Transcriptional subtyping and CD8 immunohistochemistry identifies poor prognosis stage II/III colorectal cancer patients who benefit from adjuvant chemotherapy.

PURPOSE: Transcriptomic profiling of colorectal cancer (CRC) has led to identification of four consensus molecular subtypes (CMS1-4), which have prognostic value in stage II/III disease. More recently, the Colorectal Cancer Intrinsic Subtypes (CRIS) classification system has helped to define the biology specific to the epithelial component of colorectal tumors. However, the clinical value of these classifications in predicting response to standard-of-care adjuvant chemotherapy remains unknown. PATIENTS AND METHODS: Using samples from 4 European sites, we assembled a novel stage II/III CRC patient cohort and performed transcriptomic profiling on 156 samples, targeted sequencing and generated a tissue microarray to enable integrated "multi-omics" analyses. We also accessed data from 2 published stage II/III CRC patient cohorts: GSE39582 and GSE14333 (479 and 185 samples respectively). RESULTS: The epithelial-rich CMS2 subtype of CRC benefitted significantly from adjuvant chemotherapy treatment in both stage II and III disease (p=0.02 and p<0.0001 respectively), while the CMS3 subtype significantly benefitted in stage III only (p=0.00073). Following CRIS sub-stratification of CMS2, we observed that only the CRIS-C subtype significantly benefitted from adjuvant chemotherapy in stage II and III disease (p=0.0081 and p<0.0001 respectively), while CRIS-D significantly benefitted in stage III only (p=0.0034). We also observed that CRIS-C patients with low levels of CD8+ tumor-infiltrating lymphocytes were most at risk of relapse in both stage II and III disease (p=0.0031). CONCLUSION: Patient stratification using a combination of transcriptional subtyping and CD8 immunohistochemistry analyses is capable of identifying poor prognostic stage II/III patients who benefit from adjuvant standard-of-care chemotherapy. These findings are particularly relevant for stage II disease, where the overall benefit of adjuvant chemotherapy is marginal.

Nanoencapsulation of ABT-737 and camptothecin enhances their clinical potential through synergistic antitumor effects and reduction of systemic toxicity.

The simultaneous delivery of multiple cancer drugs in combination therapies to achieve optimal therapeutic effects in patients can be challenging. This study investigated whether co-encapsulation of the BH3-mimetic ABT-737 and the topoisomerase I inhibitor camptothecin (CPT) in PEGylated polymeric nanoparticles (NPs) was a viable strategy for overcoming their clinical limitations and to deliver both compounds at optimal ratios. We found that thrombocytopenia induced by exposure to ABT-737 was diminished through its encapsulation in NPs. Similarly, CPT-associated leukopenia and gastrointestinal toxicity were reduced compared with the administration of free CPT. In addition to the reduction of dose-limiting side effects, the co-encapsulation of both anticancer compounds in a single NP produced synergistic induction of apoptosis in both in vitro and in vivo colorectal cancer models. This strategy may widen the therapeutic window of these and other drugs and may enhance the clinical efficacy of synergistic drug combinations.

Differential affinity of FLIP and procaspase 8 for FADD's DED binding surfaces regulates DISC assembly.

Death receptor activation triggers recruitment of FADD, which via its death effector domain (DED) engages the DEDs of procaspase 8 and its inhibitor FLIP to form death-inducing signalling complexes (DISCs). The DEDs of FADD, FLIP and procaspase 8 interact with one another using two binding surfaces defined by α1/α4 and α2/α5 helices, respectively. Here we report that FLIP has preferential affinity for the α1/α4 surface of FADD, whereas procaspase 8 has preferential affinity for FADD's α2/α5 surface. These relative affinities contribute to FLIP being recruited to the DISC at comparable levels to procaspase 8 despite lower cellular expression. Additional studies, including assessment of DISC stoichiometry and functional assays, suggest that following death receptor recruitment, the FADD DED preferentially engages FLIP using its α1/α4 surface and procaspase 8 using its α2/α5 surface; these tripartite intermediates then interact via the α1/α4 surface of FLIP DED1 and the α2/α5 surface of procaspase 8 DED2.

