Organoids from colorectal peritoneal metastases as a platform for improving hyperthermic intraperitoneal chemotherapy.

BACKGROUND: Patients with peritoneal metastases from colorectal cancer have a poor prognosis. If the intraperitoneal tumour load is limited, patients may be eligible for cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (HIPEC). This treatment has improved overall survival, but recurrence rates are high. The aim of this study was to create a preclinical platform for the development of more effective intraperitoneal chemotherapy strategies. METHODS: Using organoid technology, five tumour cultures were generated from malignant ascites and resected peritoneal metastases. These were used in an in vitro HIPEC model to assess sensitivity to mitomycin C (MMC) and oxaliplatin, the drugs used most commonly in HIPEC. The model was also used to test a rational combination treatment involving MMC and inhibitors of the checkpoint kinase ATR. RESULTS: MMC was more effective in eliminating peritoneal metastasis-derived organoids than oxaliplatin at clinically relevant concentrations. However, the drug concentrations required to eliminate 50 per cent of the tumour cells (IC50) were higher than the median clinical dose in two of five organoid lines for MMC, and all five lines for oxaliplatin, indicating a general resistance to monotherapy. ATR inhibition increased the sensitivity of all peritoneal metastasis-derived organoids to MMC, as the IC50 decreased 2·6-12·4-fold to well below concentrations commonly attained in clinical practice. Live-cell imaging and flow cytometric analysis showed that ATR inhibition did not release cells from MMC-induced cell cycle arrest, but caused increased replication stress and accelerated cell death. CONCLUSION: Peritoneal metastasis-derived organoids can be used to evaluate existing HIPEC regimens on an individual-patient level and for development of more effective treatment strategies. Surgical relevance Cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (HIPEC) has improved prognosis of patients with peritoneal metastases from colorectal cancer, but disease recurrence is common. More effective and personalized HIPEC is urgently needed. Organoid technology is frequently used for drug screens, as patient-derived organoids can accurately predict clinical therapeutic response in vitro. A panel of organoids was established from peritoneal metastases from colorectal cancer and used to develop a model for testing HIPEC regimens in vitro. Patient-derived organoids differed in sensitivity to commonly used chemotherapeutics, in line with variable clinical outcomes following cytoreductive surgery-HIPEC. Combining MMC with an ATR inhibitor improved the efficacy of MMC. Peritoneal metastasis-derived organoids can be used as a platform to test novel (combination) strategies that increase HIPEC efficacy. In the future, organoids could be used to select patent-tailored HIPEC regimens.

ANTECEDENTES: Los pacientes con metástasis peritoneales (peritoneal metastasis, PM) de cáncer colorrectal tienen un mal pronóstico. Si la carga tumoral intraperitoneal es reducida, los pacientes pueden ser candidatos a cirugía citorreductora seguida de quimioterapia intraperitoneal hipertérmica (hyperthermic intraperitoneal chemotherapy, HIPEC). Este tratamiento ha mejorado la supervivencia global, pero las tasas de recidiva son altas. El objetivo de este estudio fue crear una plataforma preclínica para el desarrollo de las estrategias de quimioterapia intraperitoneal más efectivas. MÉTODOS: Mediante la utilización de la tecnología de organoides, se generaron cinco cultivos tumorales a partir de ascitis maligna y PM resecadas. Se utilizó un modelo de HIPEC in vitro para evaluar la sensibilidad a la mitomicina C (mitomycin C, MMC) y al oxaliplatino, los fármacos más utilizados en la HIPEC. El modelo también se usó para probar un tratamiento combinado de MMC e inhibidores de control inmunitario de la quinasa ATR. RESULTADOS: A concentraciones clínicamente relevantes, la MMC fue más efectiva que el oxaliplatino para eliminar los organoides derivados de PM. Sin embargo, las concentraciones de fármaco necesarias para eliminar el 50% de las células tumorales (IC50) fueron más elevadas que la mediana de la dosis clínica en 2/5 (MMC) o 5/5 (oxaliplatino) de las líneas de organoides, lo que indica una resistencia general a la monoterapia. La inhibición de ATR aumentó la sensibilidad a MMC de todos los organoides derivados de PM, ya que la IC50 disminuyó (2,6-12,4 veces) a concentraciones muy por debajo de las que se alcanzan comúnmente en la práctica clínica. Los análisis de viabilidad celular y de citometría de flujo (FACS) mostraron que la inhibición de ATR no liberaba células tras la detención del ciclo celular inducida por la MMC, sino que causaba un aumento en el estrés replicativo y muerte celular acelerada. CONCLUSIÓN: Se pueden usar los organoides derivados de PM para evaluar los regímenes HIPEC existentes a nivel del paciente individual y para desarrollar estrategias terapéuticas más efectivas.

