Systematic drug screening reveals specific vulnerabilities and co-resistance patterns in endocrine-resistant breast cancer.

BACKGROUND: The estrogen receptor (ER) inhibitor tamoxifen reduces breast cancer mortality by 31 % and has served as the standard treatment for ER-positive breast cancers for decades. However, 50 % of advanced ER-positive cancers display de novo resistance to tamoxifen, and acquired resistance evolves in 40 % of patients who initially respond. Mechanisms underlying resistance development remain poorly understood and new therapeutic opportunities are urgently needed. Here, we report the generation and characterization of seven tamoxifen-resistant breast cancer cell lines from four parental strains. METHODS: Using high throughput drug sensitivity and resistance testing (DSRT) with 279 approved and investigational oncology drugs, exome-sequencing and network analysis, we for the first time, systematically determine the drug response profiles specific to tamoxifen resistance. RESULTS: We discovered emerging vulnerabilities towards specific drugs, such as ERK1/2-, proteasome- and BCL-family inhibitors as the cells became tamoxifen-resistant. Co-resistance to other drugs such as the survivin inhibitor YM155 and the chemotherapeutic agent paclitaxel also occurred. CONCLUSION: This study indicates that multiple molecular mechanisms dictate endocrine resistance, resulting in unexpected vulnerabilities to initially ineffective drugs, as well as in emerging co-resistances. Thus, combatting drug-resistant tumors will require patient-tailored strategies in order to identify new drug vulnerabilities, and to understand the associated co-resistance patterns.

Somatic STAT3 mutations in large granular lymphocytic leukemia.

BACKGROUND: T-cell large granular lymphocytic leukemia is a rare lymphoproliferative disorder characterized by the expansion of clonal CD3+CD8+ cytotoxic T lymphocytes (CTLs) and often associated with autoimmune disorders and immune-mediated cytopenias. METHODS: We used next-generation exome sequencing to identify somatic mutations in CTLs from an index patient with large granular lymphocytic leukemia. Targeted resequencing was performed in a well-characterized cohort of 76 patients with this disorder, characterized by clonal T-cell-receptor rearrangements and increased numbers of large granular lymphocytes. RESULTS: Mutations in the signal transducer and activator of transcription 3 gene (STAT3) were found in 31 of 77 patients (40%) with large granular lymphocytic leukemia. Among these 31 patients, recurrent mutational hot spots included Y640F in 13 (17%), D661V in 7 (9%), D661Y in 7 (9%), and N647I in 3 (4%). All mutations were located in exon 21, encoding the Src homology 2 (SH2) domain, which mediates the dimerization and activation of STAT protein. The amino acid changes resulted in a more hydrophobic protein surface and were associated with phosphorylation of STAT3 and its localization in the nucleus. In vitro functional studies showed that the Y640F and D661V mutations increased the transcriptional activity of STAT3. In the affected patients, downstream target genes of the STAT3 pathway (IFNGR2, BCL2L1, and JAK2) were up-regulated. Patients with STAT3 mutations presented more often with neutropenia and rheumatoid arthritis than did patients without these mutations. CONCLUSIONS: The SH2 dimerization and activation domain of STAT3 is frequently mutated in patients with large granular lymphocytic leukemia; these findings suggest that aberrant STAT3 signaling underlies the pathogenesis of this disease. (Funded by the Academy of Finland and others.).

Novel TBL1XR1, EPHA7 and SLFN12 mutations in a Sezary syndrome patient discovered by whole exome sequencing.

Sezary syndrome (SS) is an aggressive leukaemic variant of cutaneous T-cell lymphoma. Recurrent chromosomal aberrations have been found in SS, but the whole genetic mutation spectrum is unknown. To better understand the molecular pathogenesis of SS, we performed exome sequencing, copy number variation (CNV) and gene expression analysis of primary SS cells. In our index patient with typical SS, we found novel somatic missense mutations in TBL1XR1, EPHA7 and SLFN12 genes in addition to larger chromosomal changes. The mutations are located in biologically relevant genes affecting apoptosis and T-cell maturation. They may play a role in the pathobiology of the disease, but no recurrent mutations were discovered in nine additional patients with SS studied. Thus, screening of larger patient cohorts is needed to confirm their prevalence and biological significance in SS.