Fibroblast growth factor receptor 4 (FGFR4): a targetable regulator of drug resistance in colorectal cancer.

The discovery of underlying mechanisms of drug resistance, and the development of novel agents to target these pathways, is a priority for patients with advanced colorectal cancer (CRC). We previously undertook a systems biology approach to design a functional genomic screen and identified fibroblast growth factor receptor 4 (FGFR4) as a potential mediator of drug resistance. The aim of this study was to examine the role of FGFR4 in drug resistance using RNAi and the small-molecule inhibitor BGJ398 (Novartis). We found that FGFR4 is highly expressed at the RNA and protein levels in colon cancer tumour tissue compared with normal colonic mucosa and other tumours. Silencing of FGFR4 reduced cell viability in a panel of colon cancer cell lines and increased caspase-dependent apoptosis. A synergistic interaction was also observed between FGFR4 silencing and 5-fluorouracil (5-FU) and oxaliplatin chemotherapy in colon cancer cell lines. Mechanistically, FGFR4 silencing decreased activity of the pro-survival STAT3 transcription factor and expression of the anti-apoptotic protein c-FLIP. Furthermore, silencing of STAT3 resulted in downregulation of c-FLIP protein expression, suggesting that FGFR4 may regulate c-FLIP expression via STAT3. A similar phenotype and downstream pathway changes were observed following FGFR4 silencing in cell lines resistant to 5-FU, oxaliplatin and SN38 and upon exposure of parental cells to the FGFR small-molecule inhibitor BGJ398. Our results indicate that FGFR4 is a targetable regulator of chemo-resistance in CRC, and hence inhibiting FGFR4 in combination with 5-FU and oxaliplatin is a potential therapeutic strategy for this disease.

Prognostic and therapeutic relevance of FLIP and procaspase-8 overexpression in non-small cell lung cancer.

Non-small cell lung carcinoma remains by far the leading cause of cancer-related deaths worldwide. Overexpression of FLIP, which blocks the extrinsic apoptotic pathway by inhibiting caspase-8 activation, has been identified in various cancers. We investigated FLIP and procaspase-8 expression in NSCLC and the effect of HDAC inhibitors on FLIP expression, activation of caspase-8 and drug resistance in NSCLC and normal lung cell line models. Immunohistochemical analysis of cytoplasmic and nuclear FLIP and procaspase-8 protein expression was carried out using a novel digital pathology approach. Both FLIP and procaspase-8 were found to be significantly overexpressed in tumours, and importantly, high cytoplasmic expression of FLIP significantly correlated with shorter overall survival. Treatment with HDAC inhibitors targeting HDAC1-3 downregulated FLIP expression predominantly via post-transcriptional mechanisms, and this resulted in death receptor- and caspase-8-dependent apoptosis in NSCLC cells, but not normal lung cells. In addition, HDAC inhibitors synergized with TRAIL and cisplatin in NSCLC cells in a FLIP- and caspase-8-dependent manner. Thus, FLIP and procaspase-8 are overexpressed in NSCLC, and high cytoplasmic FLIP expression is indicative of poor prognosis. Targeting high FLIP expression using HDAC1-3 selective inhibitors such as entinostat to exploit high procaspase-8 expression in NSCLC has promising therapeutic potential, particularly when used in combination with TRAIL receptor-targeted agents.

SAHA overcomes FLIP-mediated inhibition of SMAC mimetic-induced apoptosis in mesothelioma.

