Adrenomedullin is a therapeutic target in colorectal cancer.

The KRAS oncogene influences angiogenesis, metastasis and chemoresistance in colorectal cancers (CRCs), and these processes are all enhanced in hypoxic conditions. To define functional activities of mutant KRAS in a hypoxic microenvironment, we first performed cDNA microarray experiments in isogenic DKs5 and DKO3 colon cancer cell lines that differ only by their expression of mutant KRAS (K-ras(D13)). Adrenomedullin (ADM) was identified as one of the most significantly upregulated genes in DKs5 cells that express the KRAS oncogene in hypoxia (3.2-fold, p = 1.47 × 10(-5)). Ectopic expression of mutant KRAS (K-ras(V12)) in Caco-2 cells (K-ras(WT)) induced ADM, whereas selective knockdown of mutant KRAS alleles (K-ras(D13) or K-ras(V12)) in HCT116, DLD1 and SW480 colon cancer cells suppressed the expression of ADM in hypoxia. Knockdown of ADM in colon tumor xenografts blocked angiogenesis and stimulated apoptosis, resulting in tumor suppression. Furthermore, ADM also regulated colon cancer cell invasion in vitro. Among 56 patients with CRC, significantly higher expression levels of ADM were observed in samples harboring a KRAS mutation. Collectively, ADM is a new target of oncogenic KRAS in the setting of hypoxia. This observation suggests that therapeutic targets may differ depending upon the specific tumor microenvironment.

Psychological impact of BRAF mutational status in advanced melanoma patients.

BACKGROUND: The aim of the study was to highlight the psychological aspects involved in patients with advanced melanoma and to describe the differences between subjects who are positive and negative for the BRAFv600e genetic mutation, a variable that leads to a different medical approach to cancer therapy. The hypothesis is that following knowledge of the genetic mutation and the therapeutic possibilities inherent to it, mutation positive patients (BRAF+) exhibit fewer negative psychological reactions than negative patients (BRAF-) at the time of diagnosis. METHODS: The tests used (SF-12, MHQ) were administered at the time of diagnosis and after three months. RESULTS: The main findings suggest a greater impairment of quality of life at T1 than at T0, regardless of the mutation; BRAF mutated patients show more favorable scores at diagnosis and a reversal of the trend at three months after diagnosis. CONCLUSIONS: The results obtained, in line with the literature under review, show a significant general psychological distress in the present oncological sample, suggesting the importance of a psychological, as well as medical, care of the patient and the family.

The national COVID-19 vaccination campaign targeting the extremely vulnerable: the Florence Medical Oncology Unit experience in patients with cancer.

BACKGROUND: International and national oncology societies had released recommendations in favor of COVID-19 vaccination in cancer patients. In the context of the national vaccination campaign targeting the so called extremely vulnerable, we aimed to assess the safety and efficacy of the mRNA vaccines in a cohort of 623 patients. METHODS: Between March 26 and April 04, 2021, the Pfizer and BioNTech BNT162b2 mRNA and the Moderna mRNA-1273 vaccines were given as a two-dose prime-boost regimen. Starting on September 25th 2021 a third dose was offered to patients in whom a suboptimal immunogenicity with COVID-19 vaccination could be expected. Safety assessments were performed by phone call 7 days after each dose. Electronic health records were accessed to review demographic information, disease history, treatment detail, and outcome events of participants patients'. FINDINGS: No toxicities were reported in 63.7%, 54%, and in 48.7% patients with cancer after each dose. Mild-to-moderate pain at the injection site was the most commonly adverse event. After the second dose, 46% of the 610 patients reported toxicity, with more systemic side-effects observed. Fever was reported in 45% of patients, with a temperature ≥ 38 °C in 21.4% of them. Of the 335 patients receiving a third vaccine dose, 51% reported toxicity, with 13% of patients reporting more than one effect. Logistic regression analysis reported mixed results, with limited variables or categories reporting a significant odd ratio. The type of vaccine reported a significant value at first dose (OR = 0.12; CI 0.52, 0.26; p = 0.00). Thirty-four cases of COVID-19 infection were reported with only one patient requiring a short-term hospitalization for monitoring. INTERPRETATION: The safety profile of the mRNA vaccines does not raise any specific concerns and support prioritization of vaccination for cancer patients.

