Organoids as a biomarker for personalized treatment in metastatic colorectal cancer: drug screen optimization and correlation with patient response.

Background: The inability to predict treatment response of colorectal cancer patients results in unnecessary toxicity, decreased efficacy and survival. Response testing on patient-derived organoids (PDOs) is a promising biomarker for treatment efficacy. The aim of this study is to optimize PDO drug screening methods for correlation with patient response and explore the potential to predict responses to standard chemotherapies.

Live imaging of cell division in 3D stem-cell organoid cultures.

Examining cell behavior in its correct tissue context is a major challenge in cell biology. The recent development of mammalian stem cell-based organoid cultures offers exciting opportunities to visualize dynamic cellular events in a 3D tissue-like setting. We describe here an approach for live imaging of cell division processes in intestinal organoid cultures derived from human and mouse adult stem cells.

Corrigendum: Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors.

This corrects the article DOI: 10.1038/nm.4219.

Genome-wide CRISPR screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43-mutant pancreatic tumors.

Forward genetic screens with CRISPR-Cas9 genome editing enable high-resolution detection of genetic vulnerabilities in cancer cells. We conducted genome-wide CRISPR-Cas9 screens in RNF43-mutant pancreatic ductal adenocarcinoma (PDAC) cells, which rely on Wnt signaling for proliferation. Through these screens, we discovered a unique requirement for a Wnt signaling circuit: engaging FZD5, one of the ten Frizzled receptors encoded in the human genome.

Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening.

Colorectal cancer (CRC) organoids can be derived from almost all CRC patients and therefore capture the genetic diversity of this disease. We assembled a panel of CRC organoids carrying either wild-type or mutant RAS, as well as normal organoids and tumor organoids with a CRISPR-introduced oncogenic mutation. Using this panel, we evaluated RAS pathway inhibitors and drug combinations that are currently in clinical trial for RAS mutant cancers.

Sequential cancer mutations in cultured human intestinal stem cells.

Crypt stem cells represent the cells of origin for intestinal neoplasia. Both mouse and human intestinal stem cells can be cultured in medium containing the stem-cell-niche factors WNT, R-spondin, epidermal growth factor (EGF) and noggin over long time periods as epithelial organoids that remain genetically and phenotypically stable. Here we utilize CRISPR/Cas9 technology for targeted gene modification of four of the most commonly mutated colorectal cancer genes (APC, P53 (also known as TP53), KRAS and SMAD4) in cultured human intestinal stem cells.

DNA damage response and transcription.

A network of DNA damage surveillance systems is triggered by sensing of DNA lesions and the initiation of a signal transduction cascade that activates genome-protection pathways including nucleotide excision repair (NER). NER operates through coordinated assembly of repair factors into pre- and post-incision complexes. Recent work identifies RPA as a key regulator of the transition from dual incision to repair-synthesis in UV-irradiated non-cycling cells, thereby averting the generation of unprocessed repair intermediates.

Combined promoter methylation analysis of CADM1 and MAL: an objective triage tool for high-risk human papillomavirus DNA-positive women.

Purpose: Screening women for high-grade cervical intraepithelial neoplasia or cervical cancer (CIN3(+)) by high-risk human papillomavirus (hrHPV) testing has as side-effect the detection of hrHPV-positive women without clinically relevant lesions. Here, we developed an objective assay assessing the methylation status of the promoter regions of CADM1 and MAL to triage hrHPV-positive women for CIN3(+).

Experimental Design: In a training set (51 women with CIN3(+) and 224 without CIN2(+)), panels consisting of one to four quantitative methylation-specific PCR (qMSP) assays (CADM1-m12,CADM1-m18,MAL-m1,MAL-m2) were analyzed.

Replication protein A safeguards genome integrity by controlling NER incision events.

Single-stranded DNA gaps that might arise by futile repair processes can lead to mutagenic events and challenge genome integrity. Nucleotide excision repair (NER) is an evolutionarily conserved repair mechanism, essential for removal of helix-distorting DNA lesions. In the currently prevailing model, NER operates through coordinated assembly of repair factors into pre- and post-incision complexes; however, its regulation in vivo is poorly understood.

UV-induced photolesions elicit ATR-kinase-dependent signaling in non-cycling cells through nucleotide excision repair-dependent and -independent pathways.

Activation of signaling pathways by UV radiation is a key event in the DNA damage response and initiated by different cellular processes. Here we show that non-cycling cells proficient in nucleotide excision repair (NER) initiate a rapid but transient activation of the damage response proteins p53 and H2AX; by contrast, NER-deficient cells display delayed but persistent signaling and inhibition of cell cycle progression upon release from G0 phase. In the absence of repair, UV-induced checkpoint activation coincides with the formation of single-strand DNA breaks by the action of the endonuclease Ape1.

Combined CADM1 and MAL promoter methylation analysis to detect (pre-)malignant cervical lesions in high-risk HPV-positive women.

Given the lower specificity for high-grade cervical lesions of high-risk human papillomavirus (hrHPV) testing compared to cytology, additional triage testing for hrHPV test-positive women is needed to detect high-grade cervical lesions. Here, we tested whether combined methylation analysis for cell adhesion molecule 1 (CADM1) and T-lymphocyte maturation associated protein (MAL), both functionally involved in cervical carcinogenesis, could serve as such a triage marker. Four quantitative methylation-specific PCRs (qMSP), two for CADM1 (regions M12 and M18) and MAL (regions M1 and M2) each, were applied to 261 cervical tissue specimens ranging from no neoplasia to carcinoma.

