PRL-3 overexpression in epithelial cells is induced by surrounding stromal fibroblasts

We isolate and culture carcinoma-associated fibroblasts (CAFs) from primary tumour (CAFpt), CAFs from corresponding synchronous liver metastasis (CAFlm) as well as normal colonic fibroblasts (NCF) from the same patient. From these cultures, conditioned media (CM) was obtained. Culture of a wide panel of colorectal and pancreatic cell lines in CM from CAFlm resulted in overexpression of mRNA PRL-3 and higher overexpression in CAFs than in non-activated fibroblasts. Moreover PRL-3 mRNA expression correlates with expression of α-SMA and deposition of collagen fibrils in the stroma. We demonstrate that products secreted by CAFs trigger PRL-3 overexpression in cancer cells. Identification of these factors may contribute to new stroma-targeted therapies for desmoplastic tumours

Hepatic Carcinoma—Associated Fibroblasts Promote an Adaptative Response in Colorectal Cancer Cells That Inhibit Proliferation and Apoptosis: Nonresistant Cells Die by Nonapoptotic Cell Death

AbstractCarcinoma-associated fibroblasts (CAFs) are important contributors of microenvironment in determining the tumor’s fate. This study aimed to compare the influence of liver microenvironment and primary tumor microenvironment on the behavior of colorectal carcinoma. Conditioned medium (CM) from normal colonic fibroblasts (NCFs), CAFs from primary tumor (CAF-PT) or liver metastasis (CAF-LM) were obtained. We performed functional assays to test the influence of each CM on colorectal cell lines. Microarray and gene set enrichment analysis (GSEA) were performed in DLD1 cells cultured in matched CM. In DLD1 cells, CAF-LM CM compared with CAF-PT CM and NCF led to a more aggressive phenotype, induced the features of an epithelial-to-mesenchymal transition more efficiently, and stimulated migration and invasion to a greater extent. Sustained stimulation with CAF-LM CM evoked a transient G2/M cell cycle arrest accompanied by a reduction of apoptosis, inhibition of proliferation, and decreased viability of SW1116, SW620, SW480, DLD1, HT-29, and Caco-2 cells and provoked nonapoptotic cell death in those cells carrying KRAS mutations. Cells resistant to CAF-LM CM completely changed their morphology in an extracellular signal-regulated protein kinase-dependent process and depicted an increased stemness capacity alongside the Wnt pathway stimulation. The transcriptomic profile of DLD1 cells treated with CAF-LM CM was associated with Wnt and mitogen-activated protein kinase pathways activation in GSEA. Therefore, the liver microenvironment induces more efficiently the aggressiveness of colorectal cancer cells than other matched microenvironments do but secondarily evokes cell death. Resistant cells displayed higher stemness capacity

Hepatic carcinoma-associated fibroblasts promote an adaptative response in colorectal cancer cells that inhibit proliferation and apoptosis: nonresistant cells die by nonapoptotic cell death

Carcinoma-associated fibroblasts (CAFs) are important contributors of microenvironment in determining the tumor's fate. This study aimed to compare the influence of liver microenvironment and primary tumor microenvironment on the behavior of colorectal carcinoma. Conditioned medium (CM) from normal colonic fibroblasts (NCFs), CAFs from primary tumor (CAF-PT) or liver metastasis (CAF-LM) were obtained. We performed functional assays to test the influence of each CM on colorectal cell lines. Microarray and gene set enrichment analysis (GSEA) were performed in DLD1 cells cultured in matched CM. In DLD1 cells, CAF-LM CM compared with CAF-PT CM and NCF led to a more aggressive phenotype, induced the features of an epithelial-to-mesenchymal transition more efficiently, and stimulated migration and invasion to a greater extent. Sustained stimulation with CAF-LM CM evoked a transient G(2)/M cell cycle arrest accompanied by a reduction of apoptosis, inhibition of proliferation, and decreased viability of SW1116, SW620, SW480, DLD1, HT-29, and Caco-2 cells and provoked nonapoptotic cell death in those cells carrying KRAS mutations. Cells resistant to CAF-LM CM completely changed their morphology in an extracellular signal-regulated protein kinase-dependent process and depicted an increased stemness capacity alongside the Wnt pathway stimulation. The transcriptomic profile of DLD1 cells treated with CAF-LM CM was associated with Wnt and mitogen-activated protein kinase pathways activation in GSEA. Therefore, the liver micro-environment induces more efficiently the aggressiveness of colorectal cancer cells than other matched micro-environments do but secondarily evokes cell death. Resistant cells displayed higher stemness capacity