Integrated breast cancer genomics.

Predicting survival and therapy responses of breast cancer patients is a significant challenge. Two studies in this issue of Cancer Cell present a novel integrated analysis of genomic and transcriptomic profiles of 145 primary breast cancers and 51 established cell lines. Data from clinical tumors highlighted mechanisms of disease and facilitated identification of potential therapeutic targets and prognostic biomarkers. An extensive well-characterized cancer cell line resource opens up opportunities to explore the determinants of cellular responses to existing and emerging therapies. Taken together, these studies illustrate how integrated molecular profiling may one day significantly impact diagnosis and therapeutic choice in human breast cancer.

Aneuploidy facilitates oncogenic transformation via specific genetic alterations, including Twist2 upregulation.

Aneuploidy, deviation from the normal chromosome number, and other chromosomal aberrations are commonly observed in cancer. Integrin-mediated adhesion and dynamic turnover of adhesion sites are required for successful cytokinesis of normal adherent cells and impaired cell division can lead to the generation of cells with abnormal chromosome contents. We find that repeated cytokinesis failure, due to impaired integrin traffic alone, is sufficient to induce chromosome aberrations resulting in the generation of aneuploid cells with malignant properties. Here, we have compared isogenic aneuploid and euploid cell lines with unravel aneuploidy-induced changes in cellular signaling. Euploid, non-transformed, and aneuploid, transformed, cell lines were investigated using genome-wide gene expression profiling, analysis of deregulated biological pathways and array-comparative genomic hybridization. We find that aneuploidy drives malignancy via inducing marked changes in gene and micro RNA expression profiles and thus imposing specific growth and survival promoting alterations in cellular signaling. Importantly, we identify Twist2 as a key regulator of survival, invasion and anchorage-independent growth in the aneuploid cells. In addition, alterations in lipid biosynthetic pathways and miR-10b upregulation are likely contributors to the malignant phenotype.

Neurocan expression associates with better survival and viral positivity in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine carcinoma that is frequently divided into Merkel cell polyomavirus negative and positive tumors due their distinct genomic and transcriptomic profiles, and disease outcomes. Although some prognostic factors in MCC are known, tumorigenic pathways, which that explain outcome differences in MCC are not fully understood. We investigated transcriptomes of 110 tissue samples of a formalin-fixed, paraffin-embedded MCC series by RNA sequencing to identify genes showing a bimodal expression pattern and predicting outcome in cancer and that potentially could play a role in tumorigenesis. We discovered 19 genes among which IGHM, IGKC, NCAN, OTOF, and USH2A were associated also with overall survival (all p-values < 0.05). From these genes, NCAN (neurocan) expression was detected in all 144 MCC samples by immunohistochemistry. Increased NCAN expression was associated with presence of Merkel cell polyomavirus DNA (p = 0.001) and viral large T antigen expression in tumor tissue (p = 0.004) and with improved MCC-specific survival (p = 0.027) and overall survival (p = 0.034). We conclude that NCAN expression is common in MCC, and further studies are warranted to investigate its role in MCC tumorigenesis.

Integrative genomic, transcriptomic, and RNAi analysis indicates a potential oncogenic role for FAM110B in castration-resistant prostate cancer.