Malignant pleural mesothelioma (MPM) is a highly pro-inflammatory malignancy that is rapidly fatal and increasing in incidence. Cytokine signaling within the pro-inflammatory tumor microenvironment makes a critical contribution to the development of MPM and its resistance to conventional chemotherapy approaches. SMAC mimetic compounds (SMCs) are a promising class of anticancer drug that are dependent on tumor necrosis factor alpha (TNFα) signaling for their activity. As circulating TNFα expression is significantly elevated in MPM patients, we examined the sensitivity of MPM cell line models to SMCs. Surprisingly, all MPM cell lines assessed were highly resistant to SMCs either alone or when incubated in the presence of clinically relevant levels of TNFα. Further analyses revealed that MPM cells were sensitized to SMC-induced apoptosis by siRNA-mediated downregulation of the caspase 8 inhibitor FLIP, an antiapoptotic protein overexpressed in several cancer types including MPM. We have previously reported that FLIP expression is potently downregulated in MPM cells in response to the histone deacetylase inhibitor (HDACi) Vorinostat (SAHA). In this study, we demonstrate that SAHA sensitizes MPM cells to SMCs in a manner dependent on its ability to downregulate FLIP. Although treatment with SMC in the presence of TNFα promoted interaction between caspase 8 and the necrosis-promoting RIPK1, the cell death induced by combined treatment with SAHA and SMC was apoptotic and mediated by caspase 8. These results indicate that FLIP is a major inhibitor of SMC-mediated apoptosis in MPM, but that this inhibition can be overcome by the HDACi SAHA.

Apoptosome-dependent caspase activation proteins as prognostic markers in Stage II and III colorectal cancer.

BACKGROUND: Critical to successful execution of mitochondrial-mediated apoptosis is apoptosome formation and subsequent activation of caspases. Defects in this pathway have an important role in colorectal carcinogenesis and chemoresistance; therefore, the expression of apoptosome-associated proteins may be associated with clinical outcome and response to chemotherapy. METHODS: Here we performed a systematic analysis of the immunohistochemical expression of the key proteins involved in apoptosome-dependent caspase activation (APAF1, Pro-caspases 9 and 3, SMAC, and XIAP) in a cohort of Stage II and III colorectal cancer patients from a Phase III trial of adjuvant 5-fluorouracil-based chemotherapy vs postoperative observation alone. RESULTS: Survival analysis indicated that of the apoptosome-associated proteins examined here, Pro-caspase 3 and APAF1 have potential clinical utility as predictive markers in Stage II and III colorectal cancer, respectively. Interestingly, we identified APAF1 staining to be associated with better recurrence-free and overall survival in patients receiving chemotherapy. CONCLUSION: These studies reveal the importance of the apoptosome-dependent caspase activation pathway, specifically Pro-caspase 3 and APAF1 proteins, for predicting both prognosis and response to therapy.

Identification of an acetylation-dependant Ku70/FLIP complex that regulates FLIP expression and HDAC inhibitor-induced apoptosis.

FLIP is a potential anti-cancer therapeutic target that inhibits apoptosis by blocking caspase 8 activation by death receptors. We report a novel interaction between FLIP and the DNA repair protein Ku70 that regulates FLIP protein stability by inhibiting its polyubiquitination. Furthermore, we found that the histone deacetylase (HDAC) inhibitor Vorinostat (SAHA) enhances the acetylation of Ku70, thereby disrupting the FLIP/Ku70 complex and triggering FLIP polyubiquitination and degradation by the proteasome. Using in vitro and in vivo colorectal cancer models, we further demonstrated that SAHA-induced apoptosis is dependant on FLIP downregulation and caspase 8 activation. In addition, an HDAC6-specific inhibitor Tubacin recapitulated the effects of SAHA, suggesting that HDAC6 is a key regulator of Ku70 acetylation and FLIP protein stability. Thus, HDAC inhibitors with anti-HDAC6 activity act as efficient post-transcriptional suppressors of FLIP expression and may, therefore, effectively act as 'FLIP inhibitors'.

The role of Cathepsin S as a marker of prognosis and predictor of chemotherapy benefit in adjuvant CRC: a pilot study.