The chromosomal instability pathway in colon cancer.

The acquisition of genomic instability is a crucial feature in tumor development and there are at least 3 distinct pathways in colorectal cancer pathogenesis: the chromosomal instability (CIN), microsatellite instability, and CpG island methylator phenotype pathways. Most cases of colorectal cancer arise through the CIN pathway, which is characterized by widespread imbalances in chromosome number (aneuploidy) and loss of heterozygosity. It can result from defects in chromosomal segregation, telomere stability, and the DNA damage response, although the full complement of genes underlying CIN remains incompletely described. Coupled with the karyotypic abnormalities observed in CIN tumors are the accumulation of a characteristic set of mutations in specific tumor suppressor genes and oncogenes that activate pathways critical for colorectal cancer initiation and progression. Whether CIN creates the appropriate milieu for the accumulation of these mutations or vice versa remains a provocative and unanswered question. The goal of this review is to provide an updated perspective on the mechanisms that lead to CIN and the key mutations that are acquired in this pathway.

Epithelial to mesenchymal transition is impaired in colon cancer cells with microsatellite instability.

BACKGROUND & AIMS: Colorectal cancers (CRCs) displaying DNA microsatellite instability (MSI) are associated with a favorable natural history, but the molecular basis for this observation has not been defined. We sought to determine whether the epithelial to mesenchymal transition (EMT) is impaired in MSI-positive CRCs that characteristically have a mutant transforming growth factor-beta receptor type II (TGFBR2) gene. METHODS: The induction of EMT by transforming growth factor-beta1 (TGF-beta1) was analyzed by phase contrast microscopy, immunofluorescence, quantitative reverse transcription polymerase chain reaction, immunoblotting, and cellular migration, and invasion assays. Expression of EMT markers was evaluated by immunohistochemistry and quantitative reverse transcription polymerase chain reaction in a series of human colorectal tumors. RESULTS: TGF-beta1 induced changes in cellular morphology, gene expression, motility, and invasion consistent with EMT in microsatellite stable (MSS) colon cancer cells, whereas cells with MSI and mutant TGFBR2 were unresponsive to TGF-beta1. These effects did not require Smad4, but depended on the recruitment of extracellular signal-regulated kinase. Tumor cells with MSI but wild-type TGFBR2 underwent EMT in response to TGF-beta1, indicating that TGFBR2 genotype is a key determinant of the EMT response in tumors with MSI. In human colorectal tumors, expression of EMT markers was significantly associated with adverse clinicopathologic features and the absence of MSI. CONCLUSIONS: These findings define a unique genotype-phenotype relationship between TGFBR2 and EMT that may contribute to the improved prognosis consistently observed in colon cancers with MSI.

Melanoma diagnosis: traumatic impact of the event on the patient.

BACKGROUND: The aim of our study was to consider the distressing impact of the diagnosis in a group of patients with metastatic melanoma, and the effects it could have on the quality of life of the patients. METHODS: We proposed an Impact Event Scale (IES-R) to a group of 31 patients. The patients were positive to the distress thermometer (DS) and accepted the psychological support. After six months from the start of the treatment we made a semi-structured interview of 10 multiple choice questions. RESULTS: Sixty-five per cent of women and 50% of men report that all the event related to the disease, cause emotions that recall the disease. Eighty-two per cent of women compared to 50% of men, report that the thought of their medical condition tends to affect their quality of sleep; the patients report feelings of anger and irritation (41% of the women and 78% of the men). CONCLUSIONS: The traumatic aspects following the diagnosis of melanoma burst powerfully into the life of these patients, who show different reactions, also according to gender.

Circulating Galectin-1 and Galectin-3 in Sera From Patients With Systemic Sclerosis: Associations With Clinical Features and Treatment.