Differences between CAFs and their paired NCF from adjacent colonic mucosa reveal functional heterogeneity of CAFs, providing prognostic information

Little is known about the difference in gene expression between carcinoma-associated fibroblasts (CAFs) and paired normal colonic fibroblasts (NCFs) in colorectal cancer. Paired CAFs and NCFs were isolated from eight primary human colorectal carcinoma specimens. In culture conditions, soluble factors secreted by CAFs in the conditioned media increased clonogenicity and migration of epithelial cancer cells lines to a greater extent than did NCF. In vivo, CAFs were more competent as tumour growth enhancers than paired NCFs when co-inoculated with colorectal cell lines. Gene expression analysis of microarrays of CAF and paired NCF populations enabled us to identify 108 deregulated genes (38 upregulated and 70 downregulated genes). Most of those genes are fibroblast-specific. This has been validated in silico in dataset GSE39396 and by qPCR in selected genes. GSEA analysis revealed a differential transcriptomic profile of CAFs, mainly involving the Wnt signallingsignalling pathway, focal adhesion and cell cycle. Both deregulated genes and biological processes involved depicted a considerable degree of overlap with deregulated genes reported in breast, lung, oesophagus and prostate CAFs. These observations suggest that similar transcriptomic programs may be active in the transition from normal fibroblast in adjacent tissues to CAFs, independently of their anatomic demarcation. Additionally NCF already depicted an activated pattern associated with inflammation. The deregulated genes signature score seemed to correlate with CAF tumour promoter abilities in vitro, suggesting a high degree of heterogeneity between CAFs, and it has also prognostic value in two independent datasets. Further characterization of the roles these biomarkers play in cancer will reveal how CAFs provide cancer cells with a suitable microenvironment and may help in the development of new therapeutic targets for cancer treatment

A 5-gene classifier from the carcinoma-associated fibroblast transcriptomic profile and clinical outcome in colorectal cancer

Based on 108 differentially expressed genes between carcinoma-associated fibroblasts (CAFs) and paired normal colonic fibroblasts we recently reported, a 5-gene classifier for relapse prediction in Stage II/III colorectal cancer (CRC ) was developed. Its predictive value was validated in datasets GSE17538, GSE33113 and GSE14095. An additional validation was performed in a metacohort (n=317) and 142 CRC patients by means of RT-PCR. The 5-gene classifier was significantly associated with increased relapse risk and death from CRC across all validation series of Stage II/III patients used. Multivariate Cox regression analyses confirmed the independent prognostic value of the stromal classifier (HR=2.67; P=0.002). Post-test probabilities provided evidence of the suitability of the 5-gene classifier in clinical practice, identifying a subgroup of Stage-II patients who were at high risk of relapse. Moreover, the a priory worst prognosis mesenchymal subtype of tumours can be stratified according to the physiological status of their carcinoma-associated fibroblasts. In conclusion the CAFs-derived 5-gene classifier provides more accurate information about outcome than conventional clinicopathological criteria and it could be useful to take clinical decisions, especially in Stage II. Additionally, the classifier put into relevance the CAF's intratumoral heterogeneity and might contribute to find relevant targets for depleting adequate CAFS subtypes

Carcinoma-associated fibroblasts affect sensitivity to oxaliplatin and 5FU in colorectal cancer cells

