Assessment of targeted therapy opportunities in sinonasal cancers using patient-derived functional tumor models.

Malignant tumors derived from the epithelium lining the nasal cavity region are termed sinonasal cancers, a highly heterogeneous group of rare tumors accounting for 3 - 5 % of all head and neck cancers. Progress with next-generation molecular profiling has improved our understanding of the complexity of sinonasal cancers and resulted in the identification of an increasing number of distinct tumor entities. Despite these significant developments, the treatment of sinonasal cancers has hardly evolved since the 1980s, and an advanced sinonasal cancer presents a poor prognosis as targeted therapies are usually not available. To gain insights into potential targeted therapeutic opportunities, we performed a multiomics profiling of patient-derived functional tumor models to identify molecular characteristics associated with pharmacological responses in the different subtypes of sinonasal cancer. METHODS: Patient-derived ex vivo tumor models representing four distinct sinonasal cancer subtypes: sinonasal intestinal-type adenocarcinoma, sinonasal neuroendocrine carcinoma, sinonasal undifferentiated carcinoma and SMARCB1 deficient sinonasal carcinoma were included in the analyses. Results of functional drug screens of 160 anti-cancer therapies were integrated with gene panel sequencing and histological analyses of the tumor tissues and the ex vivo cell cultures to establish associations between drug sensitivity and molecular characteristics including driver mutations. RESULTS: The different sinonasal cancer subtypes display considerable differential drug sensitivity. Underlying the drug sensitivity profiles, each subtype was associated with unique molecular features. The therapeutic vulnerabilities correlating with specific genomic background were extended and validated with in silico analyses of cancer cell lines representing different human cancers and with reported case studies of sinonasal cancers treated with targeted therapies. CONCLUSION: The results demonstrate the importance of understanding the differential biology and the molecular features associated with the different subtypes of sinonasal cancers. Patient-derived ex vivo tumor models can be a powerful tool for investigating these rare cancers and prioritizing targeted therapeutic strategies for future clinical development and personalized medicine.

In-vitro assays for immuno-oncology drug efficacy assessment and screening for personalized cancer therapy: scopes and challenges.

INTRODUCTION: Immunotherapies have revolutionized cancer treatment, but often fail to produce desirable therapeutic outcomes in all patients. Due to the inter-patient heterogeneity and complexity of the tumor microenvironment, personalized treatment approaches are gaining demand. Researchers have long been using a range of in-vitro assays including 2D models, organoid co-cultures, and cancer-on-a-chip platforms for cancer drug screening. A comparative analysis of these assays with their suitability, high-throughput capacity, and clinical translatability is required for optimal translational use. AREAS COVERED: The review summarized in-vitro platforms with their comparative advantages and limitations including construction strategies, and translational potential for immuno-oncology drug efficacy assessment. We also discussed end-point analysis strategies so that researchers can contextualize their usefulness and optimally design experiments for personalized immunotherapy efficacy prediction. EXPERT OPINION: Researchers developed several in-vitro platforms that can provide information on personalized immunotherapy efficacy from different angles. Image-based assays are undoubtedly more suitable to gather a wide range of information including cellular morphology and phenotypical behaviors but need significant improvement to overcome issues including background noise, sample preparation difficulty, and long duration of experiment. More studies and clinical trials are needed to resolve these issues and validate the assays before they can be used in real-life scenarios.

Defined extracellular matrix compositions support stiffness-insensitive cell spreading and adhesion signaling.