BACKGROUND: Castration-resistant prostate cancer (CRPC) represents a therapeutic challenge for current medications. METHODS: In order to explore the molecular mechanisms involved in CRPC progression and to identify new therapeutic targets, we analyzed a unique sample set of 11 CRPCs and 7 advanced tumors by array-CGH and gene expression microarrays. The genome-wide DNA and RNA data were integrated to identify genes whose overexpression was driven by their amplification. To assess the functional role of these genes, their expression was analyzed in a transcriptional data set of 329 clinical prostate cancers and the corresponding gene products were silenced using RNA interference in prostate cancer cells. RESULTS: Six recurrent genetic targets were identified in the CRPCs; ATP1B1, AR, FAM110B, LAS1L, MYC, and YIPF6. In addition to AR and MYC, FAM110B emerged as a potential key gene involved in CRPC progression in a subset of the tumors. FAM110B was able to regulate AR signaling in prostate cancer cells and FAM110B itself was regulated by androgens. FAM110B siRNA inhibited the growth of prostate cancer cells in vitro, and this effect was substantially enhanced in androgen deficient conditions. Ectopic FAM110B expression in non-cancerous epithelial prostate cells induced aneuploidy and impaired antigen presentation. CONCLUSIONS: The DNA/RNA gene outlier detection combined with siRNA cell proliferation assay identified FAM110B as a potential growth promoting key gene for CRPC. FAM110B appears to have a key role in the androgen signaling and progression of CRPC impacting multiple cancer hallmarks and therefore highlighting a potential therapeutic target.

Enhanced serine production by bone metastatic breast cancer cells stimulates osteoclastogenesis.

Since bone metastatic breast cancer is an incurable disease, causing significant morbidity and mortality, an understanding of the underlying molecular mechanisms would be highly valuable. Here, we describe in vitro and in vivo evidences for the importance of serine biosynthesis in the metastasis of breast cancer to bone. We first characterized the bone metastatic propensity of the MDA-MB-231(SA) cell line variant as compared to the parental MDA-MB-231 cells by radiographic and histological observations in the inoculated mice. Genome-wide gene expression profiling of this isogenic cell line pair revealed that all the three genes involved in the L: -serine biosynthesis pathway, phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase 1 (PSAT1), and phosphoserine phosphatase (PSPH) were upregulated in the highly metastatic variant. This pathway is the primary endogenous source for L: -serine in mammalian tissues. Consistently, we observed that the proliferation of MDA-MB-231(SA) cells in serine-free conditions was dependent on PSAT1 expression. In addition, we observed that L: -serine is essential for the formation of bone resorbing human osteoclasts and may thus contribute to the vicious cycle of osteolytic bone metastasis. High expression of PHGDH and PSAT1 in primary breast cancer was significantly associated with decreased relapse-free and overall survival of patients and malignant phenotypic features of breast cancer. In conclusion, high expression of serine biosynthesis genes in metastatic breast cancer cells and the stimulating effect of L: -serine on osteoclastogenesis and cancer cell proliferation indicate a functionally critical role for serine biosynthesis in bone metastatic breast cancer and thereby an opportunity for targeted therapeutic interventions.

Interactions between Notch- and hypoxia-induced transcriptomes in embryonic stem cells.

Interaction between key signaling mechanisms is important to generate the diversity in signaling output required for proper control of cellular differentiation and function, although the molecular manifestations of such cross-talk are only partially understood. Notch signaling and the cellular response to hypoxia intersect at different points in the signaling cascades, and in this report we analyze the consequences of this cross-talk at the transcriptome level. Mouse ES cells were subjected to various combinations of hypoxia and/or activated Notch signaling, and the transcriptome changes could be grouped into different categories, reflecting various modes of hypoxia and Notch signaling integration. Two principal categories of novel Notch- and hypoxia-induced genes were identified: (i) a larger set of Notch or hypoxic target genes which were induced by one pathway and not significantly affected by the activity status of the other pathway and (ii) a smaller set of genes co-regulated by Notch and hypoxia. In the latter category, we identified genes that were induced by hypoxia and the expression of which was enhanced by active Notch signaling and another group of genes that were induced by Notch and hypoxia independently. Several of the hypoxia- and Notch-induced genes were found to be upregulated in various forms of cancer. Identification of genes co-regulated by the two pathways may provide a molecular platform to better understand the intersection between the two signaling cascades in normal development and cancer.

