Subcellular protein expression models for microsatellite instability in colorectal adenocarcinoma tissue images

Background New bioimaging techniques capable of visualising the co-location of numerous proteins within individual cells have been proposed to study tumour heterogeneity of neighbouring cells within the same tissue specimen. These techniques have highlighted the need to better understand the interplay between proteins in terms of their colocalisation. Results We recently proposed a cellular-level model of the healthy and cancerous colonic crypt microenvironments. Here, we extend the model to include detailed models of protein expression to generate synthetic multiplex fluorescence data. As a first step, we present models for various cell organelles learned from real immunofluorescence data from the Human Protein Atlas. Comparison between the distribution of various features obtained from the real and synthetic organelles has shown very good agreement. This has included both features that have been used as part of the model input and ones that have not been explicitly considered. We then develop models for six proteins which are important colorectal cancer biomarkers and are associated with microsatellite instability, namely MLH1, PMS2, MSH2, MSH6, P53 and PTEN. The protein models include their complex expression patterns and which cell phenotypes express them. The models have been validated by comparing distributions of real and synthesised parameters and by application of frameworks for analysing multiplex immunofluorescence image data. Conclusions The six proteins have been chosen as a case study to illustrate how the model can be used to generate synthetic multiplex immunofluorescence data. Further proteins could be included within the model in a similar manner to enable the study of a larger set of proteins of interest and their interactions. To the best of our knowledge, this is the first model for expression of multiple proteins in anatomically intact tissue, rather than within cells in culture.QNRF grant NPRP 5-1345-1-228. BBSRC and University of Warwick Institute of Advanced Study

Subcellular protein expression models for microsatellite instability in colorectal adenocarcinoma tissue images

Background: New bioimaging techniques capable of visualising the co-location of numerous proteins within individual cells have been proposed to study tumour heterogeneity of neighbouring cells within the same tissue specimen. These techniques have highlighted the need to better understand the interplay between proteins in terms of their colocalisation. Results: We recently proposed a cellular-level model of the healthy and cancerous colonic crypt microenvironments. Here, we extend the model to include detailed models of protein expression to generate synthetic multiplex fluorescence data. As a first step, we present models for various cell organelles learned from real immunofluorescence data from the Human Protein Atlas. Comparison between the distribution of various features obtained from the real and synthetic organelles has shown very good agreement. This has included both features that have been used as part of the model input and ones that have not been explicitly considered. We then develop models for six proteins which are important colorectal cancer biomarkers and are associated with microsatellite instability, namely MLH1, PMS2, MSH2, MSH6, P53 and PTEN. The protein models include their complex expression patterns and which cell phenotypes express them. The models have been validated by comparing distributions of real and synthesised parameters and by application of frameworks for analysing multiplex immunofluorescence image data. Conclusions: The six proteins have been chosen as a case study to illustrate how the model can be used to generate synthetic multiplex immunofluorescence data. Further proteins could be included within the model in a similar manner to enable the study of a larger set of proteins of interest and their interactions. To the best of our knowledge, this is the first model for expression of multiple proteins in anatomically intact tissue, rather than within cells in culture

Identification of genes expressed by immune cells of the colon that are regulated by colorectal cancer-associated variants.

A locus on human chromosome 11q23 tagged by marker rs3802842 was associated with colorectal cancer (CRC) in a genome-wide association study; this finding has been replicated in case-control studies worldwide. In order to identify biologic factors at this locus that are related to the etiopathology of CRC, we used microarray-based target selection methods, coupled to next-generation sequencing, to study 103 kb at the 11q23 locus. We genotyped 369 putative variants from 1,030 patients with CRC (cases) and 1,061 individuals without CRC (controls) from the Ontario Familial Colorectal Cancer Registry. Two previously uncharacterized genes, COLCA1 and COLCA2, were found to be co-regulated genes that are transcribed from opposite strands. Expression levels of COLCA1 and COLCA2 transcripts correlate with rs3802842 genotypes. In colon tissues, COLCA1 co-localizes with crystalloid granules of eosinophils and granular organelles of mast cells, neutrophils, macrophages, dendritic cells and differentiated myeloid-derived cell lines. COLCA2 is present in the cytoplasm of normal epithelial, immune and other cell lineages, as well as tumor cells. Tissue microarray analysis demonstrates the association of rs3802842 with lymphocyte density in the lamina propria (p = 0.014) and levels of COLCA1 in the lamina propria (p = 0.00016) and COLCA2 (tumor cells, p = 0.0041 and lamina propria, p = 6 × 10(-5)). In conclusion, genetic, expression and immunohistochemical data implicate COLCA1 and COLCA2 in the pathogenesis of colon cancer. Histologic analyses indicate the involvement of immune pathways