Histopathological and molecular classification of colorectal cancer and corresponding peritoneal metastases.

BACKGROUND: Patients with colorectal peritoneal carcinomatosis have a very poor prognosis. The recently developed consensus molecular subtype (CMS) classification of primary colorectal cancer categorizes tumours into four robust subtypes, which could guide subtype-targeted therapy. CMS4, also known as the mesenchymal subtype, has the greatest propensity to form distant metastases. CMS4 status and histopathological features of colorectal peritoneal carcinomatosis were investigated in this study. METHODS: Fresh-frozen tissue samples from primary colorectal cancer and paired peritoneal metastases from patients who underwent cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy were collected. Histopathological features were analysed, and a reverse transcriptase-quantitative PCR test was used to assess CMS4 status of all collected lesions. RESULTS: Colorectal peritoneal carcinomatosis was associated with adverse histopathological characteristics, including a high percentage of stroma in both primary tumours and metastases, and poor differentiation grade and high-grade tumour budding in primary tumours. Furthermore, CMS4 was significantly enriched in primary tumours with peritoneal metastases, compared with unselected stage I-IV tumours (60 per cent (12 of 20) versus 23 per cent; P = 0.002). The majority of peritoneal metastases (75 per cent, 21 of 28) were also classified as CMS4. Considerable intrapatient subtype heterogeneity was observed. Notably, 15 of 16 patients with paired tumours had at least one CMS4-positive tumour location. CONCLUSION: Significant enrichment for CMS4 was observed in colorectal peritoneal carcinomatosis. Surgical relevance Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (CRS-HIPEC) improves survival of selected patients with colorectal peritoneal carcinomatosis, but recurrence is common. Histopathological and molecular analysis of colorectal peritoneal carcinomatosis could provide clues for development of novel therapies. In this study, colorectal peritoneal carcinomatosis was found to be enriched for tumours with high stromal content and CMS4-positive status. To further improve prognosis for patients with colorectal peritoneal carcinomatosis, therapies that target tumour-stroma interaction could be added to CRS-HIPEC.

Imatinib treatment of poor prognosis mesenchymal-type primary colon cancer: a proof-of-concept study in the preoperative window period (ImPACCT).

BACKGROUND: The identification of four Consensus Molecular Subtypes (CMS1-4) of colorectal cancer forms a new paradigm for the design and evaluation of subtype-directed therapeutic strategies. The most aggressive subtype - CMS4 - has the highest chance of disease recurrence. Novel adjuvant therapies for patients with CMS4 tumours are therefore urgently needed. CMS4 tumours are characterized by expression of mesenchymal and stem-like genes. Previous pre-clinical work has shown that targeting Platelet-Derived Growth Factor Receptors (PDGFRs) and the related KIT receptor with imatinib is potentially effective against mesenchymal-type colon cancer. In the present study we aim to provide proof for the concept that imatinib can reduce the aggressive phenotype of primary CMS4 colon cancer. METHODS: Tumour biopsies from patients with newly diagnosed stage I-III colon cancer will be analysed with a novel RT-qPCR test to pre-select patients with CMS4 tumours. Selected patients (n = 27) will receive treatment with imatinib (400 mg per day) starting two weeks prior to planned tumour resection. To assess treatment-induced changes in the aggressive CMS4 phenotype, RNA sequencing will be performed on pre- and post-treatment tissue samples. DISCUSSION: The development of effective adjuvant therapy for primary colon cancer is hindered by multiple factors. First, new drugs that may have value in the prevention of (early) distant recurrence are almost always first tested in patients with heavily pre-treated metastatic disease. Second, measuring on-target drug effects and biological consequences in tumour tissue is not commonly a part of the study design. Third, due to the lack of patient selection tools, clinical trials in the adjuvant setting require large patient populations. Finally, the evaluation of recurrence-prevention requires a long-term follow-up. In the ImPACCT trial these issues are addressed by including newly diagnosed pre-selected patients with CMS4 tumours prior to primary tumour resection, rather than non-selected patients with late-stage disease. By making use of the pre-operative window period, the biological effect of imatinib treatment on CMS4 tumours can be rapidly assessed. Delivering proof-of-concept for drug action in early stage disease should form the basis for the design of future trials with subtype-targeted therapies in colon cancer patients. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02685046 . Registration date: February 9, 2016.