Use of cancer-specific genomic rearrangements to quantify disease burden in plasma from patients with solid tumors.

Detection of recurrent somatic rearrangements routinely allows monitoring of residual disease burden in leukemias, but is not used for most solid tumors. However, next-generation sequencing now allows rapid identification of patient-specific rearrangements in solid tumors. We mapped genomic rearrangements in three cancers and showed that PCR assays for rearrangements could detect a single copy of the tumor genome in plasma without false positives. Disease status, drug responsiveness, and incipient relapse could be serially assessed. In future, this strategy could be readily established in diagnostic laboratories, with major impact on monitoring of disease status and personalizing treatment of solid tumors.

Array-based gene expression, CGH and tissue data defines a 12q24 gain in neuroblastic tumors with prognostic implication.

BACKGROUND: Neuroblastoma has successfully served as a model system for the identification of neuroectoderm-derived oncogenes. However, in spite of various efforts, only a few clinically useful prognostic markers have been found. Here, we present a framework, which integrates DNA, RNA and tissue data to identify and prioritize genetic events that represent clinically relevant new therapeutic targets and prognostic biomarkers for neuroblastoma. METHODS: A single-gene resolution aCGH profiling was integrated with microarray-based gene expression profiling data to distinguish genetic copy number alterations that were strongly associated with transcriptional changes in two neuroblastoma cell lines. FISH analysis using a hotspot tumor tissue microarray of 37 paraffin-embedded neuroblastoma samples and in silico data mining for gene expression information obtained from previously published studies including up to 445 healthy nervous system samples and 123 neuroblastoma samples were used to evaluate the clinical significance and transcriptional consequences of the detected alterations and to identify subsequently activated gene(s). RESULTS: In addition to the anticipated high-level amplification and subsequent overexpression of MYCN, MEIS1, CDK4 and MDM2 oncogenes, the aCGH analysis revealed numerous other genetic alterations, including microamplifications at 2p and 12q24.11. Most interestingly, we identified and investigated the clinical relevance of a previously poorly characterized amplicon at 12q24.31. FISH analysis showed low-level gain of 12q24.31 in 14 of 33 (42%) neuroblastomas. Patients with the low-level gain had an intermediate prognosis in comparison to patients with MYCN amplification (poor prognosis) and to those with no MYCN amplification or 12q24.31 gain (good prognosis) (P = 0.001). Using the in silico data mining approach, we identified elevated expression of five genes located at the 12q24.31 amplicon in neuroblastoma (DIABLO, ZCCHC8, RSRC2, KNTC1 and MPHOSPH9). Among these, DIABLO showed the strongest activation suggesting a putative role in neuroblastoma progression. CONCLUSIONS: The presented systematic and rapid framework, which integrates aCGH, gene expression and tissue data to obtain novel targets and biomarkers for cancer, identified a low-level gain of the 12q24.31 as a potential new biomarker for neuroblastoma progression. Furthermore, results of in silico data mining suggest a new neuroblastoma target gene, DIABLO, within this region, whose functional and therapeutic role remains to be elucidated in follow-up studies.

Array comparative genomic hybridization identifies a distinct DNA copy number profile in renal cell cancer associated with hereditary leiomyomatosis and renal cell cancer.

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a tumor predisposition syndrome with cutaneous and uterine leiomyomatosis as well as renal cell cancer (RCC) as its clinical manifestations. HLRCC is caused by heterozygous germline mutations in the fumarate hydratase (fumarase) gene. In this study, we used array comparative genomic hybridization to identify the specific copy number changes characterizing the HLRCC-associated RCCs. The study material comprised formalin-fixed paraffin-embedded renal tumors obtained from Finnish patients with HLRCC. All 11 investigated tumors displayed the papillary type 2 histopathology typical for HLRCC renal tumors. The most frequent copy number changes detected in at least 3/11 (27%) of the tumors were gains in chromosomes 2, 7, and 17, and losses in 13q12.3-q21.1, 14, 18, and X. These findings provide genetic evidence for a distinct copy number profile in HLRCC renal tumors compared with sporadic RCC tumors of the same histopathological subtype, and delineate chromosomal regions that associate with this very aggressive form of RCC.