Integrin-dependent adhesion to the extracellular matrix (ECM) mediates mechanosensing and signaling in response to altered microenvironmental conditions. In order to provide tissue- and organ-specific cues, the ECM is composed of many different proteins that temper the mechanical properties and provide the necessary structural diversity. Despite most human tissues being soft, the prevailing view from predominantly in vitro studies is that increased stiffness triggers effective cell spreading and activation of mechanosensitive signaling pathways. To address the functional coupling of ECM composition and matrix rigidity on compliant substrates, we developed a matrix spot array system to screen cell phenotypes against different ECM mixtures on defined substrate stiffnesses at high resolution. We applied this system to both cancer and normal cells and surprisingly identified ECM mixtures that support stiffness-insensitive cell spreading on soft substrates. Employing the motor-clutch model to simulate cell adhesion on biochemically distinct soft substrates, with varying numbers of available ECM-integrin-cytoskeleton (clutch) connections, we identified conditions in which spreading would be supported on soft matrices. Combining simulations and experiments, we show that cell spreading on soft is supported by increased clutch engagement on specific ECM mixtures and even augmented by the partial inhibition of actomyosin contractility. Thus, "stiff-like" spreading on soft is determined by a balance of a cell's contractile and adhesive machinery. This provides a fundamental perspective for in vitro mechanobiology studies, identifying a mechanism through which cells spread, function, and signal effectively on soft substrates.

FBXL12 degrades FANCD2 to regulate replication recovery and promote cancer cell survival under conditions of replication stress.

Fanconi anemia (FA) signaling, a key genomic maintenance pathway, is activated in response to replication stress. Here, we report that phosphorylation of the pivotal pathway protein FANCD2 by CHK1 triggers its FBXL12-dependent proteasomal degradation, facilitating FANCD2 clearance at stalled replication forks. This promotes efficient DNA replication under conditions of CYCLIN E- and drug-induced replication stress. Reconstituting FANCD2-deficient fibroblasts with phosphodegron mutants failed to re-establish fork progression. In the absence of FBXL12, FANCD2 becomes trapped on chromatin, leading to replication stress and excessive DNA damage. In human cancers, FBXL12, CYCLIN E, and FA signaling are positively correlated, and FBXL12 upregulation is linked to reduced survival in patients with high CYCLIN E-expressing breast tumors. Finally, depletion of FBXL12 exacerbated oncogene-induced replication stress and sensitized cancer cells to drug-induced replication stress by WEE1 inhibition. Collectively, our results indicate that FBXL12 constitutes a vulnerability and a potential therapeutic target in CYCLIN E-overexpressing cancers.

Cisplatin overcomes radiotherapy resistance in OCT4-expressing head and neck squamous cell carcinoma.

OBJECTIVES: Cisplatin is combined with radiotherapy for advanced head and neck squamous cell carcinoma (HNSCC). While providing a beneficial effect on survival, it also causes side effects and thus is an important target when considering treatment de-escalation. Currently, there are no biomarkers to predict its patient-selective therapeutic utility. In this study, we examined the role of the stem cell factor OCT4 as a potential biomarker to help clinicians stratify HNSCC patients between radiotherapy and chemoradiotherapy. MATERIALS AND METHODS: OCT4 immunohistochemical staining of a population-validated tissue microarray (PV-TMA) (n = 166) representative of a standard HNSCC patients was carried out, and 5-year survival was analyzed. The results were validated using ex vivo drug sensitivity analysis of HNSCC tumor samples, and further cross-validated in independent oropharyngeal (n = 118), nasopharyngeal (n = 170), and vulvar carcinoma (n = 95) clinical datasets. In vitro, genetically modified, patient-derived HNSCC cells were used. RESULTS: OCT4 expression in HNSCC tumors was associated with radioresistance. However, combination therapy with cisplatin was found to overcome thisradioresistance in OCT4-expressing HNSCC tumors. The results were validated by using several independent patient cohorts. Furthermore, CRISPRa-based OCT4 overexpression in the HNSCC cell line resulted in apoptosis resistance, and cisplatin was found to downregulate OCT4 protein expression in vitro. Ex vivo drug sensitivity analysis of HNSCC tumors confirmed the association between OCT4 expression and cisplatin sensitivity. CONCLUSION: This study introduces OCT4 immunohistochemistry as a simple and cost-effective diagnostic approach for clinical practice to identify HNSCC patients benefitting from radiosensitization by cisplatin using either full or reduced dosing.

Ex Vivo Drug Screening Informed Targeted Therapy for Metastatic Parotid Squamous Cell Carcinoma.