Maspin is a marker for early recurrence in primary stage III and IV colorectal cancer.

BACKGROUND: Little is known about the factors that drive metastasis formation in colorectal cancer (CRC). Here, we set out to identify genes and proteins in patients with colorectal liver metastases that correlate with early disease recurrence. Such factors may predict a propensity for metastasis in earlier stages of CRC. METHODS: Gene expression profiling and proteomics were used to identify differentially expressed genes/proteins in resected liver metastases that recurred within 6 months following liver surgery vs those that did not recur for >24 months. Expression of the identified genes/proteins in stage II (n=243) and III (n=176) tumours was analysed by immunohistochemistry on tissue microarrays. Correlation of protein levels with stage-specific outcome was assessed by uni- and multivariable analyses. RESULTS: Both gene expression profiling and proteomics identified Maspin to be differentially expressed in colorectal liver metastases with early (<6 months) and prolonged (>24 months) time to recurrence. Immunohistochemical analysis of Maspin expression on tumour sections revealed that it was an independent predictor of time to recurrence (log-rank P=0.004) and CRC-specific survival (P=0.000) in stage III CRC. High Maspin expression was also correlated with mucinous differentiation. In stage II CRC patients, high Maspin expression did not correlate with survival but was correlated with a right-sided tumour location. CONCLUSION: High Maspin expression correlates with poor outcome in CRC after spread to the local lymph nodes. Therefore, Maspin may have a stage-specific function possibly related to tumour cell dissemination and/or metastatic outgrowth.

Comparative proteomics of colon cancer stem cells and differentiated tumor cells identifies BIRC6 as a potential therapeutic target.

Patients with liver metastases from colon carcinoma show highly variable responses to chemotherapy and tumor recurrence is frequently observed. Therapy-resistant cancer stem cells have been implicated in drug resistance and tumor recurrence. However, the factors determining therapy resistance and tumor recurrence are poorly understood. The aim of this study was to gain insight into these mechanisms by comparing the proteomes of patient-derived cancer stem cell cultures and their differentiated isogenic offspring. We established colonosphere cultures derived from resection specimens of liver metastases in patients with colon cancer. These colonospheres, enriched for colon cancer stem cells, were used to establish isogenic cultures of stably differentiated nontumorigenic progeny. Proteomics based on one-dimensional gel electrophoresis coupled to nano liquid chromatography tandem MS was used to identify proteome differences between three of these paired cultures. The resulting data were analyzed using Ingenuity Pathway Software. Out of a total data set of 3048 identified proteins, 32 proteins were at least twofold up-regulated in the colon cancer stem cells when compared with the differentiated cells. Pathway analysis showed that "cell death " regulation is strikingly different between the two cell types. Interestingly, one of the top-up-regulated proteins was BIRC6, which belongs to the class of Inhibitor of Apoptosis Proteins. Knockdown of BIRC6 sensitized colon cancer stem cells against the chemotherapeutic drugs oxaliplatin and cisplatin. This study reveals that differentiation of colon cancer stem cells is accompanied by altered regulation of cell death pathways. We identified BIRC6 as an important mediator of cancer stem cell resistance against cisplatin and oxaliplatin. Targeting BIRC6, or other Inhibitors of Apoptosis Proteins, may help eradicating colon cancer stem cells.

