IntroductionThere is an unmet clinical need for biomarkers to identify breast cancer patients at an increased risk of developing brain metastases. The objective is to identify gene signatures and biological pathways associated with human epidermal growth factor receptor 2-positive (HER2+) brain metastasis.MethodsWe combined laser capture microdissection and gene expression microarrays to analyze malignant epithelium from HER2+ breast cancer brain metastases with that from HER2+ nonmetastatic primary tumors. Differential gene expression was performed including gene set enrichment analysis (GSEA) using publicly available breast cancer gene expression data sets.ResultsIn a cohort of HER2+ breast cancer brain metastases, we identified a gene expression signature that anti-correlates with overexpression of BRCA1. Sequence analysis of the HER2+ brain metastases revealed no pathogenic mutations of BRCA1, and therefore the aforementioned signature was designated BRCA1 Deficient-Like (BD-L). Evaluation of an independent cohort of breast cancer metastases demonstrated that BD-L values are significantly higher in brain metastases as compared to other metastatic sites. Although the BD-L signature is present in all subtypes of breast cancer, it is significantly higher in BRCA1 mutant primary tumors as compared with sporadic breast tumors. Additionally, BD-L signature values are significantly higher in HER2-/ER- primary tumors as compared with HER2+/ER + and HER2-/ER + tumors. The BD-L signature correlates with breast cancer cell line pharmacologic response to a combination of poly (ADP-ribose) polymerase (PARP) inhibitor and temozolomide, and the signature outperformed four published gene signatures of BRCA1/2 deficiency.ConclusionsA BD-L signature is enriched in HER2+ breast cancer brain metastases without pathogenic BRCA1 mutations. Unexpectedly, elevated BD-L values are found in a subset of primary tumors across all breast cancer subtypes. Evaluation of pharmacological sensitivity in breast cancer cell lines representing all breast cancer subtypes suggests the BD-L signature may serve as a biomarker to identify sporadic breast cancer patients who might benefit from a therapeutic combination of PARP inhibitor and temozolomide and may be indicative of a dysfunctional BRCA1-associated pathway.

Genetic instability, a hallmark feature of human cancers including prostatic adenocarcinomas, is considered a driver of metastasis. Somatic copy number alterations are found in most aggressive primary human prostate cancers, and the overall number of such changes is increased in metastases. Chromosome 10q23 deletions, encompassing PTEN, and amplification of 8q24, harboring MYC, are frequently observed, and the presence of both together portends a high risk of prostate cancer-specific mortality. In extant genetically engineered mouse prostate cancer models (GEMMs), isolated MYC overexpression or targeted Pten loss can each produce early prostate adenocarcinomas, but are not sufficient to induce genetic instability or metastases with high penetrance. While a previous study showed that combining Pten loss with focal MYC overexpression in a small fraction of prostatic epithelial cells exhibits cooperativity in GEMMs, additional targeted Tp53 disruption was required for formation of metastases. We hypothesized that driving combined MYC overexpression and Pten loss using recently characterized Hoxb13 transcriptional control elements that are active in prostate luminal epithelial cells would induce the development of genomic instability and aggressive disease with metastatic potential. Neoplastic lesions that developed with either MYC activation alone (Hoxb13-MYC) or Pten loss alone (Hoxb13-Cre|PtenFl/Fl) failed to progress beyond PIN and did not harbor genomic copy number alterations. By contrast, mice with both alterations (Hoxb13-MYC|Hoxb13-Cre|PtenFl/Fl or BMPC) developed lethal adenocarcinoma with distant metastases and widespread genome copy number alterations that were independent of forced disruption of Tp53 and telomere shortening. BMPC cancers lacked neuroendocrine or sarcomatoid differentiation, features uncommon in human disease but common in other models of prostate cancer that metastasize. These data show that combined MYC activation and Pten loss driven by the Hoxb13 regulatory locus synergize to induce genomic instability and aggressive prostate cancer that phenocopies the human disease at the histological and genomic levels.

BACKGROUNDWhole genome amplification (WGA) is required for single cell genotyping. Effectiveness of currently available WGA technologies in combination with next generation sequencing (NGS) and material preservation is still elusive.RESULTSIn respect to the accuracy of SNP/mutation, indel, and copy number aberrations (CNA) calling, the HiSeq2000 platform outperformed IonProton in all aspects. Furthermore, more accurate SNP/mutation and indel calling was demonstrated using single tumor cells obtained from EDTA-collected blood in respect to CellSave-preserved blood, whereas CNA analysis in our study was not detectably affected by fixation. Although MDA-based WGA yielded the highest DNA amount, DNA quality was not adequate for downstream analysis. PCR-based WGA demonstrates superiority over MDA-PCR combining technique for SNP and indel analysis in single cells. However, SNP calling performance of MDA-PCR WGA improves with increasing amount of input DNA, whereas CNA analysis does not. The performance of PCR-based WGA did not significantly improve with increase of input material. CNA profiles of single cells, amplified with MDA-PCR technique and sequenced on both HiSeq2000 and IonProton platforms, resembled unamplified DNA the most.MATERIALS AND METHODSWe analyzed the performance of PCR-based, multiple-displacement amplification (MDA)-based, and MDA-PCR combining WGA techniques (WGA kits Ampli1, REPLI-g, and PicoPlex, respectively) on single and pooled tumor cells obtained from EDTA- and CellSave-preserved blood and archival material. Amplified DNA underwent exome-Seq with the Illumina HiSeq2000 and ThermoFisher IonProton platforms.CONCLUSIONWe demonstrate the feasibility of single cell genotyping of differently preserved material, nevertheless, WGA and NGS approaches have to be chosen carefully depending on the study aims.

