Prognostic role of carcinoembryonic antigen and carbohydrate antigen 19-9 in metastatic colorectal cancer: a BRAF-mutant subset with high CA 19-9 level and poor outcome.

BACKGROUND: Mutation status of RAS and BRAF, as well as serum levels of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9), are biomarkers used in clinical management of patients with gastrointestinal cancers. This study aimed to examine the prognostic role of these biomarkers in a patient population that started first-line chemotherapy for unresectable metastatic colorectal cancer (mCRC) in the NORDIC-VII study. METHODS: CEA and CA 19-9 were measured in serum samples from 545 patients obtained before the start of chemotherapy. Four hundred and ninety-four patients had detectable levels of carbohydrate antigen 19-9 (CA 19-9). RAS (exons 2-4) and BRAF (V600E) mutation status were available from 440 patients. Overall survival (OS) was estimated in patient groups defined by serum CEA or CA 19-9 levels using cut-off values of 5 µg/L and 35 kU/L, respectively, in the total population and in subgroups according to RAS and BRAF mutation status. RESULTS: For both CEA and CA 19-9, elevated serum levels were associated with reduced OS in adjusted analyses which included RAS and BRAF mutation status, baseline World Health Organization performance status, and levels of alkaline phosphatase and C-reactive protein. The negative prognostic information provided by an elevated CA 19-9 level was particularly marked in patients with BRAF mutation (hazard ratio = 4.35, interaction P = 0.003, in an adjusted model for OS). CONCLUSIONS: High baseline serum concentrations of CEA and CA 19-9 provide independent information of impaired prognosis in mCRC. In patients with BRAF-mutant tumours, elevated serum CA 19-9 may identify a subgroup with highly aggressive disease and could contribute to improving therapeutic decisions.

p38 differentially regulates ERK, p21, and mitogenic signalling in two pancreatic carcinoma cell lines.

Whereas the p38 MAP kinase has largely been associated with anti-proliferative functions, several observations have indicated that it may also have positive effects on proliferation. In hepatocytes, we have found that p38 has opposing effects on DNA synthesis when activated by EGF and HGF. Here we have studied the function of p38 in EGF- and HGF-induced DNA synthesis in the two pancreatic carcinoma cell lines AsPC-1 and Panc-1. In Panc-1 cells, the MEK inhibitor PD98059 reduced EGF- and HGF-induced DNA synthesis, while the p38 inhibitor SB203580 strongly increased the basal DNA synthesis and reduced expression of the cyclin-dependent kinase inhibitor (CDKI) p21. In contrast, in AsPC-1 cells, EGF- and HGF-induced DNA synthesis was not significantly reduced by PD98059 but was inhibited by SB203580. Treatment with SB203580 amplified the sustained ERK phosphorylation induced by these growth factors and caused a marked upregulation of the expression of p21, which could be blocked by PD98059. These results suggest that while DNA synthesis in Panc-1 cells is enhanced by ERK and strongly suppressed by p38, in AsPC-1 cells, p38 exerts a pro-mitogenic effect through MEK/ERK-dependent downregulation of p21. Thus, p38 may have suppressive or stimulatory effects on proliferation depending on the cell type, due to differential cross-talk between the p38 and MEK/ERK pathways.

Functional heterogeneity in tumor-derived human pancreatic stellate cells: Differential expression of HGF and implications for mitogenic signaling and migration in pancreatic cancer cells.