Systemic Sclerosis (SSc) is a rheumatic disease characterized by fibrosis, microvascular damage and immune dysregulation. Two major subsets, limited cutaneous systemic sclerosis (lcSSc) and diffuse cutaneous systemic sclerosis (dcSSc) can be defined, according to the extent of skin involvement. Increasing evidence indicates a role for galectins in immune and vascular programs, extracellular matrix remodeling and fibrosis, suggesting their possible involvement in SSc. Here, we determined serum levels of galectin (Gal)-1 and Gal-3 in 83 SSc patients (dcSSc n = 17; lcSSc n = 64; ssSSc n = 2), and evaluated their association with clinical manifestations of the disease. Patients with dcSSc showed lower Gal-3 levels, compared to lcSSc (p = 0.003), whereas no considerable difference in Gal-1 levels was detected between groups. Remarkably, higher concentrations of Gal-1 were associated with the presence of telangiectasias (p = 0.015), and higher concentrations Gal-3 were associated with telangiectasias (p = 0.021), diarrhea (p = 0.039) and constipation (p = 0.038). Moreover, lower Gal-3 levels were associated with the presence of tendinous retractions (p = 0.005). Patients receiving calcium blockers (p = 0.048), methotrexate (p = 0.046) or any immunosuppressive treatment (p = 0.044) presented lower concentrations of Gal-3 compared to those not receiving such treatments. The presence of telangiectasia and the type of SSc maintained their statistical association with Gal-3 (ß 0.25; p = 0.022 and ß 0.26; p = 0.017, respectively) in multiple linear regression models. In conclusion, serum levels of Gal-3 are associated with clinical manifestations of SSc. Among them, the presence of telangiectasias could be explained by the central role of this lectin in the vascularization programs.

Capecitabine and celecoxib as second-line treatment of advanced pancreatic and biliary tract cancers.

OBJECTIVE: An increasing number of patients with advanced pancreatic or biliary tract cancer who progress after a gemcitabine-containing regimen are candidates for further chemotherapy. We therefore evaluated a fully oral regimen of capecitabine and celecoxib (CapCel) as second-line treatment in these patients. METHODS: Thirty-five patients with documented progressive disease after first-line treatment were enrolled. Capecitabine was administered at a dose of 1,000 mg/m(2) b.i.d. for 2 consecutive weeks followed by 1 week of rest; celecoxib was given continuously at 200 mg b.i.d. Progression-free survival at 3 months was the primary study endpoint. RESULTS: The CapCel combination was associated with an overall response rate of 9% and median survival duration of 19 weeks. Sixty percent of patients were free from progression 3 months after the start of treatment. Multivariate analysis identified a positive clinical benefit response and a decline in CA 19.9 serum levels >25% compared with baseline levels as independent predictors of prolonged survival. The treatment protocol was well tolerated with negligible hematological toxicity. The most common grade 3 non-hematological toxicities were hypertransaminasemia, diarrhea and asthenia. CONCLUSIONS: The CapCel combination is a safe treatment option with moderate activity in patients with pancreatic/biliary tract cancer after failure of a previous gemcitabine-containing regimen.

Human Mena+11a isoform serves as a marker of epithelial phenotype and sensitivity to epidermal growth factor receptor inhibition in human pancreatic cancer cell lines.

PURPOSE: hMena, member of the enabled/vasodilator-stimulated phosphoprotein family, is a cytoskeletal protein that is involved in the regulation of cell motility and adhesion. The aim of this study was to determine whether or not the expression of hMena isoforms correlated with sensitivity to EGFR tyrosine kinase inhibitors and could serve as markers with potential clinical use. EXPERIMENTAL DESIGN: Human pancreatic ductal adenocarcinoma cell lines were characterized for in vitro sensitivity to erlotinib, expression of HER family receptors, markers of epithelial to mesenchymal transition, and expression of hMena and its isoform hMena(+11a). The effects of epidermal growth factor (EGF) and erlotinib on hMena expression as well as the effect of hMena knockdown on cell proliferation were also evaluated. RESULTS: hMena was detected in all of the pancreatic tumor cell lines tested as well as in the majority of the human tumor samples [primary (92%) and metastatic (86%)]. Intriguingly, in vitro hMena(+11a) isoform was specifically associated with an epithelial phenotype, EGFR dependency, and sensitivity to erlotinib. In epithelial BxPC3 cells, epidermal growth factor up-regulated hMena/hMena(+11a) and erlotinib down-regulated expression. hMena knockdown reduced cell proliferation and mitogen-activated protein kinase and AKT activation in BxPC3 cells, and promoted the growth inhibitory effects of erlotinib. CONCLUSIONS: Collectively, our data indicate that the hMena(+11a) isoform is associated with an epithelial phenotype and identifies EGFR-dependent cell lines that are sensitive to the EGFR inhibitor erlotinib. The availability of anti-hMena(+11a)-specific probes may offer a new tool in pancreatic cancer management if these results can be verified prospectively in cancer patients.