Effects of tirapazamine on experimental colorectal liver metastases after radiofrequency ablation.

BACKGROUND: Radiofrequency ablation (RFA) is a common procedure for the management of colorectal liver metastases. RFA-generated lesions are surrounded by a rim of hypoxia that is associated with aggressive outgrowth of intrahepatic micrometastases. Hypoxia-activated prodrugs such as tirapazamine are designed selectively to induce apoptosis in tumour cells under hypoxic conditions. Therefore, it was hypothesized that tirapazamine may have therapeutic value in limiting hypoxia-associated tumour outgrowth following RFA. METHODS: Murine C26 and MC38 colorectal cancer cells were grown under hypoxia and normal oxygenation in vitro, and treated with different concentrations of tirapazamine. Apoptosis and cell cycle distribution were assessed by western blot and fluorescence-activated cell sorting analysis. Proliferative capacity was tested by means of colony-formation assays. Mice harbouring microscopic colorectal liver metastases were treated with RFA, followed by a single injection of tirapazamine (60 mg/kg) or saline. Tumour load was assessed morphometrically 7 days later. RESULTS: Tirapazamine induced apoptosis of colorectal tumour cells under hypoxia in vitro. Under normal oxygenation, tirapazamine caused a G2 cell cycle arrest from which cells recovered partly. This reduced, but did not abolish, colony-forming capacity. A single dose of tirapazamine largely prevented accelerated outgrowth of hypoxic micrometastases following RFA. Tirapazamine administration was associated with minimal toxicity. CONCLUSION: Tirapazamine induced apoptosis in colorectal cancer cells in a hypoxia-dependent manner and potently suppressed hypoxia-associated outgrowth of liver metastases with limited toxicity. This warrants further study to assess the potential value of tirapazamine, or other hypoxia-activated prodrugs, as adjuvant therapeutics following RFA treatment of colorectal liver metastases.

Lysophosphatidic acid: G-protein signalling and cellular responses.

Lysophosphatidic acid (LPA) is a serum-borne phospholipid that activates a specific G protein coupled receptor to evoke multiple cellular responses. Recent work has identified two cDNAs encoding putative LPA receptors, various LPA-like agonists that act on distinct receptors, and new pathways that link the receptor(s) to such diverse events as Ras signalling, cytoskeletal remodelling and membrane depolarization.

Oncogenic KRAS sensitises colorectal tumour cells to chemotherapy by p53-dependent induction of Noxa.

BACKGROUND: Oxaliplatin and 5-fluorouracil (5-FU) currently form the backbone of conservative treatment in patients with metastatic colorectal cancer. Tumour responses to these agents are highly variable, but the underlying mechanisms are poorly understood. Our previous results have indicated that oncogenic KRAS in colorectal tumour cells sensitises these cells to chemotherapy. METHODS: FACS analysis was used to determine cell-cycle distribution and the percentage of apoptotic and mitotic cells. A multiplexed RT-PCR assay was used to identify KRAS-controlled apoptosis regulators after exposure to 5-FU or oxaliplatin. Lentiviral expression of short-hairpin RNAs was used to suppress p53 or Noxa. RESULTS: Oncogenic KRAS sensitised colorectal tumour cells to oxaliplatin and 5-FU in a p53-dependent manner and promoted p53 phosphorylation at Ser37 and Ser392, without affecting p53 stabilisation, p21 induction, or cell-cycle arrest. Chemotherapy-induced expression of the p53 target gene Noxa was selectively enhanced by oncogenic KRAS. Suppression of Noxa did not affect p21 induction or cell-cycle arrest, but reduced KRAS/p53-dependent apoptosis after exposure to chemotherapy in vitro and in tumour xenografts. Noxa suppression did not affect tumour growth per se, but strongly reduced the response of these tumours to chemotherapy. CONCLUSION: Oncogenic KRAS determines the cellular response to p53 activation by oxaliplatin or 5-FU, by facilitating apoptosis induction through Noxa.

Inhibition of cyclin-dependent kinase activity triggers neuronal differentiation of mouse neuroblastoma cells.

