nucleotide variants, compared with only six in tumours from the Kras LA2 model. By contrast, the Kras LA2 tumours exhibited a significantly higher level of aneuploidy and copy number alterations compared with the carcinogen-induced tumours, suggesting that carcinogen-induced and genetically engineered models lead to tumour development through different routes. The wild-type allele of Kras has been shown to act as a tumour suppressor in mouse models of non-small-cell lung cancer. We demonstrate that urethane-induced tumours from wild-type mice carry mostly (94%) Kras Q61R mutations, whereas those from Kras heterozygous animals carry mostly (92%) Kras Q61L mutations, indicating a major role for germline Kras status in mutation selection during initiation. The exome-wide mutation spectra in carcinogen-induced tumours overwhelmingly display signatures of the initiating carcinogen, while adenocarcinomas acquire additional C > T mutations at CpG sites. These data provide a basis for understanding results from human tumour genome sequencing, which has identified two broad categories of tumours based on the relative frequency of single-nucleotide variations and copy number alterations 1 , and underline the importance of carcinogen models for understanding the complex mutation spectra seen in human cancers.

Small nucleolar RNAs (snoRNAs) are a diverse group of non-coding RNAs that direct chemical modifications at specific residues on other RNA molecules, primarily on ribosomal RNA (rRNA). SnoRNAs are altered in several cancers; however, their role in cell homeostasis as well as in cellular transformation remains poorly explored. Here, we show that specific subsets of snoRNAs are differentially regulated during the earliest cellular response to oncogenic RASG12V expression. We describe a novel function for one H/ACA snoRNA, SNORA24, which guides two pseudouridine modifications within the small ribosomal subunit, in RAS-induced senescence in vivo. We find that in mouse models, loss of Snora24 cooperates with RASG12V to promote the development of liver cancer that closely resembles human steatohepatitic hepatocellular carcinoma (HCC). From a clinical perspective, we further show that human HCCs with low SNORA24 expression display increased lipid content and are associated with poor patient survival. We next asked whether ribosomes lacking SNORA24-guided pseudouridine modifications on 18S rRNA have alterations in their biophysical properties. Single-molecule Fluorescence Resonance Energy Transfer (FRET) analyses revealed that these ribosomes exhibit perturbations in aminoacyl-transfer RNA (aa-tRNA) selection and altered pre-translocation ribosome complex dynamics. Furthermore, we find that HCC cells lacking SNORA24-guided pseudouridine modifications have increased translational miscoding and stop codon readthrough frequencies. These findings highlight a role for specific snoRNAs in safeguarding against oncogenic insult and demonstrate a functional link between H/ACA snoRNAs regulated by RAS and the biophysical properties of ribosomes in cancer. Ribosomes are cellular machines responsible for translating the genetic code into proteins. Research has shown that changes in ribosome activity can contribute to healthy cells becoming cancerous. Ribosomes consist of proteins and other molecules known as ribosomal RNAs (or rRNAs for short). Before they can become part of a ribosome, another type of molecule called snoRNAs must modify new rRNAs. Indeed, many of the modifications that allow rRNAs to accurately translate genetic information into proteins are introduced by snoRNAs. As such, it is possible that changes to snoRNAs could contribute to the creation of cancerous cells by affecting how ribosomes operate. To explore this possibility, McMahon, Contreras et al. examined snoRNAs in healthy cells grown in the laboratory that have been given pro-cancer signals, in cancer from mice, and in samples from human cancer patients. The investigation revealed that the activation of growth signals – a hallmark of many cancers – affects the abundance of some snoRNAs and changes the pattern of rRNA modifications they make on ribosomes. Reducing the levels of one such snoRNA called SNORA24 led mice to develop fatty liver cancer when combined with cancer-promoting growth signals. Analyzing why reducing the levels of SNORA24 led to liver cancer, McMahon, Contreras et al. found that ribosomes lacking rRNA modifications introduced by SNORA24 made more mistakes when producing proteins coded for by certain genes. These results contribute to the view of ribosomes as a key hub for the transformation of healthy cells into cancer cells. Increasing the error rate of ribosomes could be a key driver in further changes that drive cancer development. This study also highlights the role of snoRNAs in responding to growth signals, particularly in cancer. These findings identify snoRNAs as new potential diagnostic factors and treatment targets.

The androgen receptor (AR) antagonist enzalutamide is one of the principal treatments for men with castration-resistant prostate cancer (CRPC). However, not all patients respond, and resistance mechanisms are largely unknown. We hypothesized that genomic and transcriptional features from metastatic CRPC biopsies prior to treatment would be predictive of de novo treatment resistance. To this end, we conducted a phase II trial of enzalutamide treatment (160 mg/d) in 36 men with metastatic CRPC. Thirty-four patients were evaluable for the primary end point of a prostate-specific antigen (PSA)50 response (PSA decline ≥50% at 12 wk vs. baseline). Nine patients were classified as nonresponders (PSA decline <50%), and 25 patients were classified as responders (PSA decline ≥50%). Failure to achieve a PSA50 was associated with shorter progression-free survival, time on treatment, and overall survival, demonstrating PSA50’s utility. Targeted DNA-sequencing was performed on 26 of 36 biopsies, and RNA-sequencing was performed on 25 of 36 biopsies that contained sufficient material. Using computational methods, we measured AR transcriptional function and performed gene set enrichment analysis (GSEA) to identify pathways whose activity state correlated with de novo resistance. TP53 gene alterations were more common in nonresponders, although this did not reach statistical significance (P = 0.055). AR gene alterations and AR expression were similar between groups. Importantly, however, transcriptional measurements demonstrated that specific gene sets— including those linked to low AR transcriptional activity and a stemness program—were activated in nonresponders. Our results suggest that patients whose tumors harbor this program should be considered for clinical trials testing rational agents to overcome de novo enzalutamide resistance.