Transforming growth factor alpha expression drives constitutive epidermal growth factor receptor pathway activation and sensitivity to gefitinib (Iressa) in human pancreatic cancer cell lines.

The epidermal growth factor receptor (EGFR) is considered an important therapeutic target in pancreatic cancer, but it is currently impossible to identify those patients who are most likely to benefit from EGFR-directed therapy. We examined the biological effects of the EGFR tyrosine kinase inhibitor gefitinib (ZD1839, Iressa) in a panel of nine human pancreatic cancer cell lines. The drug strongly inhibited DNA synthesis and induced low levels of apoptosis at clinically relevant concentrations in a subset of three of the lines (L3.6pl, BxPC3, and Cfpac1). Sensitivity to gefitinib correlated directly with ligand [transforming growth factor-alpha (TGF-alpha)] expression (r(2) = 0.71, P = 0.004) but not with surface EGFR expression. The gefitinib-sensitive cells displayed constitutive baseline EGFR phosphorylation, whereas the gefitinib-resistant cells did not. Exposure to gefitinib or a small interfering RNA construct specific for TGF-alpha reversed the constitutive EGFR phosphorylation and downstream target [extracellular signal-regulated kinases (ERK), AKT] phosphorylation in the gefitinib-sensitive cells but had no effects on ERK or AKT phosphorylation in gefitinib-resistant cells. Baseline EGFR phosphorylation was lower in a subclone of L3.6pl selected for low TGF-alpha expression, and these cells were also resistant to gefitinib-mediated growth inhibition. Gefitinib blocked the growth of tumor xenografts derived from L3.6pl cells but had no effect on the growth of tumors derived from EGFR-independent MiaPaCa-2 cells. Together, our data show that TGF-alpha expression identifies a subset of human pancreatic cancer cells that is dependent on EGFR signaling in vitro and in vivo. Quantification of TGF-alpha expression may therefore represent an effective means of identifying EGFR-responsive primary tumors.

Aggresome disruption: a novel strategy to enhance bortezomib-induced apoptosis in pancreatic cancer cells.

The proteasome inhibitor bortezomib (formerly known as PS-341) recently received Food and Drug Administration approval for the treatment of multiple myeloma, and its activity is currently being evaluated in solid tumors. Bortezomib triggers apoptosis in pancreatic cancer cells, but the mechanisms involved have not been fully elucidated. Here, we show that pancreatic cancer cells exposed to bortezomib formed aggregates of ubiquitin-conjugated proteins ("aggresomes") in vitro and in vivo. Bortezomib-induced aggresome formation was determined to be cytoprotective and could be disrupted using histone deacetylase (HDAC) 6 small interfering RNA or chemical HDAC inhibitors, which resulted in endoplasmic reticulum stress and synergistic levels of apoptosis in vitro and in an orthotopic pancreatic cancer xenograft model in vivo. Interestingly, bortezomib did not induce aggresome formation in immortalized normal human pancreatic epithelial cells in vitro or in murine pancreatic epithelial cells in vivo. In addition, these cells did not undergo apoptosis following treatment with bortezomib, suberoylanilide hydroxamic acid, or the combination, showing tumor selectivity. Taken together, our study shows that inhibition of aggresome formation can strongly potentiate the efficacy of bortezomib and provides the foundation for clinical trials of bortezomib in combination with HDAC inhibitors for the treatment of pancreatic cancer.