The importance of tumor microenvironment (TME) as a relevant contributor to cancer progression and its role in the development of de novo resistance to targeted therapies has become increasingly apparent. However, the mechanisms of microenvironment-mediated drug resistance for nonspecific conventional chemotherapeutic agents, such as platinum compounds or antimetabolites, are still unclear. Here we describe a mechanism induced by soluble factors released by carcinoma-associated fibroblasts (CAFs) that induce the translocation of AKT, Survivin and P38 to the nucleus of tumor cells. These changes are guided to ensure DNA repair and the correct entrance and exit from mitosis in the presence of chemotherapy. We used conditioned media (CM) from normal-colonic fibroblasts and paired CAFs to assess dose response curves of oxaliplatin and 5-fluorouracil, separately or combined, compared with standard culture medium. We also evaluated a colony-forming assay and cell death to demonstrate the protective role of CAF-CM. Immunofluorescence confirmed the translocation of AKT, P38 and Survivin to the nucleus induced by CAF-soluble factors. We also have shown that STAT3 or P38 inhibition provides a promising strategy for overcoming microenvironment-mediated resistance. Conversely, pharmacologic AKT inhibition induces an antagonistic effect that relieves a cMET and STAT3-mediated compensatory feedback that might explain the failure of AKT inhibitors in the clinic so far

Implicació funcional i prognosi dels fibroblasts associats a carcinoma en el càncer colorectal