Studies on the molecular mechanisms underlying neuronal differentiation are frequently performed using cell lines established from neuroblastomas. In this study we have used mouse N1E-115 neuroblastoma cells that undergo neuronal differentiation in response to DMSO. During differentiation, cyclin-dependent kinase (cdk) activities decline and phosphorylation of the retinoblastoma gene product (pRb) is lost, leading to the appearance of a pRb-containing E2F DNA-binding complex. The loss of cdk2 activity is due to a decrease in cdk2 abundance whereas loss of cdk4 activity is caused by strong association with the cdk inhibitor (CKI) p27KIP1 and concurrent loss of cdk4 phosphorylation. Moreover, neuronal differentiation can be induced by overexpression of p27KIP1 or pRb, suggesting that inhibition of cdk activity leading to loss of pRb phosphorylation, is the major determinant for neuronal differentiation.

Identification of a novel, putative Rho-specific GDP/GTP exchange factor and a RhoA-binding protein: control of neuronal morphology.

The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein-coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116(Rip), that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116(Rip) stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116(Rip) fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein-coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116(Rip) inhibits RhoA-stimulated contractility and promotes neurite outgrowth.

The guanine nucleotide exchange factor Tiam1 affects neuronal morphology; opposing roles for the small GTPases Rac and Rho.

The invasion-inducing T-lymphoma invasion and metastasis 1 (Tiam1) protein functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. Differentiation-dependent expression of Tiam1 in the developing brain suggests a role for this GEF and its effector Rac1 in the control of neuronal morphology. Here we show that overexpression of Tiam1 induces cell spreading and affects neurite outgrowth in N1E-115 neuroblastoma cells. These effects are Rac-dependent and strongly promoted by laminin. Overexpression of Tiam1 recruits the alpha 6 beta 1 integrin, a laminin receptor, to specific adhesive contacts at the cell periphery, which are different from focal contacts. Cells overexpressing Tiam1 no longer respond to lysophosphatidic acid- induced neurite retraction and cell rounding, processes mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively active RhoA, which may indicate that regulation occurs at the level of Rho or upstream. Conversely, neurite formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate that Rac- and Rho-mediated pathways oppose each other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, our data underscore the potential role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process.

Molecular dissection of the Rho-associated protein kinase (p160ROCK)-regulated neurite remodeling in neuroblastoma N1E-115 cells.

A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho-ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.

A strategy for genetic modification of the spike-encoding segment of human reovirus T3D for reovirus targeting.

Human Orthoreovirus Type 3 Dearing is not pathogenic to humans and has been evaluated clinically as an oncolytic agent. Its transduction efficiency and the tumor cell selectivity may be enhanced by incorporating ligands for alternative receptors. However, the genetic modification of reoviruses has been difficult, and genetic targeting of reoviruses has not been reported so far. Here we describe a technique for generating genetically targeted reoviruses. The propagation of wild-type reoviruses on cells expressing a modified sigma 1-encoding segment embedded in a conventional RNA polymerase II transcript leads to substitution of the wild-type genome segment by the modified version. This technique was used for generating reoviruses that are genetically targeted to an artificial receptor expressed on U118MG cells. These cells lack the junction adhesion molecule-1 and therefore resist infection by wild-type reoviruses. The targeted reoviruses were engineered to carry the ligand for this receptor at the C terminus of the sigma 1 spike protein. This demonstrates that the C terminus of the sigma 1 protein is a suitable locale for the insertion of oligopeptide ligands and that targeting of reoviruses is feasible. The genetically targeted viruses can be propagated using the modified U118MG cells as helper cells. This technique may be applicable for the improvement of human reoviruses as oncolytic agents.

Regulating c-Ras function. cholesterol depletion affects caveolin association, GTP loading, and signaling.