융합유전자인 FAM174A-WWC1의 기능 연구

학위논문(박사)--서울대학교 대학원 :의과대학 의학과,2020. 2. 정승용.Introduction: Recent epidemiological studies suggest that there has been a significant increase of colorectal cancer (CRC) diagnosis in young adults, even though the overall incidence and mortality has decreased in recent years. Although these early-onset CRCs (EOCRCs) are likely to suggest a hereditary predisposition, known familial CRC syndromes account for only 20% of EOCRC cases; the genetic aberrations remain unknown for the other 80% of cases. Therefore, we aimed to establish reproducible biological resources and contribute to expanding the mutational database for EOCRC. Methods: Four cell lines were established from the original tumor tissues of individual CRC patients diagnosed prior to 30 years of age, and next-generation sequencing was used to identify the genetic features of EOCRC. Results: Analysis of the mutation profile related to CRC carcinogenesis revealed no significant mutations except for the TP53 splice mutation in the SNU-1460 cell line. Whole-exome sequencing data showed PCLO gene mutation in all EOCRC samples and four COlon ADenocarcinoma (COAD) cohort samples obtained from The Cancer Genome Atlas-Genomic Data Commons (TCGA-GDC). In addition, we identified one novel fusion gene, FAM174A-WWC1, and analyzed its functional role. FAM174A-WWC1 expression in normal fibroblasts caused alterations in cellular morphology and modulated the intercellular expression levels of E-cadherin and N-cadherin. Moreover, FAM174A-WWC1 abrogated the membrane expression of Yes-associated protein 1 (YAP1) and significantly increased the level of nuclear YAP1. Lastly, FAM174A-WWC1 expression increased oncogenic capacity and invasiveness of normal fibroblasts. These findings suggest that this fusion gene may act as a driver mutation in EOCRC. Conclusions: There were fewer mutations related to CRC carcinogenesis in 4 EOCRC cell lines. Mutations were common in the PCLO gene in our cell lines and EOCRC from TCGA-GDC. A novel fusion gene, FAM174A-WWC1 increases the oncogenic and metastatic capacity of cells in vitro. This fusion gene may represent a robust molecular and therapeutic target in EOCRC.서론: 50세 이후 대장암의 발생률과 사망률이 감소하고 있음에도 불구하고, 50세 미만의 조기 발병 대장암은 증가 추세이다. 50세 이전에 발생하는 조기발병대장암은 유전성이라고 생각할 수 있지만, 조기발병대장암의 약 20%정도만 가족성 대장암 증후군(familial colorectal cancer syndrome)이고 나머지 80%정도는 원인 유전자를 모른다. 이에 저자들은 재현 가능한 생물학적인 자원을 확립하고, 조기발병대장암의 유전자 변이에 관한 유전정보 구축에 기여하기 위해 본 연구를 시행하였다. 방법: 30세 이전에 대장암을 진단받은 네 명의 조기발병대장암 환자들의 수술 후 적출된 대장암 조직으로부터 네 종류의 세포주를 수립했다. 이 세포주들에 대한 차세대염기서열 (Next-generation sequencing)을 통해, 조기발병대장암의 유전적인 특징을 분석하였다. 결과: 전체 엑솜 염기서열 분석(whole-exome sequencing)을 통해, 대장암 발생기전에 주로 작용하는 유전자의 변이를 분석해 보았을 때, SNU-1460 세포주에서 TP53 gene의 slice 돌연변이가 발견된 것을 제외하고는 의미 있는 유전자의 변이가 발견되지 않았다. 또한, 엑솜 염기서열 분석 자료를 The Cancer Genome Atlas-Genomic Data Commons (TCGA-GDC)에서 추출한 35세 미만의 조기발병대장암 환자에서의 유전자 변이와 비교해서, PCLO 유전자에 공통적으로 변이가 있는 것을 발견하였다. RNA 염기서열 분석(RNA sequencing)을 통해서는 FAM174A-WWC1이라는 새로운 융합 유전자(fusion gene)을 발견했고, 그 기능을 알아보았다. FAM174-WWC1의 발현으로 인해, 정상 섬유아세포의 형태의 변형이 있었고, 이 형질 도입이 된 섬유아세포들이 종양 구체(tumor-sphere)모양으로 자라는 것을 확인하였다. 또한, 세포내 E-cadherin의 감소와 N-cadherin의 증가가 관찰되었다. 이 융합유전자는 세포막과 세포질에서의 YAP1단백질의 발현을 감소시키고, 세포핵에서의 YAP1 단백질의 발현을 증가시켰다. FAM174-WWC1 단백질은 주로 세포질에서 발견되며, 세포골격에서 정상 WWC1 단백질보다 많이 존재함을 발견했다. 결론: 이 연구를 통해, 조기발병대장암에 공통적으로 PCLO 유전자의 변이가 있으며, 새로운 융합유전자인 FAM174A-WWC1의 발현은 세포의 발암성(oncogenic capacity) 및 침습성(invasiveness)이 증가시키는 것을 밝혔다.Introduction 1 Materials and Methods 7 Results 19 Discussion 57 References 64 Abstract in Korean 72Docto