The purpose of ex vivo drug screening in the context of precision oncology is to serve as a functional diagnostic method for therapy efficacy modeling directly on patient-derived tumor cells. Here, we report a case study using integrated multiomics ex vivo drug screening approach to assess therapy efficacy in a rare metastatic squamous cell carcinoma of the parotid gland. Tumor cells isolated from lymph node metastasis and distal subcutaneous metastasis were used for imaging-based single-cell resolution drug screening and reverse-phase protein array-based drug screening assays to inform the treatment strategy after standard therapeutic options had been exhausted. The drug targets discovered on the basis of the ex vivo measured drug efficacy were validated with histopathology, genomic profiling, and in vitro cell biology methods, and targeted treatments with durable clinical responses were achieved. These results demonstrate the use of serial ex vivo drug screening to inform adjuvant therapy options prior to and during treatment and highlight HER2 as a potential therapy target also in metastatic squamous cell carcinoma of the salivary glands.

Ex vivo analysis of DNA repair targeting in extreme rare cutaneous apocrine sweat gland carcinoma.

Cutaneous apocrine carcinoma is an extreme rare malignancy derived from a sweat gland. Histologically sweat gland cancers resemble metastatic mammary apocrine carcinomas, but the genetic landscape remains poorly understood. Here, we report a rare metastatic case with a PALB2 aberration identified previously as a familial susceptibility gene for breast cancer in the Finnish population. As PALB2 exhibits functions in the BRCA1/2-RAD51-dependent homologous DNA recombination repair pathway, we sought to use ex vivo functional screening to explore sensitivity of the tumor cells to therapeutic targeting of DNA repair. Drug screening suggested sensitivity of the PALB2 deficient cells to BET-bromodomain inhibition, and modest sensitivity to DNA-PKi, ATRi, WEE1i and PARPi. A phenotypic RNAi screen of 300 DNA repair genes was undertaken to assess DNA repair targeting in more detail. Core members of the HR and MMEJ pathways were identified to be essential for viability of the cells. RNAi inhibition of RAD52-dependent HR on the other hand potentiated the efficacy of a novel BETi ODM-207. Together these results describe the first ever CAC case with a BRCAness genetic background, evaluate combinatorial DNA repair targeting, and provide a data resource for further analyses of DNA repair targeting in PALB2 deficient cancers.

Substrate-biased activity-based probes identify proteases that cleave receptor CDCP1.

CUB domain-containing protein 1 (CDCP1) is an oncogenic orphan transmembrane receptor and a promising target for the detection and treatment of cancer. Extracellular proteolysis of CDCP1 by poorly defined mechanisms induces pro-metastatic signaling. We describe a new approach for the rapid identification of proteases responsible for key proteolytic events using a substrate-biased activity-based probe (sbABP) that incorporates a substrate cleavage motif grafted onto a peptidyl diphenyl phosphonate warhead for specific target protease capture, isolation and identification. Using a CDCP1-biased probe, we identify urokinase (uPA) as the master regulator of CDCP1 proteolysis, which acts both by directly cleaving CDCP1 and by activating CDCP1-cleaving plasmin. We show that coexpression of uPA and CDCP1 is strongly predictive of poor disease outcome across multiple cancers and demonstrate that uPA-mediated CDCP1 proteolysis promotes metastasis in disease-relevant preclinical in vivo models. These results highlight CDCP1 cleavage as a potential target to disrupt cancer and establish sbABP technology as a new approach to identify disease-relevant proteases.

Ex vivo assessment of targeted therapies in a rare metastatic epithelial-myoepithelial carcinoma.

Epithelial-myoepithelial carcinoma (EMC) is a rare subtype of salivary gland neoplasms. Since the initial description of the cancer, just over 300 cases have been reported. EMCs occupy a biphasic cellular differentiation-state defined by the constitution of two cell types representing epithelial and myoepithelial lineages, yet the functional consequence of the differentiation-state heterogeneity with respect to therapy resistance of the tumors remains unclear. The reported local recurrence rate of the cases is approximately 30%, and while distant metastases are rare, a significant fraction of these cases are reported to receive no survival benefit from radio- or chemotherapy given in addition to surgery. Moreover, no targeted therapies have been reported for these neoplasms. We report here the first use and application of ex vivo drug screening together with next generation sequencing to assess targeted treatment strategies for a rare metastatic epithelial-myoepithelial carcinoma. Results of the ex vivo drug screen demonstrate significant differential therapeutic sensitivity between the epithelial and myoepithelial intra-tumor cell lineages suggesting that differentiation-state heterogeneity within epithelial-myoepithelial carcinomas may present an outlet to partial therapeutic responses to targeted therapies including MEK and mTOR inhibitors. These results suggest that the intra-tumor lineage composition of EMC could be an important factor to be assessed when novel treatments are being evaluated for management of metastatic EMC.