Cholesterol-rich and caveolin-containing microdomains of the plasma membrane, termed "caveolae," have been implicated in signal transduction. However, the role of caveolae in regulating the Ras-MAP kinase cascade is incompletely understood. The mammalian Ras isoforms (H, N, and K) use different membrane anchors to attach to the plasma membrane and thereby may localize to functionally distinct microdomains, which might explain isoform-specific signaling. Here, we show that, in Cos epithelial cells, endogenous K-Ras colocalizes largely with caveolin, whereas N-Ras localizes to both caveolar and noncaveolar subdomains; H-Ras localization was below detection limits. We find that epidermal growth factor (EGF) activates N-Ras but fails to activate K-Ras in these cells. Extraction of cholesterol with methyl-beta-cyclodextrin disrupts complex formation between caveolin and K- and N-Ras and, strikingly, enables EGF to activate both K-Ras and N-Ras. While cholesterol depletion enhances GTP-loading on total c-Ras, activation of the downstream MEK-MAP kinase cascade by EGF and lysophosphatidic acid but not that by phorbol ester is inhibited. Thus, plasma membrane cholesterol is essential for negative regulation of c-Ras isoforms (complexed to caveolin), as well as for mitogenic signaling downstream of receptor-activated c-Ras.

Activation of RhoA by lysophosphatidic acid and Galpha12/13 subunits in neuronal cells: induction of neurite retraction.

Neuronal cells undergo rapid growth cone collapse, neurite retraction, and cell rounding in response to certain G protein-coupled receptor agonists such as lysophosphatidic acid (LPA). These shape changes are driven by Rho-mediated contraction of the actomyosin-based cytoskeleton. To date, however, detection of Rho activation has been hampered by the lack of a suitable assay. Furthermore, the nature of the G protein(s) mediating LPA-induced neurite retraction remains unknown. We have developed a Rho activation assay that is based on the specific binding of active RhoA to its downstream effector Rho-kinase (ROK). A fusion protein of GST and the Rho-binding domain of ROK pulls down activated but not inactive RhoA from cell lysates. Using GST-ROK, we show that in N1E-115 neuronal cells LPA activates endogenous RhoA within 30 s, concomitant with growth cone collapse. Maximal activation occurs after 3 min when neurite retraction is complete and the actin cytoskeleton is fully contracted. LPA-induced RhoA activation is completely inhibited by tyrosine kinase inhibitors (tyrphostin 47 and genistein). Activated Galpha12 and Galpha13 subunits mimic LPA both in activating RhoA and in inducing RhoA-mediated cytoskeletal contraction, thereby preventing neurite outgrowth. We conclude that in neuronal cells, LPA activates RhoA to induce growth cone collapse and neurite retraction through a G12/13-initiated pathway that involves protein-tyrosine kinase activity.

Neoadjuvant chemotherapy affects molecular classification of colorectal tumors.

The recent discovery of 'molecular subtypes' in human primary colorectal cancer has revealed correlations between subtype, propensity to metastasize and response to therapy. It is currently not known whether the molecular tumor subtype is maintained after distant spread. If this is the case, molecular subtyping of the primary tumor could guide subtype-targeted therapy of metastatic disease. In this study, we classified paired samples of primary colorectal carcinomas and their corresponding liver metastases (n=129) as epithelial-like or mesenchymal-like, using a recently developed immunohistochemistry-based classification tool. We observed considerable discordance (45%) in the classification of primary tumors and their liver metastases. Discordant classification was significantly associated with the use of neoadjuvant chemotherapy. Furthermore, gene expression analysis of chemotherapy-exposed versus chemotherapy naive liver metastases revealed expression of a mesenchymal program in pre-treated tumors. To explore whether chemotherapy could cause gene expression changes influencing molecular subtyping, we exposed patient-derived colonospheres to six short cycles of 5-fluorouracil. Gene expression profiling and signature enrichment analysis subsequently revealed that the expression of signatures identifying mesenchymal-like tumors was strongly increased in chemotherapy-exposed tumor cultures. Unsupervised clustering of large cohorts of human colon tumors with the chemotherapy-induced gene expression program identified a poor prognosis mesenchymal-like subgroup. We conclude that neoadjuvant chemotherapy induces a mesenchymal phenotype in residual tumor cells and that this may influence the molecular classification of colorectal tumors.

Immunosuppression promotes reovirus therapy of colorectal liver metastases.