The Insulin Receptor Substrate 1 (Irs1) in Intestinal Epithelial Differentiation and in Colorectal Cancer

Colorectal cancer (CRC) is associated with lifestyle factors that affect insulin/IGF signaling, of which the insulin receptor substrate 1 (IRS1) is a key transducer. We investigated expression, localization and pathologic correlations of IRS1 in cancer-uninvolved colonic epithelium, primary CRCs with paired liver metastases and in vitro polarizing Caco2 and HT29 cells. IRS1 mRNA and protein resulted higher, relative to paired mucosa, in adenomas of familial adenomatous polyposis patients and in CRCs that overexpressed c-MYC, ß-catenin, InsRß, and IGF1R. Analysis of IRS1 immunostaining in 24 cases of primary CRC with paired colonic epithelium and hepatic metastasis showed that staining intensity was significantly higher in metastases relative to both primary CRC (P<0.01) and colonic epithelium (P<0.01). Primary and metastatic CRCs, compared to colonic epithelium, contained significantly higher numbers of IRS1-positive cells (P = 0.013 and P = 0.014, respectively). Pathologic correlations in 163 primary CRCs revealed that diffuse IRS1 staining was associated with tumors combining differentiated phenotype and aggressive markers (high Ki67, p53, and ß-catenin). In Caco 2 IRS1 and InsR were maximally expressed after polarization, while IGF1R was highest in pre-polarized cells. No nuclear IRS1 was detected, while, with polarization, phosphorylated IRS1 (pIRS1) shifted from the lateral to the apical plasma membrane and was expressed in surface cells only. In HT29, that carry mutations constitutively activating survival signaling, IRS1 and IGF1R decreased with polarization, while pIRS1 localized in nuclear spots throughout the course. Overall, these data provide evidence that IRS1 is modulated according to CRC differentiation, and support a role of IRS1 in CRC progression and liver metastatization

Gene mutations and losses of heterozygosity of TP53 and PTEN in colorectal cancer

This study was conducted to find out if the patterns of PTEN gene molecular aberration in colorectal carcinoma are consistent with the classic molecular features ascribed to a tumour suppressor gene. PTEN gene mutations and LOH were assayed in a series of 18 colorectal carcinomas. Three PTEN gene mutations were found in 1 tumour, complete loss of one allele(LOH)in 2 tumours and allelic imbalance (AI) in 6 tumours. LOH or AI did not target the 1 tumour with the PTEN gene mutations. The widely held belief of PTEN as a classical tumour suppressor gene is not supported by the findings from this study

Wnt/Wingless dysregulation in small intestinal adenocarcinoma : Comparison with colorectal carcinoma