The pancreatic stellate cell (PSC) is the principal cell type of the desmoplastic stroma of pancreatic ductal adenocarcinoma (PDAC). PSCs interact with cancer cells and influence the progression of the disease through a complex network of signaling molecules including hepatocyte growth factor (HGF). Functional heterogeneity of PSCs within a tumor might conceivably influence tumor progression. We investigated PSC populations isolated from different human PDACs and examined the effects of PSC-conditioned medium on BxPC-3 and AsPC-1 pancreatic cancer cells. The different PSC populations exhibited a wide range of variation (120-3,000 pg/ml) in their ability to secrete HGF. Media from high-HGF-producing PSCs stimulated phosphorylation of Met, Gab1, and ERK in the cancer cells and induced increases in DNA synthesis and migration which were blocked by the Met inhibitor SU11274, indicating a role of HGF as a mediator. HGF levels produced by PSCs and the effects of PSC media on the cancer cells were increased by IL-1α and inhibited by TGFß. The functional heterogeneity of PSCs in terms of HGF-mediated tumor-stroma interactions suggests that inhibition of the HGF pathway as a novel treatment approach in PDAC might have different effects in different subsets of patients.

Health-related quality of life in patients with metastatic colorectal cancer, association with systemic inflammatory response and RAS and BRAF mutation status.

BACKGROUND: The aim of this study was to evaluate the effect of cetuximab on health-related quality of life (HRQoL) in the NORDIC-VII trial on metastatic colorectal cancer (mCRC), and to assess HRQoL in relation to RAS and BRAF mutation status and inflammatory biomarkers. PATIENT AND METHODS: HRQoL was assessed using the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 (QLQ-C30) at baseline, after every fourth cycle of chemotherapy, and at the end of treatment. HRQoL during 12 cycles of chemotherapy was evaluated over time, compared between treatment arms, and assessed for association with tumour mutation status and inflammatory markers. RESULTS: QLQ-C30 was completed by 512 patients (90%) before start of treatment. HRQoL variables were well balanced across treatment arms at baseline, and no statistically significant differences during treatment were seen. Patients with BRAF-mutated tumours reported poorer HRQoL at baseline and subsequent time points than patients with RAS-mutated or RAS/BRAF wild-type tumours. Patients with high serum interleukin-6 (IL-6) or C-reactive protein (CRP) had markedly impaired HRQoL compared to patients with normal levels. There was a statistically significant association between reduction in IL-6 and CRP levels and improvement in HRQoL during treatment from baseline to cycle 4. CONCLUSION: The addition of cetuximab to chemotherapy did not affect HRQoL in mCRC patients. Patients with BRAF-mutated tumours have both a worse prognosis and a poor HRQoL. The associations between levels of systemic inflammatory markers and reduced HRQoL suggest that the patients might benefit from anti-inflammatory treatment.

Cetuximab in treatment of metastatic colorectal cancer: final survival analyses and extended RAS data from the NORDIC-VII study.

BACKGROUND: The NORDIC-VII study is a randomised phase III trial of cetuximab plus continuous or intermittent fluorouracil, folinic acid, and oxaliplatin (Nordic FLOX) vs FLOX alone in first-line treatment of metastatic colorectal cancer. The present report presents an updated and final survival analysis with BRAF and extended RAS mutational status, 5 years after the primary analysis. METHODS: A total of 566 patients were included in the intention-to-treat (ITT) population of the NORDIC-VII study. Updated survival status was obtained from 176 patients who were alive in the primary survival analyses. Samples from 223 tumours previously found to be KRAS (exon 2) and BRAF (V600E) wild-type, were re-analysed for KRAS (exons 3 and 4) and NRAS (exons 2-4) mutations. RESULTS: Including the extended RAS analyses, RAS and BRAF mutational status was available from 457 patients (81% of the ITT population). RAS was mutated in 46% and BRAF in 12% of the tumours. RAS and BRAF, if mutated, were negative prognostic factors. The updated analyses confirmed the finding of the primary report that cetuximab did not provide any additional benefit when added to FLOX in patients with RAS/BRAF wild-type tumours, neither on progression-free nor overall survival. However, the outcomes in a subset of patients, which, after the first eight treatment cycles, received cetuximab alone, suggested a beneficial effect of cetuximab monotherapy. CONCLUSIONS: Adding cetuximab to Nordic FLOX did not provide any clinical benefit, but the data suggested an effect of cetuximab monotherapy in patients with RAS/BRAF wild-type tumours in the NORDIC-VII cohort. The data were compatible with a negative interaction between cetuximab and the Nordic FLOX chemotherapy backbone.