TMPRSS2 fusions with oncogenic ETS factors in prostate cancer involve unbalanced genomic rearrangements and are associated with HDAC1 and epigenetic reprogramming.

Translocations fusing the strong androgen-responsive gene, TMPRSS2, with ERG or other oncogenic ETS factors may facilitate prostate cancer development. Here, we studied 18 advanced prostate cancers for ETS factor alterations, using reverse transcription-PCR and DNA and RNA array technologies, and identified putative ERG downstream gene targets from the microarray data of 410 prostate samples. Out of the 27 ETS factors, ERG was most frequently overexpressed. Seven cases showed TMPRSS2:ERG gene fusions, whereas the TMPRSS2:ETV4 fusion was seen in one case. In five out of six tumors with high ERG expression, array-CGH analysis revealed interstitial 2.8 Mb deletions between the TMPRSS2 and ERG loci, or smaller, unbalanced rearrangements. In silico analysis of the ERG gene coexpression patterns revealed an association with high expression of the histone deacetylase 1 gene, and low expression of its target genes. Furthermore, we observed increased expression of WNT-associated pathways and down-regulation of tumor necrosis factor and cell death pathways. In summary, our data indicate that the TMPRSS2:ERG translocation is common in advanced prostate cancer and occurs by virtue of unbalanced genomic rearrangements. Activation of ERG by fusion with TMPRSS2 may lead to epigenetic reprogramming, WNT signaling, and down-regulation of cell death pathways, implicating ERG in several hallmarks of cancer with potential therapeutic importance.

Chimeric NUP98-NSD1 transcripts from the cryptic t(5;11)(q35.2;p15.4) in adult de novo acute myeloid leukemia.

The t(5;11)(q35;p15.4) is a clinically significant marker of poor prognosis in acute myeloid leukemia (AML), which is difficult to detect due to sub-telomeric localization of the breakpoints. To facilitate the detection of this rearrangement, we studied NUP98-NSD1 transcript variants in patients with the t(5;11) using paired-end RNA sequencing and standard molecular biology techniques. We discovered three NUP98-NSD1 transcripts with two fusion junctions (NUP98 exon 11-12/NSD1 exon 6), alternative 5' donor site in NUP98 exon 7, and NSD1 exon 7 skipping. Two of the transcripts were in-frame and occurred in all t(5;11) samples (N = 5). The exonic splicing events were present in all samples (N = 23) regardless of the NUP98-NSD1 suggesting that these novel splice events are unassociated with t(5;11). In conclusion, we provide evidence of two different NUP98-NSD1 fusion transcripts in adult AML, which result in functional proteins and represent suitable molecular entities for monitoring t(5;11) AML patients.

RET fusions observed in lung and colorectal cancers are sensitive to ponatinib.

Genomic studies are revolutionizing clinical oncology, but bridging the lab and the bedside requires the ability to efficiently interrogate rare genetic lesions in unexpected pathological settings using preclinical models. Oncogenes can exhibit intrinsic drug resistance to targeted therapy in different cells of origin, adding complexity to clinical interpretations of genomic findings. Here, we capitalize on the flexibility of engineered cell systems to rapidly profile known multi-kinase inhibitors that harbor rearranged during transfection (RET) kinase activity across multiple RET fusions. Identifying ponatinib as the most potent RET inhibitor tested, we used ponatinib to gauge therapeutic responsiveness in RET fusion-positive patient-derived xenograft (PDX) models. Using a genomics guided outlier approach, we identified 4 RET fusion PDX models with 3 different fusion partners (KIF5B, CCDC6, and NCOA4) in both non-small cell lung cancer and colorectal cancer. By comparing ponatinib activity in RET fusion-positive and RET fusion-negative PDX models alongside a standard of care chemotherapeutic agent, we show that RET fusions in colorectal tumors are therapeutically responsive to RET inhibition. Finally, we suggest that coupling engineered cell systems and genomics guided PDX model selection provides a rapid workflow to triage rare genomics findings.