PTEN and DNA-PK determine sensitivity and recovery in response to WEE1 inhibition in human breast cancer.

Inhibition of WEE1 kinase by AZD1775 has shown promising results in clinical cancer trials, but markers predicting AZD1775 response are lacking. Here we analysed AZD1775 response in a panel of human breast cancer (BC) cell lines by global proteome/transcriptome profiling and identified two groups of basal-like BC (BLBCs): 'PTEN low' BLBCs were highly sensitive to AZD1775 and failed to recover following removal of AZD1775, while 'PTEN high' BLBCs recovered. AZD1775 induced phosphorylation of DNA-PK, protecting cells from replication-associated DNA damage and promoting cellular recovery. Deletion of DNA-PK or PTEN, or inhibition of DNA-PK sensitized recovering BLBCs to AZD1775 by abrogating replication arrest, allowing replication despite DNA damage. This was linked to reduced CHK1 activation, increased cyclin E levels and apoptosis. In conclusion, we identified PTEN and DNA-PK as essential regulators of replication checkpoint arrest in response to AZD1775 and defined PTEN as a promising biomarker for efficient WEE1 cancer therapy.

Ex vivo modelling of drug efficacy in a rare metastatic urachal carcinoma.

BACKGROUND: Ex vivo drug screening refers to the out-of-body assessment of drug efficacy in patient derived vital tumor cells. The purpose of these methods is to enable functional testing of patient specific efficacy of anti-cancer therapeutics and personalized treatment strategies. Such approaches could prove powerful especially in context of rare cancers for which demonstration of novel therapies is difficult due to the low numbers of patients. Here, we report comparison of different ex vivo drug screening methods in a metastatic urachal adenocarcinoma, a rare and aggressive non-urothelial bladder malignancy that arises from the remnant embryologic urachus in adults. METHODS: To compare the feasibility and results obtained with alternative ex vivo drug screening techniques, we used three different approaches; enzymatic cell viability assay of 2D cell cultures and image-based cytometry of 2D and 3D cell cultures in parallel. Vital tumor cells isolated from a biopsy obtained in context of a surgical debulking procedure were used for screening of 1160 drugs with the aim to evaluate patterns of efficacy in the urachal cancer cells. RESULTS: Dose response data from the enzymatic cell viability assay and the image-based assay of 2D cell cultures showed the best consistency. With 3D cell culture conditions, the proliferation rate of the tumor cells was slower and potency of several drugs was reduced even following growth rate normalization of the responses. MEK, mTOR, and MET inhibitors were identified as the most cytotoxic targeted drugs. Secondary validation analyses confirmed the efficacy of these drugs also with the new human urachal adenocarcinoma cell line (MISB18) established from the patient's tumor. CONCLUSIONS: All the tested ex vivo drug screening methods captured the patient's tumor cells' sensitivity to drugs that could be associated with the oncogenic KRASG12V mutation found in the patient's tumor cells. Specific drug classes however resulted in differential dose response profiles dependent on the used cell culture method indicating that the choice of assay could bias results from ex vivo drug screening assays for selected drug classes.

Image-based ex vivo drug screen to assess targeted therapies in recurrent thymoma.