Interleukin-6 and C-reactive protein as prognostic biomarkers in metastatic colorectal cancer.

OBJECTIVES: The aim was to explore the prognostic significance of IL-6 and markers of systemic inflammatory response (SIR), in particular C-reactive protein (CRP), in metastatic colorectal cancer (mCRC) patients, in the total study population and according to RAS and BRAF mutation status. RESULTS: High levels of pretreatment serum IL-6 or CRP were associated with impaired outcome, in terms of reduced PFS and OS. Patients with low versus high serum IL-6 levels had median OS of 26.0 versus 16.6 months, respectively (P < 0.001). Stratified according to increasing CRP levels, median OS varied from 24.3 months to 12.3 months, (P < 0.001). IL-6 and CRP levels affected overall prognosis also in adjusted analyses. The effect of IL-6 was particularly pronounced in patients with BRAF mutation (interaction P = 0.004). MATERIALS AND METHODS: IL-6 and CRP were determined in pre-treatment serum samples from 393 patients included in the NORDIC-VII trial, in which patients with mCRC received first line treatment. The effect of serum IL-6 and CRP on progression-free survival (PFS) and overall survival (OS) was estimated. CONCLUSIONS: High baseline serum consentrations of IL-6 or CRP were associated with impaired prognosis in mCRC. IL-6 and CRP give independent prognostic information in addition to RAS and BRAF mutation status.

The TGFß-SMAD3 pathway inhibits IL-1α induced interactions between human pancreatic stellate cells and pancreatic carcinoma cells and restricts cancer cell migration.

BACKGROUND: The most abundant cells in the extensive desmoplastic stroma of pancreatic adenocarcinomas are the pancreatic stellate cells, which interact with the carcinoma cells and strongly influence the progression of the cancer. Tumor stroma interactions induced by IL-1α/IL-1R1 signaling have been shown to be involved in pancreatic cancer cell migration. TGFß and its receptors are overexpressed in pancreatic adenocarcinomas. We aimed at exploring TGFß and IL-1α signaling and cross-talk in the stellate cell cancer cell interactions regulating pancreatic adenocarcinoma cell migration. METHODS: Human pancreatic stellate cells were isolated from surgically resected pancreatic adenocarcinomas and cultured in the presence of TGFß or pancreatic adenocarcinoma cell lines. The effects of TGFß were blocked by inhibitors or amplified by silencing the endogenous inhibitor of SMAD signaling, SMAD7. Pancreatic stellate cell responses to IL-1α or to IL-1α-expressing pancreatic adenocarcinoma cells (BxPC-3) were characterized by their ability to stimulate migration of cancer cells in a 2D migration model. RESULTS: In pancreatic stellate cells, IL-1R1 expression was found to be down-regulated by TGFß and blocking of TGFß signaling re-established the expression. Endogenous inhibition of TGFß signaling by SMAD7 was found to correlate with the levels of IL-1R1, indicating a regulatory role of SMAD7 in IL-1R1 expression. Pancreatic stellate cells cultured in the presence of IL-1α or in co-cultures with BxPC-3 cells enhanced the migration of cancer cells. This effect was blocked after treatment of the pancreatic stellate cells with TGFß. Silencing of stellate cell expression of SMAD7 was found to suppress the levels of IL-1R1 and reduce the stimulatory effects of IL-1α, thus inhibiting the capacity of pancreatic stellate cells to induce cancer cell migration. CONCLUSIONS: TGFß signaling suppressed IL-1α mediated pancreatic stellate cell induced carcinoma cell migration. Depletion of SMAD7 upregulated the effects of TGFß and reduced the expression of IL-1R1, leading to inhibition of IL-1α induced stellate cell enhancement of carcinoma cell migration. SMAD7 might represent a target for inhibition of IL-1α induced tumor stroma interactions.