Response to ERBB3-Directed Targeted Therapy in NRG1-Rearranged Cancers.

NRG1 rearrangements are oncogenic drivers that are enriched in invasive mucinous adenocarcinomas (IMA) of the lung. The oncoprotein binds ERBB3-ERBB2 heterodimers and activates downstream signaling, supporting a therapeutic paradigm of ERBB3/ERBB2 inhibition. As proof of concept, a durable response was achieved with anti-ERBB3 mAb therapy (GSK2849330) in an exceptional responder with an NRG1-rearranged IMA on a phase I trial (NCT01966445). In contrast, response was not achieved with anti-ERBB2 therapy (afatinib) in four patients with NRG1-rearranged IMA (including the index patient post-GSK2849330). Although in vitro data supported the use of either ERBB3 or ERBB2 inhibition, these clinical results were consistent with more profound antitumor activity and downstream signaling inhibition with anti-ERBB3 versus anti-ERBB2 therapy in an NRG1-rearranged patient-derived xenograft model. Analysis of 8,984 and 17,485 tumors in The Cancer Genome Atlas and MSK-IMPACT datasets, respectively, identified NRG1 rearrangements with novel fusion partners in multiple histologies, including breast, head and neck, renal, lung, ovarian, pancreatic, prostate, and uterine cancers.Significance: This series highlights the utility of ERBB3 inhibition as a novel treatment paradigm for NRG1-rearranged cancers. In addition, it provides preliminary evidence that ERBB3 inhibition may be more optimal than ERBB2 inhibition. The identification of NRG1 rearrangements across various solid tumors supports a basket trial approach to drug development. Cancer Discov; 8(6); 686-95. ©2018 AACR.See related commentary by Wilson and Politi, p. 676This article is highlighted in the In This Issue feature, p. 663.

A new look towards BAC-based array CGH through a comprehensive comparison with oligo-based array CGH.

BACKGROUND: Currently, two main technologies are used for screening of DNA copy number; the BAC (Bacterial Artificial Chromosome) and the recently developed oligonucleotide-based CGH (Chromosomal Comparative Genomic Hybridization) arrays which are capable of detecting small genomic regions with amplification or deletion. The correlation as well as the discriminative power of these platforms has never been compared statistically on a significant set of human patient samples. RESULTS: In this paper, we present an exhaustive comparison between the two CGH platforms, undertaken at two independent sites using the same batch of DNA from 19 advanced prostate cancers. The comparison was performed directly on the raw data and a significant correlation was found between the two platforms. The correlation was greatly improved when the data were averaged over large chromosomic regions using a segmentation algorithm. In addition, this analysis has enabled the development of a statistical model to discriminate BAC outliers that might indicate microevents. These microevents were validated by the oligo platform results. CONCLUSION: This article presents a genome-wide statistical validation of the oligo array platform on a large set of patient samples and demonstrates statistically its superiority over the BAC platform for the Identification of chromosomic events. Taking advantage of a large set of human samples treated by the two technologies, a statistical model has been developed to show that the BAC platform could also detect microevents.

Drug-screening and genomic analyses of HER2-positive breast cancer cell lines reveal predictors for treatment response.