Replication factor C recruits DNA polymerase delta to sites of nucleotide excision repair but is not required for PCNA recruitment.

Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood.

hTERT promoter activity and CpG methylation in HPV-induced carcinogenesis.

Background: Activation of telomerase resulting from deregulated hTERT expression is a key event during high-risk human papillomavirus (hrHPV)-induced cervical carcinogenesis. In the present study we examined hTERT promoter activity and its relation to DNA methylation as one of the potential mechanisms underlying deregulated hTERT transcription in hrHPV-transformed cells.

Methods: Using luciferase reporter assays we analyzed hTERT promoter activity in primary keratinocytes, HPV16- and HPV18-immortalized keratinocyte cell lines and cervical cancer cell lines.

Three DNA polymerases, recruited by different mechanisms, carry out NER repair synthesis in human cells.

Nucleotide excision repair (NER) is the most versatile DNA repair system that deals with the major UV photoproducts in DNA, as well as many other DNA adducts. The early steps of NER are well understood, whereas the later steps of repair synthesis and ligation are not. In particular, which polymerases are definitely involved in repair synthesis and how they are recruited to the damaged sites has not yet been established.

RNF4 and VHL regulate the proteasomal degradation of SUMO-conjugated Hypoxia-Inducible Factor-2alpha.

Hypoxia-inducible factors (HIFs) are critical transcription factors that mediate cell survival during reduced oxygen conditions (hypoxia). At regular oxygen conditions (normoxia), HIF-1alpha and HIF-2alpha are continuously synthesized in cells and degraded via the ubiquitin-proteasome pathway. During hypoxia, these proteins are stabilized and translocate to the nucleus to activate transcription of target genes that enable cell survival at reduced oxygen levels.

Repression of MAL tumour suppressor activity by promoter methylation during cervical carcinogenesis.

We recently identified MAL (T-lymphocyte maturation associated protein) as the most down-regulated gene in cervical oncogenesis. Here, we examined the mechanism underlying MAL silencing, its functional role in cervical carcinogenesis, and the relevance of detecting MAL alterations for risk assessment of hrHPV-positive women. MAL mRNA expression and promoter methylation were analysed in primary keratinocytes, hrHPV-immortalized keratinocytes, cervical cancer cell lines, biopsies, and scrapings by quantitative (methylation-specific) PCR.

MAL promoter hypermethylation as a novel prognostic marker in gastric cancer.

T-lymphocyte maturation associated protein, MAL, has been described as a tumour-suppressor gene with diagnostic value in colorectal and oesophageal cancers, and can be inactivated by promoter hypermethylation. The aim of this study was to analyse the prevalence of MAL promoter hypermethylation and the association with mRNA expression in gastric cancers and to correlate methylation status to clinicopathological data. Bisulphite-treated DNA isolated from formalin-fixed and paraffin-embedded samples of 202 gastric adenocarcinomas and 22 normal gastric mucosae was subjected to real-time methylation-specific PCR (Q-MSP).

Association between dense CADM1 promoter methylation and reduced protein expression in high-grade CIN and cervical SCC.

We previously showed that silencing of TSLC1, recently renamed CADM1, is functionally involved in high-risk HPV-mediated cervical carcinogenesis. CADM1 silencing often results from promoter methylation. Here, we determined the extent of CADM1 promoter methylation in cervical (pre)malignant lesions and its relation to anchorage-independent growth and gene silencing to select a CADM1-based methylation marker for identification of women at risk of cervical cancer.

Differential effects of combined Ad5- delta 24RGD and radiation therapy in in vitro versus in vivo models of malignant glioma.

Purpose: The integrin-targeted conditionally replicating adenovirus Ad5-delta 24RGD has been shown to possess strong oncolytic activity in experimental tumors and is currently being developed toward phase I clinical evaluation for ovarian cancer and malignant glioma. Previously, we reported that combination therapy of Ad5-delta 24RGD with irradiation led to synergistic antitumor activity in s.c.

Sequential gene promoter methylation during HPV-induced cervical carcinogenesis.

We aimed to link DNA methylation events occurring in cervical carcinomas to distinct stages of HPV-induced transformation. Methylation specific-multiplex ligation-dependent probe amplification (MS-MLPA) analysis of cervical carcinomas revealed promoter methylation of 12 out of 29 tumour suppressor genes analysed, with MGMT being most frequently methylated (92%). Subsequently, consecutive stages of HPV16/18-transfected keratinocytes (n=11), ranging from pre-immortal to anchorage-independent phenotypes, were analysed by MS-MLPA.

Validity of bioluminescence measurements for noninvasive in vivo imaging of tumor load in small animals.

A relatively new strategy to longitudinally monitor tumor load in intact animals and the effects of therapy is noninvasive bioluminescence imaging (BLI). The validity of BLIf or quantitative assessment of tumor load in small animals is critically evaluated in the present review. Cancer cells are grafted in mice or rats after transfection with a luciferase gene--usually that of a firefly.

Chinese hamster cell mutant, V-C8, a model for analysis of Brca2 function.

The previously described Chinese hamster cell mutant V-C8 that is defective in Brca2 shows a very complex phenotype, including increased sensitivity towards a wide variety of DNA damaging agents, chromosomal instability, abnormal centrosomes and impaired formation of Rad51 foci in response to DNA damage. Here, we demonstrate that V-C8 cells display biallelic nonsense mutations in Brca2, one in exon 15 and the other in exon 16, both resulting in truncated Brca2 proteins. We generated several independent mitomycin C (MMC)-resistant clones from V-C8 cells that had acquired an additional mutation leading to the restoration of the open reading frame of one of the Brca2 alleles.