BACKGROUND: The aim of this pilot retrospective study was to investigate the immunohistochemical expression of Cathepsin S (CatS) in three cohorts of colorectal cancer (CRC) patients (n=560). METHODS: Prevalence and association with histopathological variables were assessed across all cohorts. Association with clinical outcomes was investigated in the Northern Ireland Adjuvant Chemotherapy Trial cohort (n=211), where stage II/III CRC patients were randomised between surgery-alone or surgery with adjuvant fluorouracil/folinic acid (FU/FA) treatment. RESULTS: Greater than 95% of tumours had detectable CatS expression with significantly increased staining in tumours compared with matched normal colon (P>0.001). Increasing CatS was associated with reduced recurrence-free survival (RFS; P=0.03) among patients treated with surgery alone. Adjuvant FU/FA significantly improved RFS (hazard ratio (HR), 0.33; 95% CI, 0.12-0.89) and overall survival (OS; HR, 0.25; 95% CI, 0.08-0.81) among 36 patients with high CatS. Treatment did not benefit the 66 patients with low CatS, with a RFS HR of 1.34 (95% CI, 0.60-3.19) and OS HR of 1.33 (95% CI, 0.56-3.15). Interaction between CatS and treatment status was significant for RFS (P=0.02) and OS (P=0.04) in a multivariate model adjusted for known prognostic markers. CONCLUSION: These results signify that CatS may be an important prognostic biomarker and predictive of response to adjuvant FU/FA in CRC.

The expression and prognostic impact of CXC-chemokines in stage II and III colorectal cancer epithelial and stromal tissue.

BACKGROUND: The CXC-chemokine expression is linked with colorectal cancer (CRC) progression but their significance in resected CRC is unclear. We explored the prognostic impact of such expression in stage II and III CRC. METHODS: Tissue microarrays were constructed from stage II and III CRC biopsies (n=254), and the expression of CXCL1 and CXCL8, and their receptors CXCR1 and CXCR2, in malignant and adjacent normal tissue was graded by immunohistochemistry and was correlated with prognostic factors. RESULTS: Expression of CXCL1, CXCR1 and CXCR2 was elevated in tumour epithelium relative to normal adjacent tissue (P<0.001). CXCL8 expression was detectable in the peritumoural inflammatory infiltrate. There was no overall association between CXCL1, CXCR1 or CXCR2 expression and prognostic endpoints; however, univariate subgroup survival analysis demonstrated an inverse association between CXCL1 and recurrence-free survival (RFS) in stage III patients (P=0.041). The CXCL8 positivity in the tumour infiltrate, however, correlated with earlier disease stage (P<0.001) and improved relapse-free survival across the cohort (P<0.001). Disease stage (P<0.001) and tumour infiltrate CXCL8 positivity (P=0.007) were associated with enhanced RFS in multivariate Cox regression analysis. CONCLUSION: Autocrine CXC-chemokine signalling may have adverse prognostic effects in early CRC. Conversely, CXCL8 positivity within the immune infiltrate may have good prognostic significance.

T-box 2 represses NDRG1 through an EGR1-dependent mechanism to drive the proliferation of breast cancer cells.

T-box 2 (TBX2) is a transcription factor involved in mammary development and is known to be overexpressed in a subset of aggressive breast cancers. TBX2 has previously been shown to repress growth control genes such as p14(ARF) and p21(WAF1/cip1). In this study we show that TBX2 drives proliferation in breast cancer cells and this is abrogated after TBX2 small interfering RNA (siRNA) knockdown or after the expression of a dominant-negative TBX2 protein. Using microarray analysis we identified a large cohort of novel TBX2-repressed target genes including the breast tumour suppressor NDRG1 (N-myc downregulated gene 1). We show that TBX2 targets NDRG1 through a previously undescribed mechanism involving the recruitment of early growth response 1 (EGR1). We show EGR1 is required for the ability of TBX2 to repress NDRG1 and drive cell proliferation. We show that TBX2 interacts with EGR1 and that TBX2 requires EGR1 to target the NDRG1 proximal promoter. Abrogation of either TBX2 or EGR1 expression is accompanied by the upregulation of cell senescence and apoptotic markers. NDRG1 can recapitulate these effects when transfected into TBX2-expressing cells. Together, these data identify a novel mechanism for TBX2-driven oncogenesis and highlight the importance of NDRG1 as a growth control gene in breast tissue.

Interferon-induced transmembrane 3 binds osteopontin in vitro: expressed in vivo IFITM3 reduced OPN expression.