Genomic sequencing of thousands of tumors has revealed many genes associated with specific types of cancer. Similarly, large scale CRISPR functional genomics efforts have mapped genes required for cancer cell proliferation or survival in hundreds of cell lines. Despite this, for specific disease subtypes, such as metastatic prostate cancer, there are likely a number of undiscovered tumor specific driver genes that may represent potential drug targets. To identify such genetic dependencies, we performed genome-scale CRISPRi screens in metastatic prostate cancer models. We then created a pipeline in which we integrated pan-cancer functional genomics data with our metastatic prostate cancer functional and clinical genomics data to identify genes that can drive aggressive prostate cancer phenotypes. Our integrative analysis of these data reveals known prostate cancer specific driver genes, such as AR and HOXB13 , as well as a number of top hits that are poorly characterized. In this study we highlight the strength of an integrated clinical and functional genomics pipeline and focus on two top hit genes, KIF4A and WDR62 . We demonstrate that both KIF4A and WDR62 drive aggressive prostate cancer phenotypes in vitro and in vivo in multiple models, irrespective of AR-status, and are also associated with poor patient outcome. It is hypothesized that there are a number of tumor specific driver genes for metastatic prostate cancer. Here, the authors perform genome-wide CRISPRi screens and integrate these data with metastatic prostate cancer functional and clinical genomics data to show that KIF4A and WDR62 drive aggressive prostate cancer phenotypes.

BACKGROUNDAndrogen receptor signaling inhibitors (ARSIs) have improved outcomes for patients with metastatic castration-resistant prostate cancer (mCRPC), but their clinical benefit is limited by treatment resistance.METHODSTo investigate the mechanisms of ARSI resistance, we analyzed the whole-genome (n = 45) and transcriptome (n = 31) sequencing data generated from paired metastatic biopsies obtained before initiation of first-line ARSI therapy for mCRPC and after radiographic disease progression. We investigated the effects of genetic and pharmacologic modulation of SSTR1 in 22Rv1 cells, a representative mCRPC cell line.RESULTSWe confirmed the predominant role of tumor genetic alterations converging on augmenting androgen receptor (AR) signaling and the increased transcriptional heterogeneity and lineage plasticity during the emergence of ARSI resistance. We further identified amplifications involving a putative enhancer downstream of the AR and transcriptional downregulation of SSTR1, encoding somatostatin receptor 1, in ARSI-resistant tumors. We found that patients with SSTR1-low mCRPC tumors derived less benefit from subsequent ARSI therapy in a retrospective cohort. We showed that SSTR1 was antiproliferative in 22Rv1 cells and that the FDA-approved drug pasireotide suppressed 22Rv1 cell proliferation.CONCLUSIONOur findings expand the knowledge of ARSI resistance and point out actionable next steps, exemplified by potentially targeting SSTR1, to improve patient outcomes.FUNDINGNational Cancer Institute (NCI), NIH; Prostate Cancer Foundation; Conquer Cancer, American Society of Clinical Oncology Foundation; UCSF Benioff Initiative for Prostate Cancer Research; Netherlands Cancer Institute.

Transcription factor (TF) proteins regulate gene activity by binding to regulatory regions, most importantly at gene promoters. Many genes have alternative promoters (APs) bound by distinct TFs. The role of differential TF activity at APs during tumour development is poorly understood. Here we show, using deep RNA sequencing in 274 biopsies of benign prostate tissue, localized prostate tumours and metastatic castration-resistant prostate cancer, that AP usage increases as tumours progress and APs are responsible for a disproportionate amount of tumour transcriptional activity. Expression of the androgen receptor (AR), the key driver of prostate tumour activity, is correlated with elevated AP usage. We identified AR , FOXA1 and MYC as potential drivers of AP activation. DNA methylation is a likely mechanism for AP activation during tumour progression and lineage plasticity. Our data suggest that prostate tumours activate APs to magnify the transcriptional impact of tumour drivers, including AR and MYC . With ultra-deep RNA sequencing, Zhang et al. report increased usage of alternative promoters driven by AR, FOXA1 and MYC during prostate cancer progression and suggest altered DNA methylation as a potential underlying mechanism.

Prostate-specific membrane antigen (PSMA) is an important cell surface target in prostate cancer. There are limited data on the heterogeneity of PSMA tissue expression in metastatic castration-resistant prostate cancer (mCRPC). Furthermore, the mechanisms regulating PSMA expression (encoded by the FOLH1 gene) are not well understood. Here, we demonstrate that PSMA expression is heterogeneous across different metastatic sites and molecular subtypes of mCRPC. In a rapid autopsy cohort in which multiple metastatic sites per patient were sampled, we found that 13 of 52 (25%) cases had no detectable PSMA and 23 of 52 (44%) cases showed heterogeneous PSMA expression across individual metastases, with 33 (63%) cases harboring at least 1 PSMA-negative site. PSMA-negative tumors displayed distinct transcriptional profiles with expression of druggable targets such as MUC1. Loss of PSMA was associated with epigenetic changes of the FOLH1 locus, including gain of CpG methylation and loss of histone 3 lysine 27 (H3K27) acetylation. Treatment with histone deacetylase (HDAC) inhibitors reversed this epigenetic repression and restored PSMA expression in vitro and in vivo. Collectively, these data provide insights into the expression patterns and regulation of PSMA in mCRPC and suggest that epigenetic therapies - in particular, HDAC inhibitors - can be used to augment PSMA levels.