The prostate requires androgens for development and homeostasis. Prostate cancer shares this dependence, however progression to androgen-independence is common after androgen deprivation. There is considerable interest in achieving therapeutic gene expression after androgen ablation using prostate-specific promoters. Paradoxically, known prostate-restricted cis-regulatory elements are androgen dependent. Hoxb13 expression is restricted in adults to the prostate and colon, and robust Hoxb13 expression persists after castration. To locate regulatory elements conferring this expression pattern, a lacZ reporter was inserted into the Hoxb13 locus on a mouse genomic bacterial artificial chromosome. In transgenic mice, this construct recapitulated the Hoxb13 expression pattern, including expression after castration. Reporter gene activity was maintained during carcinogenesis in a prostate cancer model. Hoxb13 cis-regulatory elements provide a powerful tool to achieve androgen-independent transgene expression in the prostate and distal colon-specific expression in the gastrointestinal tract. These data establish a framework for high-resolution analyses of factors regulating Hoxb13.

Peng and A. Molina were employees of Janssen Research & Development at the time of this analysis.Author contributions: Eleni Efstathiou had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Efstathiou, Logothetis.

Purpose To identify the molecular signature associated with abiraterone acetate (AA) response and mechanisms underlying AA resistance in castration-resistant prostate cancer patient-derived xenografts (PDXs). Experimental Design SCID mice bearing LuCaP 136CR, 77CR, 96CR, and 35CR PDXs were treated with AA. Tumor volume and prostate-specific antigen were monitored, and tumors were harvested 7 days post-treatment or at end of study for gene expression and immunohistochemical studies. Results Three phenotypic groups were observed based on AA response. An ultraresponsive phenotype was identified in LuCaP 136CR with significant inhibition of tumor progression and increased survival, intermediate responders LuCaP 77CR and LuCaP 96CR with a modest tumor inhibition and survival benefit, and LuCaP 35CR with minimal tumor inhibition and no survival benefit upon AA treatment. We identified a molecular signature of secreted proteins associated with the AA ultraresponsive phenotype. Upon resistance, AA ultraresponder LuCaP 136CR displayed reduced androgen receptor (AR) signaling and sustainably low nuclear glucocorticoid receptor (nGR) localization, accompanied by steroid metabolism alteration and epithelial-mesenchymal transition phenotype enrichment with increased expression of NF-κB-regulated genes; intermediate and minimal responders maintained sustained AR signaling and increased tumoral nGR localization. Conclusions We identified a molecular signature of secreted proteins associated with AA ultraresponsiveness and sustained AR/GR signaling upon AA resistance in intermediate or minimal responders. These data will inform development of noninvasive biomarkers predicting AA response, and suggest further inhibition along the AR/GR signaling axis may be effective only in AA-resistant patients who are intermediate or minimal responders. These findings require verification in prospective clinical trials.

Hoxb13 is robustly transcribed in derivatives of posterior endoderm including the colon, rectum, and the prostate gland. Transcriptional activity in the prostate persists unabated under conditions of androgen deprivation and throughout the course of disease progression in a mouse prostate cancer model. To elucidate the molecular basis of prostate-restricted transcriptional activation of Hoxb13, a bacterial artificial chromosome (BAC)-based reporter gene deletion analysis was performed in transgenic mice. Two regions downstream of the Hoxb13 coding region were found to be required to support transcriptional activity in the prostate but were completely dispensable for expression in the colon and rectum. Bioinformatic analyses of one region identified a 37-bp element conserved in mammals. This element, which bears two potential binding sites for Forkhead class transcription factors, is occupied by FOXA1 in a human prostate cancer cell line. Precise replacement of this enhancer with an extended LoxP site in the context of a 218,555-bp BAC reporter nearly extinguished Hoxb13-mediated transcriptional activity in the mouse prostate. These data demonstrate that FOXA1 directly regulates HOXB13 in human prostate epithelial cells, and show that this prostate-specific regulatory mechanism is conserved in mice.androgen independent | recombineering | prostate-specific | Homeobox | Forkhead

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