Osteopontin is a secreted, integrin-binding and phosphorylated acidic glycoprotein, which has an important role in tumour progression. We have shown that Wnt, Ets, AP-1, c-jun and beta-catenin/Lef-1/Tcf-1 stimulates OPN transcription in rat mammary carcinoma cells by binding to a specific promoter sequence. However, co-repressors of OPN have not been identified. In this study, we have used the bacterial two-hybrid system to isolate cDNA-encoding proteins that bind to OPN and modulate its role in malignant transformation. Using this approach we isolated interferon-induced transmembrane protein 3 gene (IFITM3) as a potential protein partner. We show that IFITM3 and OPN interact in vitro and in vivo and that IFITM3 reduces osteopontin (OPN) mRNA expression, possibly by affecting OPN mRNA stability. Stable transfection of IFITM3 inhibits OPN, which mediates anchorage-independent growth, cell adhesion and cell invasion. Northern blot analysis revealed an inverse mRNA expression pattern of IFITM3 and OPN in human mammary cell lines. Inhibition of IFITM3 by antisense RNA promoted OPN protein expression, enhanced cell invasion by parental benign non-invasive Rama 37 cells, indicating that the two proteins interact functionally as well. We also identified an IFITM3 DNA-binding domain, which interacts with OPN, deletion of which abolished its inhibitive effect on OPN. This work has shown for the first time that IFITM3 physically interacts with OPN and reduces OPN mRNA expression, which mediates cell adhesion, cell invasion, colony formation in soft agar and metastasis in a rat model system.

Cells deficient in the base excision repair protein, DNA polymerase beta, are hypersensitive to oxaliplatin chemotherapy.

A significant proportion of human cancers overexpress DNA polymerase beta (Pol beta), the major DNA polymerase involved in base excision repair. The underlying mechanism and biological consequences of overexpression of this protein are unknown. We examined whether Pol beta, expressed at levels found in tumor cells, is involved in the repair of DNA damage induced by oxaliplatin treatment and whether the expression status of this protein alters the sensitivity of cells to oxaliplatin. DNA damage induced by oxaliplatin treatment of HCT116 and HT29 colon cancer cells was observed to be associated with the stabilization of Pol beta protein on chromatin. In comparison with HCT116 colon cancer cells, isogenic oxaliplatin-resistant (HCT-OR) cells were found to have higher constitutive levels of Pol beta protein, faster in vitro repair of a DNA substrate containing a single nucleotide gap and faster repair of 1,2-GG oxaliplatin adduct levels in cells. In HCT-OR cells, small interfering RNA knockdown of Pol beta delayed the repair of oxaliplatin-induced DNA damage. In a different model system, Pol beta-deficient fibroblasts were less able to repair 1,2-GG oxaliplatin adducts and were hypersensitive to oxaliplatin treatment compared with isogenic Pol beta-expressing cells. Consistent with previous studies, Pol beta-deficient mouse fibroblasts were not hypersensitive to cisplatin treatment. These data provide the first link between oxaliplatin sensitivity and DNA repair involving Pol beta. They demonstrate that Pol beta modulates the sensitivity of cells to oxaliplatin treatment.

Procaspase 8 overexpression in non-small-cell lung cancer promotes apoptosis induced by FLIP silencing.

We found that procaspase 8 was overexpressed in non-small-cell lung cancers (NSCLCs) compared with matched normal tissues. The caspase 8 inhibitor FLICE-inhibitory protein (FLIP) was also overexpressed in the majority of NSCLCs. Silencing FLIP induced caspase 8 activation and apoptosis in NSCLC cell lines, but not in normal lung cell lines. Apoptosis induced by FLIP silencing was mediated by the TRAIL death receptors DR4 and DR5, but was not dependent on ligation of the receptors by TRAIL. Furthermore, the apoptosis induced by FLIP silencing was dependent on the overexpression of procaspase 8 in NSCLC cells. Moreover, in NSCLC cells, but not in normal cells, FLIP silencing induced co-localization of DR5 and ceramide, and disruption of this co-localization abrogated apoptosis. FLIP silencing supra-additively increased TRAIL-induced apoptosis of NSCLC cells; however, normal lung cells were resistant to TRAIL, even when FLIP was silenced. Importantly, FLIP silencing sensitized NSCLC cells but not normal cells to chemotherapy in vitro, and silencing FLIP in vivo retarded NSCLC xenograft growth and enhanced the anti-tumour effects of cisplatin. Collectively, our results suggest that due to frequent procaspase 8 overexpression, NSCLCs may be particularly sensitive to FLIP-targeted therapies.