Die arretierte Aktivierung des wnt/wingless Signalweges durch eine Mutation ist eine der häufigsten Veränderungen bei intestinalen Karzinomen. Unabhängig von der wnt/wingless Komponente, die von der Mutation betroffen ist, kommt es zu einer Stabilisierung des transkriptionell aktiven wnt/wingless Mediators β-catenin. Die Stablisierung des β-catenin beim Karzinom des Dickdarms beruht in der Regel auf Mutationen des degradierenden APC, dies ist bei Karzinomen des Dünndarms, die ebenfalls eine Stabilisierung des β-catenin aufweisen, nicht der Fall. Ziel dieser Arbeit war zum einen, die Ursache der β-catenin Stabilisierung bei Tumoren ohne APC Mutationen zu identifizieren. Hierzu wurden 20 Adenokarzinome des Dünndarms und 20 früh manifestierte Kolonkarzinome, die ebenfalls häufig keine APC Mutation aufweisen, auf Mutationen im β-catenin Gen (CTNNB1) untersucht. Auf diese Weise fanden wir in beiden untersuchten Gruppen einen ungewöhnlichen Mutationstyp, der zu einem Verlust von Teilen der für die Degradierung des β-catenin essentiellen n-terminalen Domäne führte. Der Umfang der Deletionen war hierbei variabel und hatte einen Einfluß weniger auf die Intensität der β-catenin Stabilisierung als auf die Lokalisation des β-catenin im Zellkern und im Zytoplasma. Darüber hinaus fand sich, daß gleichartige Deletions-Mutanten des β-catenin in Dünn- und Dickdarmkarzinomen zu unterschiedlichen Akkumulations Typen führten. Dies weist auf eine divergente Regulierung des β-catenin in Dünn- und Dickdarmmukosa hin. Um die identifizierten, ungewöhnlichen Deletionsmutanten des β-catenin näher zu charakterisieren und einen Rückschluß auf die Auswirkungen von erweiterten Deletionen, die zusätzlich zu der Degradierungsdomäne noch c-terminal lokalisierte Domänen verloren haben, ziehen zu können, wurden entsprechende Mutanten mit Hilfe der „PCR-driven overlap extension“ Methode generiert und in die Zellkulturen SW480 (Kolonkarzinomzelllinie) und MDCK (Nierenepithel Zelllinie) transfiziert. Die Ergebnisse für die Deletionsmutanten wurden mit denen von Punktmutationen an Phosphorylierungsstellen der Degaradationsbox des β-catenin verglichen. Hierbei zeigte sich, daß das Akkumulationsverhalten von β-catenin im Zellkulturmodell den Befunden in den humanen Tumoren durchaus vergleichbar ist. In der Zelllinie SW480, die bereits eine APC Mutation besitzt, führte die Transfektion jedoch zur Apoptose. Dies ist ein Hinweis für einen negativen Seletionsdruck einer zu starken β-catenin Akkumulation und erklärt, warum tumorassoziierte Mutationen entweder im APC oder im β-catenin, niemals aber in beiden Molekülen gleichzeitig gefunden werden. In der MDCK Zelllinie hingegen, einer Zelllinie ohne β-catenin oder APC Mutation, zeigte sich eine onkogene Wirkung der Transfektion der Mutanten. Diese ließ sich einerseits in Form einer erhöhten Proliferation nachweisen, andererseits fanden sich phenotypische Zellveränderungen die unter dem Begriff des „metastasierungsfähigen Phänotyps“ summiert werden. Die onkogenen Veränderungen zeigten sich jedoch in gleicher Weise für Deletionsmutanten und Punktmutationen des β-catenin, so dass eine höhere onkogene Potenz von Deletionsmutanten nicht postuliert werden kann. Die Tumordaten und die molekularen Daten weisen jedoch darauf hin, daß funktionelle Unterschiede zwischen den Mutationstypen bestehen und dass eine Ursache für die Häufung der Deletionsmutanten des β-catenin existiert. Zukünftige Analysen der Deletionsmutanten könnten dabei helfen, eine neue, Domänen-abhängige Interaktion des β-catenin aufzudecken