El procés tumorigènic ha estat descrit com un procés gradual en el que les cèl·lules normals van acumulant alteracions genètiques que els hi confereixen avantatges selectives de creixement i les transformen cap a un fenotip maligne. Aquesta visió tant focalitzada en la cèl·lula tumoral ha anat evolucionant i actualment un tumor ja no és considerat exclusivament com una massa de cèl·lules tumorals, sinó que és vist com un teixit complex compost per multitud de cèl·lules no malignes que constitueixen l’estroma tumoral, imprescindible en les diferents etapes del procés carcinogènic. Constituït per una matriu extracel·lular que envolta diferents subtipus cel·lulars (cèl·lules angiogèniques, immunitàries i fibroblasts associats a carcinoma), les interaccions directes o indirectes que s’estableixen entre els seus components i les cèl·lules neoplàsiques són claus per a l’evolució del procés. Els fibroblasts associats a carcinoma o CAFs (“Carcinoma associated fibroblasts”), són els principals components de tumors sòlids com el de mama, pulmó o còlon; caracteritzats per un elevat grau de desmoplasia. Els CAFs, denominats també miofibroblasts per l’expressió de l’alfa actina del múscul llis (α-SMA), presenten una elevada heterogeneïtat tant a nivell molecular com genètic. La identificació de biomarcadors específics que en permetin la seva distinció de la resta de fibroblasts aliens al tumor, es presenta com un repte important tant en el pronòstic com en el tractament d’aquest tipus de tumors. El present treball es centra en els CAFs del càncer colorectal. Compara tant a nivell funcional com transcripcional els fibroblasts presents en les diferents demarcacions anatòmiques amb els que es pot trobar la cèl·lula neoplàsica al llarg de la progressió tumoral: els fibroblasts de la mucosa colònica normal adjacent al tumor primari (NCF), els fibroblasts associats al tumor primari (CAF-PT), i els fibroblasts associats a la metàstasi hepàtica (CAF-LM). Fent ús de la tecnologia dels microarrays, s’han analitzat els perfils d’expressió dels diferents fibroblasts per a identificar biomarcadors moleculars diferencials, útils com a possibles dianes terapèutiques. També s’han definit un seguit de signatures genètiques estromals amb valor pronòstic. Són dues les aproximacions que s’han dut a terme en aquesta tesi. Per una banda, tot i que s’ha vist que els miofibroblasts de les diferents localitzacions anatòmiques del carcinoma colorectal presenten un perfil transcriptòmic diferent, s’ha definit una signatura d’expressió gènica comú a les tres demarcacions i gradual al llarg de la seqüència de progressió tumoral. Així mateix, amb només 19 gens d’aquesta signatura comuna s’ha vist que es poden classificar els pacients afectats de carcinoma colorectal estadis II/III en funció del seu risc de recurrència. Per altra banda, i tenint en compte l’elevat grau d’heterogeneïtat dels miofibroblasts, l’ús de mostres aparellades de fibroblasts de tumor primari i de la mucosa normal, ha permès la identificació d’una signatura de gens diferencialment expressats entre ambdues poblacions. Conèixer gens diferencialment expressats pot ser molt útil alhora d’identificar possibles dianes terapèutiques, a l’estar presents en els fibroblasts associats a carcinoma del càncer colorectal però no en la resta de fibroblasts. A partir dels 108 gens desregulats, a més, s’ha identificat també un classificador genètic constituït únicament per 5 gens estromals i que proporciona una informació més acurada del risc de recurrència que les variables clíniques convencionals. L’anàlisi de la seva expressió sembla facilitar la selecció de pacients estadi II d’alt risc que podrien beneficiar-se amb el tractament de quimioteràpia adjuvant. Aquest classificador ha estat validat tant en dades d’expressió de microarrays independents (validació in silico), com mitjançant la tècnica de la qRT-PCR (PCR quantitativa a temps real); molt més manejable en la pràctica clínica diària. Així doncs, en aquesta tesi es mostra la importància que té l’estroma tumoral en el càncer colorectal, especialment els CAFs (independentment de la seva quantitat), i remarca la rellevància que té el seu estat transcripcional en la prognosi. Així mateix es corrobora que és un tipus cel·lular molt heterogeni i per tant, múltiples i diferents respostes biològiques es poden trobar presents alhora en un mateix tumor; sent el balanç net final entre aquestes respostes el que proveeix el valor pronòstic.Tumorogenesis has been described as a multistep process where epithelial normal cells accumulate genetic alterations that confer them with selective advantages that transform them into a malignant phenotype. However, in recent years, the role of microenvironment has being getting more important and a tumour is not longer considered as a tumour cell mass, but as a complex tissue. Tumour microenvironment is the cellular environment in which the tumour exists, including angiogenic cells, immune cells, carcinoma associated fibroblasts and the extracellular matrix. The tumour and the surrounding microenvironment are closely related and interact with each other influencing and determining the tumour’s fate. Carcinoma associated fibroblasts (CAFs) are the main constitutors of that tumour stroma, especially in solid tumours such as breast, lung and colon; characterized by a high degree of desmoplasia. CAFs, also known as myofibroblasts due to their expression of α-smooth muscle actin (α-SMA), are highly heterogeneous both at molecular and genetic level. Identification of genetic/molecular markers specific of CAFs but not expressed in other fibroblasts, could be useful both in treatment and prognosis. The current work is focus on carcinoma associated fibroblasts from colorectal cancer (CAFs). It compares both at functional and transcriptional level, fibroblasts from different anatomic demarcations involved in colorectal tumour progression: fibroblasts from normal colonic mucosa adjacent to primary tumour (NCF), carcinoma associated fibroblasts from primary tumour (CAF-PT), and carcinoma associated fibroblasts from liver metastases (CAF-LM). Using microarray technology, analysis of gene expression profile from different fibroblasts has been performed in order to identify for differentially molecular biomarkers that could be useful as a therapeutic targets. As well as, prognostic stromal gene signatures have been defined. Two main approximations have been performed in this work. Firstly, although we have seen that myofibroblasts from different anatomic demarcations in colorectal cancer show different gene expression profile; we have been able to identify a common gene expression signature make up of genes whose expression is sequential and scalable in tumour progression (NCF-CAF-PT and CAF-LM). Only 19 of these common genes from the fibroblast progression signature are enough to classify colorectal cancer patients stage II/III in terms of risk recurrence. Additionally, and considering high heterogeneity of myofibroblasts, using matched samples of carcinoma associated fibroblasts from primary tumour and from normal colonic mucosa, allows us to identify a list of differentially expressed genes between both populations. Identification of differentially expressed genes in one population but not in the other could be useful to identify new molecular biomarkers and therapeutic targets specific for CAFs. We have refined the list of de-regulated gens defining a 5-gene stromal classifier which provides risk recurrence information more accurately than conventional clinical variables do; and also allows better identification of stage II colorectal cancer patients at high risk of recurrence, who could benefit with adjuvant treatment. The stromal classifier has been validated in microarray data from independent datasets (in silico validation) and also by means of qRT-PCR (quantitative real time PCR), more affordable in clinical practice. To summarize, this work shows how important tumour stroma is in colorectal cancer, specially, carcinoma associated fibroblasts. Their transcriptional state seems to be more relevant in colorectal cancer prognosis than their proportion in the tumour stroma. As well as, we confirm that CAFs are highly heterogeneous and different biological responses could be present at the same time in the same tumour determining the final prognosis