PI3K is required for both basal and LPA-induced DNA synthesis in oral carcinoma cells.

BACKGROUND: The glycerophospholipid lysophosphatidic acid (LPA), which is present in most tissues and in high concentrations in saliva, may exert profound effects on oral cancer cells. We have investigated mitogenic signalling induced by LPA in the two oral carcinoma cell lines, D2 and E10, focusing on the role of EGFR transactivation and downstream pathways. METHODS: Two oral squamous carcinoma cell lines, D2 and E10, were analysed for effects of LPA on signalling pathways and induction of DNA synthesis. Pathway activation was investigated by examining phosphorylation of signalling proteins and by the use of specific pathway inhibitors. RESULTS: The D2 cells had higher levels of activated signalling proteins and higher DNA synthesis activity in the basal condition than E10 cells. EGF did not induce proliferation in D2 cells, whereas LPA induced proliferation in both cell lines, by mechanisms depending on EGFR transactivation. Release of EGFR ligands was involved in basal and LPA-induced proliferation in both D2 and E10 cells. The proliferation in D2 cells was dependent on the PI3K/Akt pathway, but not the MEK/ERK pathway. In E10 cells, the PI3K/Akt, MEK/ERK and p38 pathways were all involved in the proliferation. CONCLUSION: Transactivation of EGFR is required for LPA-induced DNA synthesis in D2 and E10 cells. Our results also show that although proliferation of oral carcinoma cells is regulated by several pathways, and differentially in E10 and D2 cells, the PI3K pathway has a crucial role in both cell lines.

HGF-induced DNA synthesis in hepatocytes is suppressed by p38.

Previous studies in rat hepatocytes have shown that the MEK/ERK, PI3K/Akt and p38 pathways are all involved in the activation of DNA synthesis by EGF and that sustained activation of MEK/ERK is required. Here, we show that although HGF stimulated DNA synthesis and activated signaling in the same manner as EGF, the contribution of the signaling pathways to the induction of DNA synthesis differed. While HGF-induced DNA synthesis was dependent on MEK/ERK, with no significant contribution from PI3K/Akt, p38 suppressed HGF-induced DNA synthesis. The p38 inhibitor SB203580 increased HGF-induced DNA synthesis and enhanced the phosphorylation of ERK. In contrast, SB203580 decreased EGF-induced ERK phosphorylation. This suggests that p38 has distinct effects on DNA synthesis induced by EGF and HGF. Due to differential regulation of signaling through the MEK/ERK pathway, p38 acts as an enhancer of EGF-induced DNA synthesis and as a suppressor of HGF-induced DNA synthesis.

Lysophosphatidic acid induces both EGFR-dependent and EGFR-independent effects on DNA synthesis and migration in pancreatic and colorectal carcinoma cells.

Lysophosphatidic acid (LPA) is a small glycerophospholipid ubiquitously present in tissues and plasma. It acts through receptors belonging to the G-protein-coupled receptor (GPCR) family, is involved in several biological processes, and is strongly implicated in different cancers. In this paper, we have investigated the effects of LPA on DNA synthesis and migration in a panel of pancreatic and colon cancer cells, with particular focus on the involvement of the epidermal growth factor (EGF) receptor (EGFR) in LPA-induced signaling. LPA stimulated DNA synthesis and/or migration in all the cell lines included in this study. In five of the six cell lines, LPA induced phosphorylation of the EGFR, and the effects on EGFR and Akt, and in some of the cells also ERK, were sensitive to the EGFR tyrosine kinase inhibitor gefitinib, strongly suggesting LPA-induced EGFR transactivation in these cells. In contrast, in one of the pancreatic carcinoma cell lines (Panc-1), we found no evidence of transactivation of the EGFR. In the pancreatic carcinoma cell lines where transactivation took place (BxPC3, AsPC1, HPAFII), gefitinib reduced LPA-induced DNA synthesis and/or migration. However, we also found evidence of transactivation in the two colon carcinoma cell lines (HT29, HCT116) although gefitinib did not inhibit LPA-induced DNA synthesis or migration in these cells. Taken together, the data indicate that in many gastrointestinal carcinoma cells, LPA uses EGFR transactivation as a mechanism when exerting such effects as stimulation of cell proliferation and migration, but EGFR-independent pathways may be involved instead of, or in concerted action with, the EGFR transactivation.