Regulation of the alternative splicing of RAC1b in tumour cells

Tese de mestrado em Bioquímica, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, em 2018A expressão génica é o mecanismo pelo qual a informação codificada num gene é convertida num produto funcional. A regulação deste processo permite que as células expressem genes diferencialmente consoante o seu tipo, fase de desenvolvimento ou mesmo em resposta a estímulos externos. Portanto, através da regulação da expressão génica, as células conseguem ativar genes seletivamente dependendo das suas necessidades e funções. Um dos processos cruciais envolvidos neste processo regulatório é o splicing do pré-mRNA, que consiste na remoção dos intrões e junção dos exões numa sequência codificante contígua. Esta reação de splicing é catalisada pelo spliceossoma, um complexo ribonucleoproteico que reconhece e interage com as sequências de consenso nos limites dos exões e dos intrões, de forma a direcionar a excisão dos intrões e a ligação dos exões do RNA. Na maioria dos genes humanos a inclusão ou exclusão dos exões no transcrito final ocorre de forma diferencial, tornando-se assim possível a produção de múltiplos mRNAs diferentes a partir de um único gene. Este processo é denominado de splicing alternativo e é um dos mecanismos que modula a regulação da expressão proteica e a produção de um proteoma complexo e diversificado em eucariotas mais complexos. Durante o processo de splicing alternativo a decisão de qual exão é removido e qual é incluído no mRNA final, para além das sequencias de consenso é também fortemente influenciada pela interação entre elementos regulatórios cis e os fatores trans. Os elementos cis ocorrem tanto nas regiões exónicas como nas regiões intrónicas e podem promover a inclusão do exão através do recrutamento do spliceossoma (sequências promotoras) ou promover a sua exclusão por interferência com a ligação do spliceossoma às sequências de consenso (sequências silenciadoras). As sequências promotoras são, geralmente, ligadas por fatores que atuam em trans positivos, como é o caso das proteínas SR, enquanto que as sequências silenciadoras são normalmente ligadas por fatores que atuam em trans negativos, como é o caso das proteínas hnRNPs. Para além da presença de elementos regulatórios cis e fatores trans, uma variedade de outros fatores podem influenciar o splicing alternativo, como é o caso da presença de estruturas secundárias no mRNA, da presença de microRNAs, da arquitetura dos exões e intrões, da força relativa das sequências de consenso no local de splicing e ainda da velocidade de elongação durante a síntese do pré-mRNA pela RNA polimerase II. A soma das contribuições de cada um destes elementos define o potencial de reconhecimento de um exão e a sua respetiva afinidade pelo spliceossoma. Devido ao seu papel central na expressão e função de proteínas, é expectável que problemas ao nível da regulação do splicing alternativo possam resultar no desenvolvimento de doenças. Um exemplo disso é a sobreexpressão da variante de splicing hiperativa do gene RAC1, RAC1b, em diversos tumores malignos. A variante RAC1b é caracterizada pela inserção de um exão (exão 3b) extra entre os exões 3 e 4 de RAC1. Estes 19 aminoácidos extra codificados pelo exão 3b, para além de uma regulação diferente também conferem uma seletividade na sinalização a jusante de RAC1b. Esta variante promove a produção de ROS e a via de sinalização do NF-kB em detrimento de outras vias clássicas ativadas por RAC1, promovendo a progressão do ciclo celular e a sobrevivência das células. Em cancro colorretal, a variante RAC1b encontra-se sobre expressa num subgrupo específico de tumores que também apresentam uma mutação oncogénica em BRAF (BRAFV600E), tendo sido demonstrado a existência de uma cooperação entre estes eventos no sentido de promover a sobrevivência das células tumorais. Até agora, sabe-se que o splicing alternativo de RAC1 é regulado por duas proteínas SR, SRSF1 e SRSF3. SRSF1 promove a inclusão do exão alternativo 3b, ao contrário de SRSF3 que promove a sua exclusão. Ambos os fatores são regulados por vias de sinalização a montante, nomeadamente, os níveis proteicos de SRSF1 aumentam quando a via de sinalização PI3K é inibida, enquanto que a via β-catenin/TCF4 