Bortezomib sensitizes pancreatic cancer cells to endoplasmic reticulum stress-mediated apoptosis.

Bortezomib (PS-341, Velcade) is a potent and selective inhibitor of the proteasome that is currently under investigation for the treatment of solid malignancies. We have shown previously that bortezomib has activity in pancreatic cancer models and that the drug induces endoplasmic reticulum (ER) stress but also suppresses the unfolded protein response (UPR). Because the UPR is an important cytoprotective mechanism, we hypothesized that bortezomib would sensitize pancreatic cancer cells to ER stress-mediated apoptosis. Here, we show that bortezomib promotes apoptosis triggered by classic ER stress inducers (tunicamycin and thapsigargin) via a c-Jun NH(2)-terminal kinase (JNK)-dependent mechanism. We also show that cisplatin stimulates ER stress and interacts with bortezomib to increase ER dilation, intracellular Ca(2+) levels, and cell death. Importantly, combined therapy with bortezomib plus cisplatin induced JNK activation and apoptosis in orthotopic pancreatic tumors resulting in a reduction in tumor burden. Taken together, our data establish that bortezomib sensitizes pancreatic cancer cells to ER stress-induced apoptosis and show that bortezomib strongly enhances the anticancer activity of cisplatin.

Nuclear factor-kappaB maintains TRAIL resistance in human pancreatic cancer cells.

Although it displays promising activity in other tumor models, the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on human pancreatic cancer cells have not been comprehensively explored. We report that a majority of human pancreatic cancer cell lines (seven of nine) underwent apoptosis when they were exposed to recombinant human TRAIL in vitro. Characterization of surface TRAIL receptors by fluorescence-activated cell sorting showed that TRAIL-resistant cells (Panc-1 and HS766T) expressed lower levels of DR4 and DR5 than did TRAIL-sensitive cells. The proteasome inhibitor bortezomib (PS-341, Velcade) further increased TRAIL responsiveness in the TRAIL-sensitive cells and synergized with TRAIL to reverse resistance in Panc-1 and HS776T cells. The effects of bortezomib were mimicked by transfection with a small interfering RNA construct specific for the p65 subunit of nuclear factor-kappaB (NF-kappaB) or exposure to a selective chemical inhibitor of IKK (PS-1145). Silencing IkappaBalpha prevented TRAIL sensitization by PS-1145, confirming that IkappaBalpha mediated the effects of PS-1145. NF-kappaB inhibition resulted in down-regulation of BCL-XL and XIAP, and silencing either restored TRAIL sensitivity in TRAIL-resistant cells. Finally, therapy with TRAIL plus PS-1145 reversed TRAIL resistance in vivo to produce synergistic growth inhibition in orthotopic Panc-1 tumors. Together, our results show that NF-kappaB inhibits TRAIL-induced apoptosis in human pancreatic cancer cells and suggest that combination therapy with TRAIL and NF-kappaB inhibitors, such as bortezomib, PS-1145, or curcumin, should be considered as a possible treatment strategy in patients with pancreatic cancer.

Prostaglandin E2 drives cyclooxygenase-2 expression via cyclic AMP response element activation in human pancreatic cancer cells.

Cyclooxygenase-2 (COX-2) is constitutively expressed in most human primary carcinomas and with its synthesized product, prostaglandin E2 (PGE2), appears to play important roles in tumor invasion, angiogenesis, resistance to apoptosis and suppression of host immunity. However, the molecular mechanisms that control COX-2 expression are unclear. The purpose of this study was to clarify the mechanism of basal and PGE2-mediated COX-2 expression in the highly metastatic L3.6pl human pancreatic cancer cell line. Using RNA interference to disrupt the expression of CREB and the NF-kappaB p65 subunit, we found that both are involved in maintaining basal COX-2 expression in L3.6pl cells. We also demonstrated that PGE2 increased the cyclic AMP concentration, thereby activating protein kinase A (PKA), which in turn phosphorylated the cyclic AMP response element binding protein (CREB), leading to interaction with the cyclic AMP response element in the promoter region of the COX-2 gene. Immunocytochemical analysis confirmed that PGE2 stimulated the translocation of PKA to the nucleus and increased the immuno-reactivity of phosphorylated CREB. Pretreatment with the PKA selective inhibitor H 89 and the E-prostanoid receptor 2 inhibitor AH 6809 reduced COX-2 upregulation by PGE2. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay results further suggested a role for CREB in COX-2 transcriptional control. Understanding the pathways that control COX-2 expression may lead to a better understanding of its dysregulation in pancreatic carcinomas and facilitate the development of novel therapeutic approaches.

