Neuroendocrine Differentiation in Prostate Cancer Requires ASCL1.

Most patients with prostate adenocarcinoma develop resistance to therapies targeting the androgen receptor (AR). Consequently, a portion of these patients develop AR-independent neuroendocrine prostate cancer (NEPC), a rapidly progressing cancer with limited therapies and poor survival outcomes. Current research to understand the progression to NEPC suggests a model of lineage plasticity whereby AR-dependent luminal-like tumors progress towards an AR-independent NEPC state. Genetic analysis of human NEPC identified frequent loss of RB1 and TP53, and the loss of both genes in experimental models mediates the transition to a neuroendocrine lineage. Transcriptomics studies have shown that lineage transcription factors ASCL1 and NEUROD1 are present in NEPC. In this study, we modeled the progression of prostate adenocarcinoma to NEPC by establishing prostate organoids and subsequently generating subcutaneous allograft tumors from genetically-engineered mouse models harboring Cre-induced loss of Rb1 and Trp53 with Myc overexpression (RPM). These tumors were heterogeneous and displayed adenocarcinoma, squamous, and neuroendocrine features. ASCL1 and NEUROD1 were expressed within neuroendocrine-defined regions, with ASCL1 being predominant. Genetic loss of Ascl1 in this model did not decrease tumor incidence, growth, or metastasis; however, there was a notable decrease in neuroendocrine identity and an increase in basal-like identity. This study provides an in vivo model to study progression to NEPC and establishes the requirement for ASCL1 in driving neuroendocrine differentiation in prostate cancer.

Evasion of Innate Immunity Contributes to Small Cell Lung Cancer Progression and Metastasis.

Small cell lung cancer (SCLC) is a pulmonary neuroendocrine cancer with very poor prognosis and limited effective therapeutic options. Most patients are diagnosed at advanced stages, and the exact reason for the aggressive and metastatic phenotype of SCLC is completely unknown. Despite a high tumor mutational burden, responses to immune checkpoint blockade are minimal in patients with SCLC. This may reflect defects in immune surveillance. Here we illustrate that evading natural killer (NK) surveillance contributes to SCLC aggressiveness and metastasis, primarily through loss of NK-cell recognition of these tumors by reduction of NK-activating ligands (NKG2DL). SCLC primary tumors expressed very low level of NKG2DL mRNA and SCLC lines express little to no surface NKG2DL at the protein level. Chromatin immunoprecipitation sequencing showed NKG2DL loci in SCLC are inaccessible compared with NSCLC, with few H3K27Ac signals. Restoring NKG2DL in preclinical models suppressed tumor growth and metastasis in an NK cell-dependent manner. Likewise, histone deacetylase inhibitor treatment induced NKG2DL expression and led to tumor suppression by inducing infiltration and activation of NK and T cells. Among all the common tumor types, SCLC and neuroblastoma were the lowest NKG2DL-expressing tumors, highlighting a lineage dependency of this phenotype. In conclusion, these data show that epigenetic silencing of NKG2DL results in a lack of stimulatory signals to engage and activate NK cells, highlighting the underlying immune avoidance of SCLC and neuroblastoma. SIGNIFICANCE: This study discovers in SCLC and neuroblastoma impairment of an inherent mechanism of recognition of tumor cells by innate immunity and proposes that this mechanism can be reactivated to promote immune surveillance.

Subtype-specific secretomic characterization of pulmonary neuroendocrine tumor cells.