Gab1 amplifies signaling in response to low-intensity stimulation by HGF.

The receptor tyrosine kinases EGFR and Met induce phosphorylation of the docking protein Gab1, and there is evidence that Gab1 may have a role in the signaling from these receptors. Studying hepatocytes, we previously found that although Gab1 mechanistically interacted in different ways with EGFR and Met, it was involved in mitogenic signaling induced by both EGF and HGF. It has been reported that in EGFR, Gab1 is required particularly at a low dose of EGF. Whether this also applies to HGF/Met signaling has not been investigated. We have studied the role of Gab1 in activation of the Akt and ERK pathways at low- and high-intensity stimulation with EGF and HGF in cultured hepatocytes. In cells where Gab1 was depleted by a specific Gab1-directed siRNA, the EGF-induced phosphorylation of ERK was lowered and HGF-induced phosphorylation of both ERK and Akt was substantially reduced. These effects were more marked at low-dose HGF stimulation. The inhibitory consequence of Gab1 depletion was particularly pronounced for HGF-induced Akt phosphorylation. The results suggest that Gab1 is an important signal amplifier for low-intensity stimulation by HGF.

Inhibitory effects of prostaglandin E2 on collagen synthesis and cell proliferation in human stellate cells from pancreatic head adenocarcinoma.

BACKGROUND: Several studies have described an increased cyclooxygenase-2 (COX-2) expression in pancreatic cancer, but the role of COX-2 in tumour development and progression is not clear. The aim of the present study was to examine expression of COX-2 in cancer cells and stromal cells in pancreatic cancer specimens, and to explore the role of PGE2 in pancreatic stellate cell proliferation and collagen synthesis. METHODS: Immunohistochemistry and immunofluorescence was performed on slides from whole sections of tissue blocks using antibodies against COX-2 and α-smooth muscle actin (αSMA). Pancreatic stellate cells (PSC) were isolated from surgically resected tumour tissue by the outgrowth method. Cells were used between passages 4 and 8. Collagen synthesis was determined by [(3)H]-proline incorporation, or by enzyme immunoassay measurement of collagen C-peptide. DNA synthesis was measured by incorporation of [(3)H]-thymidine in DNA. Cyclic AMP (cAMP) was determined by radioimmunoassay. Collagen 1A1 mRNA was determined by RT-qPCR. RESULTS: Immunohistochemistry staining showed COX-2 in pancreatic carcinoma cells, but not in stromal cells. All tumours showed positive staining for αSMA in the fibrotic stroma. Cultured PSC expressed COX-2, which could be further induced by interleukin-1ß (IL-1ß), epidermal growth factor (EGF), thrombin, and PGE2, but not by transforming growth factor-ß1 (TGFß). Indirect coculture with the adenocarcinoma cell line BxPC-3, but not HPAFII or Panc-1, induced COX-2 expression in PSC. Treatment of PSC with PGE2 strongly stimulated cAMP accumulation, mediated by EP2 receptors, and also stimulated phosphorylation of extracellular signal-regulated kinase (ERK). Treatment of PSC with PGE2 or forskolin suppressed both TGFß-stimulated collagen synthesis and PDGF-stimulated DNA synthesis. CONCLUSIONS: The present results show that COX-2 is mainly produced in carcinoma cells and suggest that the cancer cells are the main source of PGE2 in pancreatic tumours. PGE2 exerts a suppressive effect on proliferation and fibrogenesis in pancreatic stellate cells. These effects of PGE2 are mediated by the cAMP pathway and suggest a role of EP2 receptors.