Molecularly targeted therapy: toxicity and quality of life considerations in advanced colorectal cancer.

Colorectal cancer (CRC) is the third most common malignancy and cause of death from cancer among adults worldwide. In recent years, the use of 5-fluorouracil-based regimens in combination with molecularly targeted agents has greatly expanded treatment options for patients with metastatic disease. With a more capillary use of this new class of agents comes the recognition of diverse adverse events related to disturbance of critical biological pathways involved in physiological functions. Proactive management and prevention of adverse events, with a focus on the necessary compromise between adverse events and tumor control, are often effective and allows for uninterrupted, full-dose therapy with targeted agents. Quality of life does not appear deteriorated, rather improved due to efficacy in prolonging wellness.

Cables1 is a tumor suppressor gene that regulates intestinal tumor progression in Apc(Min) mice.

The transformation of colonic mucosal epithelium to adenocarcinoma requires progressive oncogene activation and tumor suppressor gene inactivation. Loss of chromosome 18q is common in colon cancer but not in precancerous adenomas. A few candidate tumor suppressor genes have been identified in this region, including CABLES1 at 18q11.2-12.1. This study investigates the role of CABLES1 in an in vivo mouse model of intestinal adenocarcinoma and in human colon cancer cell culture. Apc(Min/+) mice were crossed with mice harboring targeted inactivation of the Cables1 gene (Cables1(-/-)). The intestinal tumor burden and tumor expression of ß-catenin and PCNA was compared in Cables1(+/+)Apc(Min/+) and Cables1(-/-)Apc(Min/+) mice. ß-catenin activity in human colon cancer cells with CABLES1 inactivation and intestinal progenitor cell function in Cables1(-/-) mice were assayed in vitro. The mean number of small intestinal tumors per mouse was 3.1 ± 0.6 in Cables1(+/+)Apc(Min/+) mice, compared with 32.4 ± 3.5 in the Cables1(-/-)Apc(Min/+) mice (P < 0.0001). Fewer colonic tumors were observed in Cables1(+/+)Apc(Min/+) mice (mean 0.6 ± 0.1) compared with the Cables1(-/-)Apc(Min/+) mice (mean 1.3 ± 0.3, P = 0.01). Tumors from Cables1(-/-)Apc(Min/+) mice demonstrated increased nuclear expression of ß-catenin and an increased number of PCNA-positive cells. In vitro studies revealed that CABLES1 deficiency increased ß-catenin dependent transcription and increased intestinal progenitor cell activity. Loss of Cables1 enhances tumor progression in the Apc(Min/+) mouse model and activates the Wnt/ß-catenin signaling pathway. Cables1 is a tumor suppressor gene on chromosome 18q in this in vivo mouse model and likely has a similar role in human colon cancer.

Microsatellite instability in the management of colorectal cancer.

Microsatellite instability (MSI) is a form of genetic instability caused by alterations in the DNA mismatch repair system. Approximately 15% of colorectal cancers display MSI due to a germline mutation in one of the mismatch repair genes (MLH1, MSH2, MSH6 and PMS2) or to epigenetic silencing of MLH1. Colorectal cancers with MSI have distinctive features, including a tendency to arise in the proximal colon, poor differentiation, lymphocytic infiltration and mucinous or signet-ring histology. Patients with MSI tumors appear to have a better prognosis than those with microsatellite stable tumors, but curiously the responses to 5-fluorouracil-based chemotherapy regimens are poorer with MSI tumors. Preliminary data suggest possible advantages of irinotecan-based regimens, but these findings need validation in well-designed clinical trials.