BACKGROUND: Approximately 15%-20% of all diagnosed breast cancers are characterized by amplified and overexpressed HER2 (= ErbB2). These breast cancers are aggressive and have a poor prognosis. Although improvements in treatment have been achieved after the introduction of trastuzumab and lapatinib, many patients do not benefit from these drugs. Therefore, in-depth understanding of the mechanisms behind the treatment responses is essential to find alternative therapeutic strategies. MATERIALS AND METHODS: Thirteen HER2 positive breast cancer cell lines were screened with 22 commercially available compounds, mainly targeting proteins in the ErbB2-signaling pathway, and molecular mechanisms related to treatment sensitivity were sought. Cell viability was measured, and treatment responses between the cell lines were compared. To search for response predictors and genomic and transcriptomic profiling, PIK3CA mutations and PTEN status were explored and molecular features associated with drug sensitivity sought. RESULTS: The cell lines were divided into three groups according to the growth-retarding effect induced by trastuzumab and lapatinib. Interestingly, two cell lines insensitive to trastuzumab (KPL4 and SUM190PT) showed sensitivity to an Akt1/2 kinase inhibitor. These cell lines had mutation in PIK3CA and loss of PTEN, suggesting an activated and druggable Akt-signaling pathway. Expression levels of five genes (CDC42, MAPK8, PLCG1, PTK6, and PAK6) were suggested as predictors for the Akt1/2 kinase-inhibitor response. CONCLUSION: Targeting the Akt-signaling pathway shows promise in cell lines that do not respond to trastuzumab. In addition, our results indicate that several molecular features determine the growth-retarding effects induced by the drugs, suggesting that parameters other than HER2 amplification/expression should be included as markers for therapy decisions.

NMD microarray analysis for rapid genome-wide screen of mutated genes in cancer.

Gene mutations play a critical role in cancer development and progression, and their identification offers possibilities for accurate diagnostics and therapeutic targeting. Finding genes undergoing mutations is challenging and slow, even in the post-genomic era. A new approach was recently developed by Noensie and Dietz to prioritize and focus the search, making use of nonsense-mediated mRNA decay (NMD) inhibition and microarray analysis (NMD microarrays) in the identification of transcripts containing nonsense mutations. We combined NMD microarrays with array-based CGH (comparative genomic hybridization) in order to identify inactivation of tumor suppressor genes in cancer. Such a "mutatomics" screening of prostate cancer cell lines led to the identification of inactivating mutations in the EPHB2 gene. Up to 8% of metastatic uncultured prostate cancers also showed mutations of this gene whose loss of function may confer loss of tissue architecture. NMD microarray analysis could turn out to be a powerful research method to identify novel mutated genes in cancer cell lines, providing targets that could then be further investigated for their clinical relevance and therapeutic potential.

Suppression of early hematogenous dissemination of human breast cancer cells to bone marrow by retinoic Acid-induced 2.

UNLABELLED: Regulatory pathways that drive early hematogenous dissemination of tumor cells are insufficiently defined. Here, we used the presence of disseminated tumor cells (DTC) in the bone marrow to define patients with early disseminated breast cancer and identified low retinoic acid-induced 2 (RAI2) expression to be significantly associated with DTC status. Low RAI2 expression was also shown to be an independent poor prognostic factor in 10 different cancer datasets. Depletion of RAI2 protein in luminal breast cancer cell lines resulted in dedifferentiation marked by downregulation of ERα, FOXA1, and GATA3, together with increased invasiveness and activation of AKT signaling. Functional analysis of the previously uncharacterized RAI2 protein revealed molecular interaction with CtBP transcriptional regulators and an overlapping function in controlling the expression of a number of key target genes involved in breast cancer. These results suggest that RAI2 is a new metastasis-associated protein that sustains differentiation of luminal breast epithelial cells. SIGNIFICANCE: We identified downregulation of RAI2 as a novel metastasis-associated genetic alteration especially associated with early occurring bone metastasis in ERα-positive breast tumors. We specified the role of the RAI2 protein to function as a transcriptional regulator that controls the expression of several key regulators of breast epithelial integrity and cancer.