OBJECTIVES: Thymoma is a rare malignancy derived from the thymic epithelial cells. No standard salvage treatments are available for recurrent thymoma and due to the low number of cases, alternative treatment regimens have been assessed only in small case series with varying success. The aim of this study was to use an image-based ex vivo drug screening strategy to assess efficacy of a large panel of anti-cancer agents for thymoma using patient derived tumor cells. MATERIALS AND METHODS: Vital tumor and tumor associated cells were used to assess the efficacy of 147 anti-cancer drugs including approved and experimental agents. Drug efficacy was analyzed at single cell resolution using image-based high content drug screening to assess tumor cell specific responses. Molecular profiling and histopathology was used to confirm the drug targets identified by the screen. RESULTS: The ex vivo drug screen identified selective sensitivity of the cancerous epithelial thymoma cells to EGFR-, HDAC- and mTOR-inhibition. Histopathology confirmed high protein level expression of EGFR in the patient's tumor. Patient was initiated treatment with Cetuximab resulting in stable disease after relapse on five different chemotherapy regimens. CONCLUSION: The results show that the image-based ex vivo therapy efficacy screening strategy can be used to identify patient and tumor relevant drug sensitivity patterns in thymoma. The results also warrant continued research on EGFR as a biomarker and therapy target in recurrent thymomas.

Clonal Evolution of MEK/MAPK Pathway Activating Mutations in a Metastatic Colorectal Cancer Case.

BACKGROUND/AIM: The aim of this study was to examine clonal heterogeneity, to test the utility of liquid biopsy in monitoring disease progression and to evaluate the usefulness of ex vivo drug screening in a BRAF L597Q-mutated colorectal cancer (CRC) patient developing metastases during adjuvant therapy. MATERIALS AND METHODS: Next generation sequencing (NGS) and droplet digital PCR (ddPCR) were performed in samples from tumor tissues and liquid biopsies. Live cancer cells from a metastatic lesion were used in ex vivo drug sensitivity assays. RESULTS: We found evidence of continued dependence of MEK/MAPK pathway activation, but different activating mutations in primary tumor and metastases. Liquid biopsy based BRAF L597Q ddPCR testing was a sensitive personalized biomarker predicting the rise of clinically aggressive metastatic disease. Ex vivo drug sensitivity assays with BRAF L597Q mutated cells showed response to MEK/MAPK targeted therapies. CONCLUSION: The rare BRAF L597Q mutation may be associated with aggressive tumor behavior in CRC. Liquid biopsy can be used to capture clinically relevant tumor features.

MHC class I loaded ligands from breast cancer cell lines: A potential HLA-I-typed antigen collection.

To build a catalog of peptides presented by breast cancer cells, we undertook systematic MHC class I immunoprecipitation followed by elution of MHC class I-loaded peptides in breast cancer cells. We determined the sequence of 3196 MHC class I ligands representing 1921 proteins from a panel of 20 breast cancer cell lines. After removing duplicate peptides, i.e., the same peptide eluted from more than one cell line, the total number of unique peptides was 2740. Of the unique peptides eluted, more than 1750 had been previously identified, and of these, sixteen have been shown to be immunogenic. Importantly, half of these immunogenic peptides were shared between different breast cancer cell lines. MHC class I binding probability was used to plot the distribution of the eluted peptides in accordance with the binding score for each breast cancer cell line. We also determined that the tested breast cancer cells presented 89 mutation-containing peptides and peptides derived from aberrantly translated genes, 7 of which were shared between four or two different cell lines. Overall, the high throughput identification of MHC class I-loaded peptides is an effective strategy for systematic characterization of cancer peptides, and could be employed for design of multi-peptide anticancer vaccines. SIGNIFICANCE: By employing proteomic analyses of eluted peptides from breast cancer cells, the current study has built an initial HLA-I-typed antigen collection for breast cancer research. It was also determined that immunogenic epitopes can be identified using established cell lines and that shared immunogenic peptides can be found in different cancer types such as breast cancer and leukemia. Importantly, out of 3196 eluted peptides that included duplicate peptides in different cells 89 peptides either contained mutation in their sequence or were derived from aberrant translation suggesting that mutation-containing epitopes are on the order of 2-3% in breast cancer cells. Finally, our results suggest that interfering with MHC class I function is one of the mechanisms of how tumor cells escape immune system attack.

Metabolic reprogramming ensures cancer cell survival despite oncogenic signaling blockade.