estimula a expressão de SRSF3. É provável que existam outros elementos que regulem o splicing alternativo de RAC1, e recentemente, as proteínas PTBP1 e ESRP1 foram descritas como possíveis modeladoras deste evento em diferentes tipos de células, enquanto que a nucleoporina RANBP2 foi relacionada com a distribuição de proteínas SR fosforiladas, as quais são responsáveis pela regulação da expressão de RAC1b. Neste trabalho experimental foram estudados estes três possíveis modeladores do splicing alternativo de RAC1 em células colorretais. Para estudar estes possíveis novos mecanismos de regulação da expressão de RAC1b em células colorretais, começámos por construir vetores de expressão para PTBP1 e ESRP1, sendo que o vetor de expressão para RANBP2 já se encontrava disponível no laboratório de acolhimento. Posteriormente, os possíveis efeitos da sobreexpressão de PTBP1, de ESRP1 e de RANBP2 no splicing alternativo de RAC1 foram determinados através do uso de um minigene RAC1. Basicamente, cada plasmídeo que codificava as proteínas em estudo foi co-transfetado em células com o minigene RAC1. Os resultados foram observados através de um RT-PCR semi-quantitativo, com primers específicos para os transcritos RAC1 e RAC1b derivados do minigene RAC1. A expressão endógena de RAC1b foi também avaliada por Western blot (WB) através do uso de um anticorpo específico contra RAC1b. De acordo com os resultados, os efeitos significativos que foram observados para ESRP1 e RANBP2 foram confirmados ao nível endógeno, através do uso de siRNAs comercialmente disponíveis de forma a silenciar a sua expressão. Os resultados foram mais uma vez obtidos através de um RT-PCR semi-quantitativo, mas desta vez os primers utilizados foram específicos para os transcritos endógenos. Os níveis proteicos de RAC1b endógeno foram também avaliados através de WB. As experiências foram realizadas principalmente em células epiteliais normais do cólon, NCM460, mas confirmadas com células HeLa e HT29 para determinar se os resultados observados eram dependentes da linha celular. A localização celular das proteínas transfetadas foi visualizada através de ensaios de imunofluorescência em células NCM460 e HeLa. A sobreexpressão de PTBP1, nas células NCM460, não afetou significativamente a inclusão do exão 3b no transcrito final. No entanto, a sobreexpressão de ESRP1 e de RANBP2 promoveu a exclusão do exão 3b. As experiências de imunofluorescência mostraram que a expressão de PTBP1 e ESRP1 ocorre tanto no núcleo como no citoplasma, enquanto que a localização de RANBP2 se restringe essencialmente à membrana nuclear. Esta observação vai de encontro com o esperado, dado que PTBP1 e ESRP1 são ambos fatores de splicing, enquanto que RANBP2 faz parte do complexo do poro nuclear. Nas células NCM460, o silenciamento de ESRP1 diminuiu a expressão endógena de RAC1b, enquanto que a depleção de RANBP2 levou ao aumento de RAC1b. O efeito da depleção de ESRP1 no splicing de RAC1 não corroborou os resultados obtidos nas experiências de sobreexpressão, dando assim a indicação de que este fator de splicing, devido ao seu papel na manutenção do fenótipo epitelial, pode ser altamente regulado por um mecanismo de feedback negativo, no qual regula a sua própria expressão. Os resultados obtidos para as células HeLa e HT29 seguiram a mesma tendência observada nas células NCM460. Assim, no geral, o silenciamento de ESRP1 diminuiu a expressão endógena de RAC1b, enquanto que a depleção de RANBP2 levou ao seu aumento, ambos independentemente da linha celular utilizada, sugerindo que tanto ESRP1 como RANBP2 são reguladores gerais da expressão de RAC1b. Em conclusão, esta tese forneceu fortes evidências de que ESRP1 e RANBP2 estão envolvidos na regulação da expressão de RAC1b e identificou pela primeira vez outros fatores, para além de SRSF1 e de SRSF3, envolvidos na regulação da expressão de RAC1b em células colorretais. Mais experiências são necessárias para esclarecer como é que estas proteínas estão a regular o splicing alternativo de RAC1. Este conhecimento será útil na caracterização dos mecanismos de regulação de splicing alternativo de RAC1 e, eventualmente, para desenvolver moduladores farmacológicos eficazes que possam restaurar a sinalização normal de RAC1 em células tumorais.Regulation