Pulmonary neuroendocrine (NE) cancer, including small cell lung cancer (SCLC), is a particularly aggressive malignancy. The lineage-specific transcription factors Achaete-scute homolog 1 (ASCL1), NEUROD1 and POU2F3 have been reported to identify the different subtypes of pulmonary NE cancers. Using a large-scale mass spectrometric approach, here we perform quantitative secretome analysis in 13 cell lines that signify the different NE lung cancer subtypes. We quantify 1,626 proteins and identify IGFBP5 as a secreted marker for ASCL1High SCLC. ASCL1 binds to the E-box elements in IGFBP5 and directly regulates its transcription. Knockdown of ASCL1 decreases IGFBP5 expression, which, in turn, leads to hyperactivation of IGF-1R signaling. Pharmacological co-targeting of ASCL1 and IGF-1R results in markedly synergistic effects in ASCL1High SCLC in vitro and in mouse models. We expect that this secretome resource will provide the foundation for future mechanistic and biomarker discovery studies, helping to delineate the molecular underpinnings of pulmonary NE tumors.

ASCL1 and NEUROD1 Reveal Heterogeneity in Pulmonary Neuroendocrine Tumors and Regulate Distinct Genetic Programs.

Small cell lung carcinoma (SCLC) is a high-grade pulmonary neuroendocrine tumor. The transcription factors ASCL1 and NEUROD1 play crucial roles in promoting malignant behavior and survival of human SCLC cell lines. Here, we find that ASCL1 and NEUROD1 identify heterogeneity in SCLC, bind distinct genomic loci, and regulate mostly distinct genes. ASCL1, but not NEUROD1, is present in mouse pulmonary neuroendocrine cells, and only ASCL1 is required in vivo for tumor formation in mouse models of SCLC. ASCL1 targets oncogenic genes including MYCL1, RET, SOX2, and NFIB while NEUROD1 targets MYC. ASCL1 and NEUROD1 regulate different genes that commonly contribute to neuronal function. ASCL1 also regulates multiple genes in the NOTCH pathway including DLL3. Together, ASCL1 and NEUROD1 distinguish heterogeneity in SCLC with distinct genomic landscapes and distinct gene expression programs.

The comparative pathology of genetically engineered mouse models for neuroendocrine carcinomas of the lung.

INTRODUCTION: Because small-cell lung carcinomas (SCLC) are seldom resected, human materials for study are limited. Thus, genetically engineered mouse models (GEMMs) for SCLC and other high-grade lung neuroendocrine (NE) carcinomas are crucial for translational research. METHODS: The pathologies of five GEMMs were studied in detail and consensus diagnoses reached by four lung cancer pathology experts. Hematoxylin and Eosin and immunostained slides of over 100 mice were obtained from the originating and other laboratories and digitalized. The GEMMs included the original Rb/p53 double knockout (Berns Laboratory) and triple knockouts from the Sage, MacPherson, and Jacks laboratories (double knockout model plus loss of p130 [Sage laboratory] or loss of Pten [MacPherson and Jacks laboratories]). In addition, a GEMM with constitutive co-expression of SV40 large T antigen and Ascl1 under the Scgb1a1 promoter from the Linnoila laboratory were included. RESULTS: The lung tumors in all of the models had common as well as distinct pathological features. All three conditional knockout models resulted in multiple pulmonary tumors arising mainly from the central compartment (large bronchi) with foci of in situ carcinoma and NE cell hyperplasia. They consisted of inter- and intra-tumor mixtures of SCLC and large-cell NE cell carcinoma in varying proportions. Occasional adeno- or large-cell carcinomas were also seen. Extensive vascular and lymphatic invasion and metastases to the mediastinum and liver were noted, mainly of SCLC histology. In the Rb/p53/Pten triple knockout model from the MacPherson and Jacks laboratories and in the constitutive SV40/T antigen model many peripherally arising non-small-cell lung carcinoma tumors having varying degrees of NE marker expression were present (non-small-cell lung carcinoma-NE tumors). The resultant histological phenotypes were influenced by the introduction of specific genetic alterations, by inactivation of one or both alleles of specific genes, by time from Cre activation and by targeting of lung cells or NE cell subpopulations. CONCLUSION: The five GEMM models studied are representative for the entire spectrum of human high-grade NE carcinomas and are also useful for the study of multistage pathogenesis and the metastatic properties of these tumors. They represent one of the most advanced forms of currently available GEMM models for the study of human cancer.