There is limited knowledge about the metabolic reprogramming induced by cancer therapies and how this contributes to therapeutic resistance. Here we show that although inhibition of PI3K-AKT-mTOR signaling markedly decreased glycolysis and restrained tumor growth, these signaling and metabolic restrictions triggered autophagy, which supplied the metabolites required for the maintenance of mitochondrial respiration and redox homeostasis. Specifically, we found that survival of cancer cells was critically dependent on phospholipase A2 (PLA2) to mobilize lysophospholipids and free fatty acids to sustain fatty acid oxidation and oxidative phosphorylation. Consistent with this, we observed significantly increased lipid droplets, with subsequent mobilization to mitochondria. These changes were abrogated in cells deficient for the essential autophagy gene ATG5 Accordingly, inhibition of PLA2 significantly decreased lipid droplets, decreased oxidative phosphorylation, and increased apoptosis. Together, these results describe how treatment-induced autophagy provides nutrients for cancer cell survival and identifies novel cotreatment strategies to override this survival advantage.

High-throughput RNAi screen in Ewing sarcoma cells identifies leucine rich repeats and WD repeat domain containing 1 (LRWD1) as a regulator of EWS-FLI1 driven cell viability.

A translocation leading to the formation of an oncogenic EWS-ETS fusion protein defines Ewing sarcoma. The most frequent gene fusion, present in 85 percent of Ewing sarcomas, is EWS-FLI1. Here, a high-throughput RNA interference screen was performed to identify genes whose function is critical for EWS-FLI1 driven cell viability. In total, 6781 genes were targeted by siRNA molecules and the screen was performed both in presence and absence of doxycycline-inducible expression of the EWS-FLI1 shRNA in A673/TR/shEF Ewing sarcoma cells. The Leucine rich repeats and WD repeat Domain containing 1 (LRWD1) targeting siRNA pool was the strongest hit reducing cell viability only in EWS-FLI1 expressing Ewing sarcoma cells. LRWD1 had been previously described as a testis specific gene with only limited information on its function. Analysis of LRWD1 mRNA levels in patient samples indicated that high expression associated with poor overall survival in Ewing sarcoma. Gene ontology analysis of LRWD1 co-expressed genes in Ewing tumors revealed association with DNA replication and analysis of differentially expressed genes in LRWD1 depleted Ewing sarcoma cells indicated a role in connective tissue development and cellular morphogenesis. Moreover, EWS-FLI1 repressed genes with repressive H3K27me3 chromatin marks were highly enriched among LRWD1 target genes in A673/TR/shEF Ewing sarcoma cells, suggesting that LRWD1 contributes to EWS-FLI1 driven transcriptional regulation. Taken together, we have identified LRWD1 as a novel regulator of EWS-FLI1 driven cell viability in A673/TR/shEF Ewing sarcoma cells, shown association between high LRWD1 mRNA expression and aggressive disease and identified processes by which LRWD1 may promote oncogenesis in Ewing sarcoma.

Androgen receptor-interacting protein HSPBAP1 facilitates growth of prostate cancer cells in androgen-deficient conditions.

Hormonal therapies targeting androgen receptor (AR) are effective in prostate cancer (PCa), but often the cancers progress to fatal castrate-resistant disease. Improved understanding of the cellular events during androgen deprivation would help to identify survival and stress pathways whose inhibition could synergize with androgen deprivation. Toward this aim, we performed an RNAi screen on 2,068 genes, including kinases, phosphatases, epigenetic enzymes and other druggable gene targets. High-content cell spot microarray (CSMA) screen was performed in VCaP cells in the presence and absence of androgens with detection of Ki67 and cleaved ADP-ribose polymerase (cPARP) as assays for cell proliferation and apoptosis. Thirty-nine candidate genes were identified, whose silencing inhibited proliferation or induced apoptosis of VCaP cells exclusively under androgen-deprived conditions. One of the candidates, HSPB (heat shock 27 kDa)-associated protein 1 (HSPBAP1), was confirmed to be highly expressed in tumor samples and its mRNA expression levels increased with the Gleason grade. We found that strong HSPBAP1 immunohistochemical staining (IHC) was associated with shorter disease-specific survival of PCa patients compared with negative to moderate staining. Furthermore, we demonstrate that HSPBAP1 interacts with AR in the nucleus of PCa cells specifically during androgen-deprived conditions, occupies chromatin at PSA/klk3 and TMPRSS2/tmprss2 enhancers and regulates their expression. In conclusion, we suggest that HSPBAP1 aids in sustaining cell viability by maintaining AR signaling during androgen-deprived conditions.