Anti-apoptotic mechanisms of drug resistance in cancer.

Resistance to chemotherapy is a major obstacle in the treatment of cancer. Despite the advent of new chemotherapies and molecular-targeted therapies, approximately 90% of patients with metastatic cancer succumb to their disease. Drug resistance, either acquired or intrinsic, often prevents tumour cells from undergoing sufficient levels of programmed cell death or apoptosis, resulting in cancer cell survival and treatment failure. In pre-clinical disease models, agents that target the apoptotic pathway have been shown to sensitize tumour cells to chemotherapy and radiotherapy. Such therapies include small molecule inhibitors and antisense strategies that inhibit the activity of anti-apoptotic proteins, or treatment with recombinant pro-apoptotic proteins or antibodies that can activate the apoptotic pathway. This review will discuss apoptosis and the mechanisms by which it can become dysregulated in human cancer. In addition, novel therapeutic strategies that target key components of the apoptotic machinery will be discussed.

Combined inhibition of FLIP and XIAP induces Bax-independent apoptosis in type II colorectal cancer cells.

Death receptors can directly (type I cells) or indirectly induce apoptosis by activating mitochondrial-regulated apoptosis (type II cells). The level of caspase 8 activation is thought to determine whether a cell is type I or II, with type II cells less efficient at activating this caspase following death receptor activation. FLICE-inhibitory protein (FLIP) blocks death receptor-mediated apoptosis by inhibiting caspase 8 activation; therefore, we assessed whether silencing FLIP could convert type II cells into type I. FLIP silencing-induced caspase 8 activation in Bax wild-type and null HCT116 colorectal cancer cells; however, complete caspase 3 processing and apoptosis were only observed in Bax wild-type cells. Bax-null cells were also more resistant to chemotherapy and tumor necrosis factor-related apoptosis inducing ligand and, unlike the Bax wild-type cells, were not sensitized to these agents by FLIP silencing. Further analyses indicated that release of second mitochondrial activator of caspases from mitochondria and subsequent inhibition of X-linked inhibitor of apoptosis protein (XIAP) was required to induce full caspase 3 processing and apoptosis following FLIP silencing. These results indicate that silencing FLIP does not necessarily bypass the requirement for mitochondrial involvement in type II cells. Furthermore, targeting FLIP and XIAP may represent a therapeutic strategy for the treatment of colorectal tumors with defects in mitochondrial-regulated apoptosis.

Dexamethasone potentiates the antiangiogenic activity of docetaxel in castration-resistant prostate cancer.

We sought to characterise whether dexamethasone (DEX) may enhance tumour response to docetaxel in in vitro and in vivo models of metastatic prostate cancer (CaP). In vitro experiments conducted on PC3 and human bone marrow endothelial cells (hBMECs) determined that administration of DEX (10 nM) reduced constitutive nuclear factor-kappaB (NF-kappaB) activity, decreasing interleukin (IL)-8, CXCL1 and VEGF gene expression in PC3 cells. Dexamethasone also attenuated docetaxel-induced NF-kappaB and activator protein-1 transcription and reduced docetaxel-promoted expression/secretion of IL-8 and CXCL1 in PC3 and hBMECs. Although DEX failed to enhance docetaxel cytotoxicity on PC3 cells, DEX potentiated the antiangiogenic activity of docetaxel in vitro, further reducing vessel area and vessel length in developing endothelial tubes (P<0.05). Docetaxel had a potent antiangiogenic activity in the dorsal skin flap-implanted PC3 tumours in vivo. Small blood vessel formation was further suppressed in tumours co-treated with docetaxel and DEX, substantiated by an increased average vessel diameter and segment length and a decreased number of branch points in the residual tumour vasculature (P<0.001). Our data show that DEX potentiates the antiangiogenic activity of docetaxel, suggesting a putative mechanism for the palliative and survival benefits of these agents in metastatic CaP.