Application of molecular diagnostics for the detection of Lynch syndrome.

Lynch syndrome (LS), or hereditary nonpolyposis colorectal cancer, is the most common hereditary colorectal cancer (CRC) syndrome, accounting for approximately 2-5% of all newly diagnosed cases of CRC. Patients with LS have an increased lifetime risk of colorectal (52.2% in women and 68.7% in men) and endometrial cancer (15-70%), as well as certain extra-colonic cancers. Germline mutations in one of several DNA mismatch repair genes underlie LS. Molecular testing has emerged as an indispensable strategy for the diagnosis of LS. The diagnostic work-up of at-risk individuals includes a careful family history evaluation, microsatellite instability, immunohistochemistry and germline DNA analysis. A positive test result can guide clinicians in formulating the appropriate screening, surveillance and management strategies. However, because of the absence of an overt phenotype, such as a diffuse polyposis, it is not always straightforward to recognize LS clinically.

Hypoxia activates the K-ras proto-oncogene to stimulate angiogenesis and inhibit apoptosis in colon cancer cells.

The KRAS proto-oncogene plays a key role in the development of many human tumors and is commonly activated by somatic mutation or signaling through specific growth factor receptors. However, the interaction between the micro-environment and K-ras activity has not been defined. Hypoxia invariably develops as tumors outgrow their supply of oxygen. A series of well-orchestrated cellular adaptations occur that stimulate angiogenesis and enhance survival of the tumor in hypoxic conditions. Our previous studies demonstrated that mutant KRAS alleles can interact with hypoxia to induce vascular endothelial growth factor (VEGF) in colon cancer. We sought to determine whether similar hypoxic responses are also present in tumors without a KRAS mutation. Hypoxia consistently increased the levels of activated, GTP-bound K-ras in colon cancer cell lines with a wild-type KRAS gene, and this depended upon the activation of c-Src. Inhibition of c-Src by PP2 treatment or siRNA knockdown blocked the hypoxic activation of K-ras. This activation of K-ras did not depend upon EGFR and resulted in the phosphorylation of Akt and induction of VEGF expression. In addition, activation of K-ras significantly blocked apoptosis in hypoxic conditions. These studies reveal a unique adaptive mechanism in hypoxia that activates K-ras signaling in the absence of a mutant KRAS oncogene.

Oncogenic KRAS and BRAF differentially regulate hypoxia-inducible factor-1alpha and -2alpha in colon cancer.

KRAS and BRAF mutations are frequently observed in human colon cancers. These mutations occur in a mutually exclusive manner, and each is associated with distinctive biological features. We showed previously that K-ras can interact with hypoxia to activate multiple signaling pathways. Many hypoxic responses are mediated by hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha, and we sought to define the roles of mutant KRAS and BRAF in the induction of HIF-1alpha and HIF-2alpha in colon cancer cells. Ectopic expression of mutant K-ras in Caco2 cells enhanced the hypoxic induction of only HIF-1alpha, whereas mutant BRAF enhanced both HIF-1alpha and HIF-2alpha. Knockout or knockdown of mutant KRAS in DLD-1 and HCT116 cells impaired the hypoxic induction of only HIF-1alpha. HIF-1alpha mRNA levels were comparable in cells with and without a KRAS mutation. However, the rate of HIF-1alpha protein synthesis was higher in cells with a KRAS mutation, and this was suppressed by the phosphoinositide 3-kinase inhibitor LY294002. In contrast, knockdown of mutant BRAF in HT29 cells suppressed both HIF-1alpha and HIF-2alpha. Although BRAF regulated mRNA levels of both HIF-1alpha and HIF-2alpha, knockdown of BRAF or treatment with the MEK inhibitor PD98059 impaired the translation of only HIF-2alpha. Our data reveal that oncogenic KRAS and BRAF mutations differentially regulate the hypoxic induction of HIF-1alpha and HIF-2alpha in colon cancer, and this may potentially contribute to the phenotypic differences of KRAS and BRAF mutations in colon tumors.