Break-induced replication repair of damaged forks induces genomic duplications in human cells.

In budding yeast, one-ended DNA double-strand breaks (DSBs) and damaged replication forks are repaired by break-induced replication (BIR), a homologous recombination pathway that requires the Pol32 subunit of DNA polymerase delta. DNA replication stress is prevalent in cancer, but BIR has not been characterized in mammals. In a cyclin E overexpression model of DNA replication stress, POLD3, the human ortholog of POL32, was required for cell cycle progression and processive DNA synthesis. Segmental genomic duplications induced by cyclin E overexpression were also dependent on POLD3, as were BIR-mediated recombination events captured with a specialized DSB repair assay. We propose that BIR repairs damaged replication forks in mammals, accounting for the high frequency of genomic duplications in human cancers.

Expanding the Diversity of Imaging-Based RNAi Screen Applications Using Cell Spot Microarrays.

Over the past decade, great strides have been made in identifying gene aberrations and deregulated pathways that are associated with specific disease states. These association studies guide experimental studies aimed at identifying the aberrant genes and networks that cause the disease states. This requires functional manipulation of these genes and networks in laboratory models of normal and diseased cells. One approach is to assess molecular and biological responses to high-throughput RNA interference (RNAi)-induced gene knockdown. These responses can be revealed by immunofluorescent staining for a molecular or cellular process of interest and quantified using fluorescence image analysis. These applications are typically performed in multiwell format, but are limited by high reagent costs and long plate processing times. These limitations can be mitigated by analyzing cells grown in cell spot microarray (CSMA) format. CSMAs are produced by growing cells on small (~200 mm diameter) spots with each spot carrying an siRNA with transfection reagent. The spacing between spots is only a few hundred micrometers, thus thousands of cell spots can be arranged on a single cell culture surface. These high-density cell cultures can be immunofluorescently stained with minimal reagent consumption and analyzed quickly using automated fluorescence microscopy platforms. This review covers basic aspects of imaging-based CSMA technology, describes a wide range of immunofluorescence assays that have already been implemented successfully for CSMA screening and suggests future directions for advanced RNAi screening experiments.

MiR-9, -31, and -182 deregulation promote proliferation and tumor cell survival in colon cancer.

Several microRNAs (miRNAs) are known to be deregulated in colon cancer, but the mechanisms behind their potential involvement on proliferation and tumor cell survival are unclear. The present study aimed to identify miRNAs with functional implications for development of colon cancer. The cell proliferation and apoptosis were examined following perturbations of miRNA levels by employing a comprehensive miRNA library screen. miRNAs nominated for relevance to colon cancer were validated on expression and functional levels. By integrating the effect of miRNA up-regulation with the endogenous miRNA expression levels within the HT29, HCT116, and SW480 colon cancer cell lines, we identified miRNAs controlling cell proliferation (n = 53) and apoptosis (n = 93). From these functionally nominated miRNAs, we narrowed the list to 10 oncogene- and 20 tumor suppressor-like miRNAs that were also differentially expressed between colon cancer (n = 80) and normal colonic mucosa (n = 20). The differential expressions of miR-9, miR-31, and miR-182 were successfully validated in a series of colon carcinomas (n = 30) and polyps (n = 10) versus normal colonic mucosa (n = 10), whereas the functional effect was confirmed in an in-depth validation using different cell viability and apoptotic markers. Several transcription factors and genes regulating cell proliferation were identified as putative target genes by integrative miRNA/mRNA expression analysis obtained from the same colon cancer patient samples. This study suggests that deregulated expression of miR-9, miR-31, and miR-182 during carcinogenesis plays a significant role in the development of colon cancer by promoting proliferation and tumor cell survival.