Mortality due to colorectal cancer (CRC) is high and is associated with the development of liver metastases. Approximately 40% of human CRCs harbor an activating mutation in the KRAS oncogene. Tumor cells with activated KRAS are particularly sensitive to Reovirus T3D, a non-pathogenic oncolytic virus. The efficacy of virus-based therapies may be positively or negatively modulated by the host immune system. This study was designed to assess the effect of immunosuppression on Reovirus T3D oncolysis of established colorectal micrometastases in the liver. Mouse C26 CRC cells harbor a mutant Kras gene and are susceptible to Kras-dependent oncolysis by Reovirus T3D in vitro. Isolated C26 liver tumors were established in syngenic immunocompetent BALB/c mice by intrahepatic injection. Reovirus T3D therapy was given as a single intratumoral injection in control mice and in cyclosporin A-treated immunosuppressed mice. Tumor growth was analyzed over time by non-invasive bioluminescence imaging. The outgrowth of established CRC liver metastases in immunocompetent mice was efficiently but temporarily inhibited with a single injection of Reovirus T3D. Immunosuppression with cyclosporin A markedly increased and prolonged the therapeutic effect and allowed complete Reovirus T3D-induced tumor eradication in a subpopulation of the mice. We conclude that Reovirus T3D is an effective therapeutic agent against established C26 colorectal liver metastases and that immunosuppression enhances treatment efficacy. Cancer Gene Therapy (2006) 13, 815-818. doi:10.1038/sj.cgt.7700949; published online 10 March 2006.

Collagen-rich stroma in aggressive colon tumors induces mesenchymal gene expression and tumor cell invasion.

Gene expression-based classification systems have identified an aggressive colon cancer subtype with mesenchymal features, possibly reflecting epithelial-to-mesenchymal transition (EMT) of tumor cells. However, stromal fibroblasts contribute extensively to the mesenchymal phenotype of aggressive colon tumors, challenging the notion of tumor EMT. To separately study the neoplastic and stromal compartments of colon tumors, we have generated a stroma gene filter (SGF). Comparative analysis of stromahigh and stromalow tumors shows that the neoplastic cells in stromahigh tumors express specific EMT drivers (ZEB2, TWIST1, TWIST2) and that 98% of differentially expressed genes are strongly correlated with them. Analysis of differential gene expression between mesenchymal and epithelial cancer cell lines revealed that hepatocyte nuclear factor 4α (HNF4α), a transcriptional activator of intestinal (epithelial) differentiation, and its target genes are highly expressed in epithelial cancer cell lines. However, mesenchymal-type cancer cell lines expressed only part of the mesenchymal genes expressed by tumor-derived neoplastic cells, suggesting that external cues were lacking. We found that collagen-I dominates the extracellular matrix in aggressive colon cancer. Mimicking the tumor microenvironment by replacing laminin-rich Matrigel with collagen-I was sufficient to induce tumor-specific mesenchymal gene expression, suppression of HNF4α and its target genes, and collective tumor cell invasion of patient-derived colon tumor organoids. The data connect collagen-rich stroma to mesenchymal gene expression in neoplastic cells and to collective tumor cell invasion. Targeting the tumor-collagen interface may therefore be explored as a novel strategy in the treatment of aggressive colon cancer.

Ras-MAP kinase signaling by lysophosphatidic acid and other G protein-coupled receptor agonists.

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are extracellular lipid mediators that signal through distinct members of the Edg/LP subfamily of G protein-coupled receptors (GPCRs). LPA and S1P receptors are expressed in almost every cell type and can couple to multiple G proteins (G(i), G(q) and G(12/13)) to mediate a great variety of responses, ranging from rapid morphological changes to long-term stimulation of cell proliferation. LPA serves as the prototypic GPCR agonist that activates the small GTPases Ras (via G(i)) and RhoA (via G(12/13)), leading to activation of the mitogen-activated protein kinase (MAPK) cascade and reorganization of the actin cytoskeleton, respectively. This review focuses on our current insights into how Ras-MAPK signaling is regulated by GPCR agonists in general, and by LPA in particular.