RAN GTPase is an effector of the invasive/metastatic phenotype induced by osteopontin.

Osteopontin (OPN) is a phosphorylated glycoprotein that binds to alpha v-containing integrins and is important in malignant transformation and cancer. Previously, we have utilized suppressive subtractive hybridization between mRNAs isolated from the Rama 37 (R37) rat mammary cell line and a subclone rendered invasive and metastatic by stable transfection with an expression vector for OPN to identify RAN GTPase (RAN) as the most overexpressed gene, in addition to that of OPN. Here we show that transfection of noninvasive R37 cells with an expression vector for RAN resulted in increased anchorage-independent growth, cell attachment and invasion through Matrigel in vitro, and metastasis in syngeneic rats. This induction of a malignant phenotype was induced independently of the expression of OPN, and was reversed by specifically reducing the expression of RAN using small-interfering RNAs. By using a combination of mutant protein and inhibitors, it was found that RAN signal transduction occurred through the c-Met receptor and PI3 kinase. This study therefore identifies RAN as a novel effector of OPN-mediated malignant transformation and some of its downstream signaling events in a mammary epithelial model of cancer invasion/metastasis.

The management of pancreatic cancer. Current expert opinion and recommendations derived from the 8th World Congress on Gastrointestinal Cancer, Barcelona, 2006.

This article summarizes the expert discussion on the management of pancreatic cancer, which took place during the 8th World Congress on Gastrointestinal Cancer in June 2006 in Barcelona. A multidisciplinary approach to a patient with pancreatic cancer is essential, in order to guarantee an optimal staging, surgery, selection of the appropriate (neo-)adjuvant strategy and chemotherapeutic choice management. Moreover, optimal symptomatic management requires a dedicated team of health care professionals. Quality control of surgery and pathology is especially important in this disease with a high locoregional failure rate. There is now solid evidence in favour of chemotherapy in both the adjuvant and palliative setting, and gemcitabine combined with erlotinib, capecitabine or platinum compounds seems to be slightly more active than gemcitabine alone in advanced pancreatic cancer. There is a place for chemoradiotherapy in selected patients with locally advanced disease, while the role in the adjuvant setting remains controversial. Those involved in the care for patients with pancreatic cancer should be encouraged to participate in well-designed clinical trials, in order to increase the evidence-based knowledge and to make further progress.

HIF-1 and NF-kappaB-mediated upregulation of CXCR1 and CXCR2 expression promotes cell survival in hypoxic prostate cancer cells.

Hypoxic cancer cells are resistant to treatment, leading to the selection of cells with a more malignant phenotype. The expression of interleukin-8 (IL-8) plays an important role in the tumorigenesis and metastasis of solid tumors including prostate cancer. Recently, we detected elevated expression of IL-8 and IL-8 receptors in human prostate cancer tissue. The objective of the current study was to determine whether hypoxia increases IL-8 and IL-8 receptor expression in prostate cancer cells and whether this contributes to a survival advantage in hypoxic cells. IL-8, CXCR1 and CXCR2 messenger RNA (mRNA) expression in PC3 cells was upregulated in response to hypoxia in a time-dependent manner. Elevated IL-8 secretion following hypoxia was detected by enzyme-linked immunosorbent assay, while immunoblotting confirmed elevated receptor expression. Attenuation of hypoxia-inducible factor (HIF-1) and nuclear factor-kappaB (NF-kappaB) transcriptional activity using small interfering RNA (siRNA), a HIF-1 dominant-negative and pharmacological inhibitors, abrogated hypoxia-induced transcription of CXCR1 and CXCR2 in PC3 cells. Furthermore, chromatin-IP analysis demonstrated binding of HIF-1 and NF-kappaB to CXCR1. Finally, inhibition of IL-8 signaling potentiated etoposide-induced cell death in hypoxic PC3 cells. These results suggest that IL-8 signaling confers a survival advantage to hypoxic prostate cancer cells, and therefore, strategies to inhibit IL-8 signaling may sensitize hypoxic tumor cells to conventional treatments.