Implicació funcional i prognosi dels fibroblasts associats a carcinoma en el càncer colorectal

El procés tumorigènic ha estat descrit com un procés gradual en el que les cèl·lules normals van acumulant alteracions genètiques que els hi confereixen avantatges selectives de creixement i les transformen cap a un fenotip maligne. Aquesta visió tant focalitzada en la cèl·lula tumoral ha anat evolucionant i actualment un tumor ja no és considerat exclusivament com una massa de cèl·lules tumorals, sinó que és vist com un teixit complex compost per multitud de cèl·lules no malignes que constitueixen l'estroma tumoral, imprescindible en les diferents etapes del procés carcinogènic. Constituït per una matriu extracel·lular que envolta diferents subtipus cel·lulars (cèl·lules angiogèniques, immunitàries i fibroblasts associats a carcinoma), les interaccions directes o indirectes que s'estableixen entre els seus components i les cèl·lules neoplàsiques són claus per a l'evolució del procés. Els fibroblasts associats a carcinoma o CAFs ("Carcinoma associated fibroblasts"), són els principals components de tumors sòlids com el de mama, pulmó o còlon; caracteritzats per un elevat grau de desmoplasia. Els CAFs, denominats també miofibroblasts per l'expressió de l'alfa actina del múscul llis (α-SMA), presenten una elevada heterogeneïtat tant a nivell molecular com genètic. La identificació de biomarcadors específics que en permetin la seva distinció de la resta de fibroblasts aliens al tumor, es presenta com un repte important tant en el pronòstic com en el tractament d'aquest tipus de tumors. El present treball es centra en els CAFs del càncer colorectal. Compara tant a nivell funcional com transcripcional els fibroblasts presents en les diferents demarcacions anatòmiques amb els que es pot trobar la cèl·lula neoplàsica al llarg de la progressió tumoral: els fibroblasts de la mucosa colònica normal adjacent al tumor primari (NCF), els fibroblasts associats al tumor primari (CAF-PT), i els fibroblasts associats a la metàstasi hepàtica (CAF-LM). Fent ús de la tecnologia dels microarrays, s'han analitzat els perfils d'expressió dels diferents fibroblasts per a identificar biomarcadors moleculars diferencials, útils com a possibles dianes terapèutiques. També s'han definit un seguit de signatures genètiques estromals amb valor pronòstic. Són dues les aproximacions que s'han dut a terme en aquesta tesi. Per una banda, tot i que s'ha vist que els miofibroblasts de les diferents localitzacions anatòmiques del carcinoma colorectal presenten un perfil transcriptòmic diferent, s'ha definit una signatura d'expressió gènica comú a les tres demarcacions i gradual al llarg de la seqüència de progressió tumoral. Així mateix, amb només 19 gens d'aquesta signatura comuna s'ha vist que es poden classificar els pacients afectats de carcinoma colorectal estadis II/III en funció del seu risc de recurrència. Per altra banda, i tenint en compte l'elevat grau d'heterogeneïtat dels miofibroblasts, l'ús de mostres aparellades de fibroblasts de tumor primari i de la mucosa normal, ha permès la identificació d'una signatura de gens diferencialment expressats entre ambdues poblacions. Conèixer gens diferencialment expressats pot ser molt útil alhora d'identificar possibles dianes terapèutiques, a l'estar presents en els fibroblasts associats a carcinoma del càncer colorectal però no en la resta de fibroblasts. A partir dels 108 gens desregulats, a més, s'ha identificat també un classificador genètic constituït únicament per 5 gens estromals i que proporciona una informació més acurada del risc de recurrència que les variables clíniques convencionals. L'anàlisi de la seva expressió sembla facilitar la selecció de pacients estadi II d'alt risc que podrien beneficiar-se amb el tractament de quimioteràpia adjuvant. Aquest classificador ha estat validat tant en dades d'expressió de microarrays independents (validació in silico), com mitjançant la tècnica de la qRT-PCR (PCR quantitativa a temps real); molt més manejable en la pràctica clínica diària. Així doncs, en aquesta tesi es mostra la importància que té l'estroma tumoral en el càncer colorectal, especialment els CAFs (independentment de la seva quantitat), i