of gene expression allows cells to differentially express genes in different cell types, developmental stages or even in response to external conditions. Alternative splicing is a crucial regulatory process in the pathway of gene expression and the mechanism by which multiple protein isoforms can be generated from a single gene. This way, complex organisms can regulate protein expression and generate a more diverse proteome from a given gene number within the genome. Due to its central role in modulating gene expression, it is not surprising that aberrant regulation of alternative splicing is associated with human disease. On example is the selective overexpression of a hyperactive splice variant of the RAC1 gene, RAC1b, in several malignant tumours. RAC1b promotes reactive oxygen species production and the NF-kB pathway activation, but not other classical RAC1 signalling pathways, and stimulates cell cycle progression and cell survival. In colorectal cancer, RAC1b was found to be overexpressed in a specific subgroup, namely in 80% of tumours with mutation in the oncogene BRAF, suggesting that both events cooperate to promote the survival of colorectal cells. Previous studies in colorectal cells showed that the splicing factor SRSF1 acts as an enhancer of RAC1 alternative splicing by promoting the inclusion of alternative exon 3b, while SRSF3 acts as a silencer by promoting the skipping of the exon 3b. Besides SRSF1 and SRSF3 it is likely that RAC1 alternative splicing can be regulated by additional factors, and recently, PTBP1 and ESRP1 were described as possible modulators of the alternative splicing of RAC1b in different cell types while RANBP2 was shown to be a modulator of the distribution of phosphorylated SR proteins, which are known to regulate RAC1b expression. To study whether these factors regulate RAC1b expression in colorectal cells, we started by analysing the effects of PTBP1, ESRP1 and RANBP2 overexpression on RAC1 alternative splicing. For that, each expression vector encoding the proteins in study was co-transfected with the RAC1 minigene into cells. The results were then assessed through a semi-quantitative RT-PCR, with specific primers for RAC1 and RAC1b transcripts derived from the RAC1 minigene. Effects on endogenous RAC1b expression was also assayed by Western blot and cellular localization of the transfected proteins assessed by immunofluorescence. Positive effects were then confirmed at the endogenous protein level through the use of commercially available siRNAs to deplete the regulators. The experiments were mainly performed in NCM460 colon cells but confirmed using HeLa and HT29 cells to determine if the observed results were cell line dependent. As expected, immunofluorescence experiments showed that both PTBP1 and ESRP1 can be found in the nucleus and cytoplasm, while RANBP2 is found at the nuclear membrane and also at the cytoplasm. RANBP2 overexpression was found to promote the skipping of the alternative exon 3b, while its depletion promoted exon 3b inclusion. Thus, RANBP2 emerged as a candidate negative regulator of RAC1 alternative splicing that promotes the skipping of exon 3b. In the case of ESRP1, both overexpression and depletion promoted the skipping of the alternative exon 3b. ESRP1 overexpression might interfere with a negative feedback mechanism, in which ESRP1 regulates its own expression, due to its role in maintaining the epithelial phenotype. Based on the depletion experiment, ESRP1 can also be considered a candidate positive regulator of RAC1 alternative splicing, which promotes the inclusion of exon 3b, unlike RANBP2. No significant effect of PTBP1 on RAC1 alternative splicing was observed. In conclusion, this thesis provided strong evidence that ESRP1 and RANBP2 are involved in RAC1b expression regulation and identified for the first time other factors besides SRSF1 and SRSF3 that are involved in the regulation of RAC1b expression in colorectal cells. Further experiments are needed to clarify how these proteins are regulating RAC1 alternative splicing. This knowledge will be useful to characterize RAC1 alternative splicing regulation mechanisms and eventually to develop effective pharmacological modulators that can restore normal RAC1 signalling in tumour cells