Role of LPAR3, PKC and EGFR in LPA-induced cell migration in oral squamous carcinoma cells.

BACKGROUND: Oral squamous cell carcinoma is an aggressive neoplasm with serious morbidity and mortality, which typically spreads through local invasive growth. Lysophosphatidic acid (LPA) is involved in a number of biological processes, and may have a role in cancer cell migration and invasiveness. LPA is present in most tissues and can activate cells through six different LPA receptors (LPAR1-6). Although LPA is predominantly promigratory, some of the receptors may have antimigratory effects in certain cells. The signalling mechanisms of LPA are not fully understood, and in oral carcinoma cells the specific receptors and pathways involved in LPA-stimulated migration are unknown. METHODS: The oral carcinoma cell lines E10, SCC-9, and D2 were investigated. Cell migration was studied in a scratch wound assay, and invasion was demonstrated in organotypic three dimensional co-cultures. Protein and mRNA expression of LPA receptors was studied with Western blotting and qRT-PCR. Activation of signalling proteins was examined with Western blotting and isoelectric focusing, and signalling mechanisms were further explored using pharmacological agents and siRNA directed at specific receptors and pathways. RESULTS: LPA stimulated cell migration in the two oral carcinoma cell lines E10 and SCC-9, but was slightly inhibitory in D2. The receptor expression profile and the effects of specific pharmacological antagonist and agonists indicated that LPA-stimulated cell migration was mediated through LPAR3 in E10 and SCC-9. Furthermore, in both these cell lines, the stimulation by LPA was dependent on PKC activity. However, while LPA induced transactivation of EGFR and the stimulated migration was blocked by EGFR inhibitors in E10 cells, LPA did not induce EGFR transactivation in SCC-9 cells. In D2 cells, LPA induced EGFR transactivation, but this was associated with slowing of a very high inherent migration rate in these cells. CONCLUSION: The results demonstrate LPA-stimulated migration in oral carcinoma cells through LPAR3, mediated further by PKC, which acts either in concert with or independently of EGFR transactivation.

The involvement of the docking protein Gab1 in mitogenic signalling induced by EGF and HGF in rat hepatocytes.

Grb2-associated binder (Gab) family proteins are docking molecules that can interact with receptor tyrosine kinases (RTKs) and cytokine receptors and bind several downstream signalling proteins. Studies in several cell types have shown that Gab1 may have a role in signalling mediated by the two RTKs epidermal growth factor (EGF) receptor (EGFR) and Met, the receptor for hepatocyte growth factor (HGF), but the involvement of Gab1 in EGFR and Met signalling has not been directly compared in the same cell. We have studied mechanisms of activation and role in mitogenic signalling of Gab1 in response to EGF and HGF in cultured rat hepatocytes. Gab1, but not Gab2, was expressed in the hepatocytes and was phosphorylated upon stimulation with EGF or HGF. Depletion of Gab1, using siRNA, decreased the ERK and Akt activation, cyclin D1 expression, and DNA synthesis in response to both EGF and HGF. Studies of mechanisms of recruitment to the receptors showed that HGF induced co-precipitation of Gab1 and Met while EGF induced binding of Gab1 to Grb2 but not to EGFR. Gab1 activation in response to both EGF and HGF was dependent on PI3K. While EGF activated Gab1 and Shc equally, within the same concentration range, HGF very potently and almost exclusively activated Gab1, having only a minimal effect on Shc. Collectively, our results strongly suggest that although Gab1 interacts differently with EGFR and Met, it is involved in mitogenic signalling mediated by both these growth factor receptors in hepatocytes.