remarca la rellevància que té el seu estat transcripcional en la prognosi. Així mateix es corrobora que és un tipus cel·lular molt heterogeni i per tant, múltiples i diferents respostes biològiques es poden trobar presents alhora en un mateix tumor; sent el balanç net final entre aquestes respostes el que proveeix el valor pronòstic.Tumorogenesis has been described as a multistep process where epithelial normal cells accumulate genetic alterations that confer them with selective advantages that transform them into a malignant phenotype. However, in recent years, the role of microenvironment has being getting more important and a tumour is not longer considered as a tumour cell mass, but as a complex tissue. Tumour microenvironment is the cellular environment in which the tumour exists, including angiogenic cells, immune cells, carcinoma associated fibroblasts and the extracellular matrix. The tumour and the surrounding microenvironment are closely related and interact with each other influencing and determining the tumour's fate. Carcinoma associated fibroblasts (CAFs) are the main constitutors of that tumour stroma, especially in solid tumours such as breast, lung and colon; characterized by a high degree of desmoplasia. CAFs, also known as myofibroblasts due to their expression of α-smooth muscle actin (α-SMA), are highly heterogeneous both at molecular and genetic level. Identification of genetic/molecular markers specific of CAFs but not expressed in other fibroblasts, could be useful both in treatment and prognosis. The current work is focus on carcinoma associated fibroblasts from colorectal cancer (CAFs). It compares both at functional and transcriptional level, fibroblasts from different anatomic demarcations involved in colorectal tumour progression: fibroblasts from normal colonic mucosa adjacent to primary tumour (NCF), carcinoma associated fibroblasts from primary tumour (CAF-PT), and carcinoma associated fibroblasts from liver metastases (CAF-LM). Using microarray technology, analysis of gene expression profile from different fibroblasts has been performed in order to identify for differentially molecular biomarkers that could be useful as a therapeutic targets. As well as, prognostic stromal gene signatures have been defined. Two main approximations have been performed in this work. Firstly, although we have seen that myofibroblasts from different anatomic demarcations in colorectal cancer show different gene expression profile; we have been able to identify a common gene expression signature make up of genes whose expression is sequential and scalable in tumour progression (NCF-CAF-PT and CAF-LM). Only 19 of these common genes from the fibroblast progression signature are enough to classify colorectal cancer patients stage II/III in terms of risk recurrence. Additionally, and considering high heterogeneity of myofibroblasts, using matched samples of carcinoma associated fibroblasts from primary tumour and from normal colonic mucosa, allows us to identify a list of differentially expressed genes between both populations. Identification of differentially expressed genes in one population but not in the other could be useful to identify new molecular biomarkers and therapeutic targets specific for CAFs. We have refined the list of de-regulated gens defining a 5-gene stromal classifier which provides risk recurrence information more accurately than conventional clinical variables do; and also allows better identification of stage II colorectal cancer patients at high risk of recurrence, who could benefit with adjuvant treatment. The stromal classifier has been validated in microarray data from independent datasets (in silico validation) and also by means of qRT-PCR (quantitative real time PCR), more affordable in clinical practice. To summarize, this work shows how important tumour stroma is in colorectal cancer, specially, carcinoma associated fibroblasts. Their transcriptional state seems to be more relevant in colorectal cancer prognosis than their proportion in the tumour stroma. As well as, we confirm that CAFs are highly heterogeneous and different biological responses could be present at the same time in the same tumour determining the final prognosis