Molecular subtypes of intestinal-type gastric cancer: Association with T-lymphocytes and formins

Despite the decline of gastric cancer (GC) incidence in Western countries over the last decade, it is still one of the most significant causes of cancer mortality worldwide. The traditional morphology-based grading systems, including the world health organization (WHO) and Lauren's grading systems, have limited applicability in managing treatment choices, as they poorly catch the molecular heterogeneity of GC. Thus, classifications based on molecular features are needed. Recent genome analyses have shown that GC consists of several molecular subtypes characterized by distinct alterations. In our study, we used tissue-based methods, i.e., immunohistochemistry and in situ hybridization, in the molecular classification of GC, emphasizing the intestinal subtype. Our results show that GC can be divided into four nonoverlapping subtypes based on Epstein-Barr virus (EBV) positivity, mismatch repair protein (MMR), and TP53 aberration status. In conclusion, GC molecular subtyping can be performed with a simple methodology applicable to clinical routine. Host immune response is an important predictive and prognostic factor in many cancer types, including GC. Detailed information on the accumulation of tumorinfiltrating T lymphocytes in the different molecular GC subtypes and their prognostic correlation is scarce. We analyzed the presence of CD3+, CD8+, and FOXP3+ (Forkhead box P3) T lymphocytes in the molecular subtypes of intestinal-type GC. We found that EBV+ cancers harbor increased lymphocyte infiltration and a high CD8+/FOXP3+ ratio. In addition, we found that high numbers of CD8+ and CD3+ T lymphocytes are associated with better survival, and their accumulation is an independent prognostic factor. Formin proteins regulate the actin cytoskeleton and cell migration and play an essential role in cancer call functions. However, the expression and clinical association of formins in GC remains largely undiscovered. Here we analyzed the expression of FHOD1 and FMNL1 formins in GC cell lines and clinical samples of intestinal- type GC. We found that FHOD1 expression in cancer cells correlated with high intratumoral CD8+ T lymphocyte infiltration. Reduced FHOD1 expression was seen in the tumors with aberrant TP53. FMNL1 expression in cancer cells was associated with the size of the tumors and the stage of the disease. The results demonstrate a link between FHOD1 and FMNL1 expression with biological features of GC. However, we did not find a correlation between formin expression and GC prognosis.Suolistotyyppisen mahasyövän molekyylityypit: Assosiaatio T-lymfosyyttien ja forminien kanssa Vaikka mahasyövän esiintyminen on viime vuosikymmeninä laskenut kehittyneissä maissa, se on edelleen yksi tärkeimmistä syöpäkuolemien aiheuttajista. Mahasyöpä on biologialtaan monimuotoinen. Tästä syystä perinteinen kudosmorfologiaan perustua luokittelu, kuten WHO:n tai Laurénin luokitus, hyödyttää hoitopäätöksien tekoa vain rajallisesti. Molekulaarisiin piirteisiin perustuvan luokituksen kehittäminen olisikin tärkeää. Genomianalyyseihin perustuva tutkimus on osoittanut, että mahasyöpä koostuu useista molekulaarisista alatyypeistä. Analyysimenetelmät ovat kuitenkin monimutkaisia ja vaativat erityisosaamista. Tutkimuksessamme käytimme kliiniseen käyttöön soveltuvia menetelmiä; immunohistokemiaa ja in situ-hybridisaatiota mahasyöpien luokitteluun. Tulosten perusteella yksinkertaisen algoritmin avulla mahasyövät voidaan luokitella eri alaryhmiin Ebstain-Barr virus-positiivisuuden, TP53 poikkeavuuden ja MMR-puutoksen perusteella. Kliinisesti eroaviin alaryhmiin tapahtuva jaottelu voidaan siis toteuttaa yksinkertaisella ja kliiniseen diagnostiikkaan soveltuvalla menetelmällä Immuunijärjestelmän toiminta on tärkeä syövän ennusteen kannalta. Yksityiskohtaista tietoa siitä, miten T solut hakeutuvat eri mahasyövän alatyyppeihin ja liittyvät taudin käyttäytymiseen ei toistaiseksi ole. Tässä tutkimuksessa analysoimme CD3, CD8 ja FOXP3 antigeenejä ilmentävien T-lymfosyyttien esiintymistä intestinaalisen mahasyövän alatyypeissä. Tulosten perusteella EBV+ syövissä on runsaimmin T-lymfosyyttejä ja korkein CD8+/FOXP3-suhde. Totesimme myös, että runsas T-lymfosyyttien määrä korreloi intestinaalisen mahasyövän parempaan ennusteeseen ja toimii itsenäisenä ennustetekijänä. Formiiniproteiinit säätelevät solujen aktiinitukirankaa ja solujen migraatiota, ja ovat tärkeitä syöpäsolujen toiminnassa. Kuitenkin formiinien esiintymistä ja tehtäviä mahasyövässä tunnetaan huonosti. Tässä tutkimuksessa analysoimme FHOD1 ja FMNL1 formiineja mahasyöpäsolulinjoissa ja intestinaalista alatyyppiä edustavien mahasyöpien kudosnäytteissä. Kasvainsolujen FHOD1:n ilmentyminen oli yhteydessä korkeaan intratumoraalisten T-lymfosyyttien määrään. FHOD1:n alentunutta ekspressiota nähtiin syövän alatyypissä, johon liittyy TP53 mutaatio. FMNL1:n ilmentyminen puolestaan korreloi kasvaimen kokoon ja taudin leviämisasteeseen. Tulosten perusteella FHOD1 ja FMNL1-formiinien ilmentyminen liittyy mahasyövän biologisiin piirteisiin, joskaan ilmentymisellä ei näytä olevan yhteyttä mahasyövän ennusteeseen

Tumor expression of cyclin-dependent kinase 5 (Cdk5) is a prognostic biomarker and predicts outcome of oxaliplatin-treated metastatic colorectal cancer patients

In recent years, an increasing number of studies have shown that elevated expression of cyclin dependent kinase (Cdk5) contributes to the oncogenic initiation and progression of many types of cancers. In this study, we investigated the expression pattern of Cdk5 in colorectal cancer (CRC) cell lines and in a large number of tumor samples in order to evaluate its relevance in this pathogenesis and possible use as a prognostic marker. We found that Cdk5 is highly expressed and activated in CRC cell lines and that silencing of the kinase decreases their migration ability. In tumor tissues, Cdk5 is overexpressed compared to normal tissues due to a copy number gain. In patients with localized disease, we found that high Cdk5 levels correlate with poor prognosis, while in the metastatic setting, this was only the case for patients receiving an oxaliplatin-based treatment. When exploring the Cdk5 levels in the consensus molecular subtypes (CMS), we found the lowest levels in subtype 1, where high Cdk5 again was associated with a poorer prognosis. In conclusion, we confirm that Cdk5 is involved in CRC and disease progression and that it could serve as a prognostic and predictive biomarker in this disease