Margherita Gallicchio

Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer

A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here we show that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance, but resistant cells remained sensitive to combinatorial inhibition of EGFR and mitogen-activated protein-kinase kinase (MEK). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

Deregulation of the PI3K and KRAS signaling pathways in human cancer cells determines their response to everolimus

Personalized cancer medicine is based on the concept that targeted therapies are effective on subsets of patients whose tumors carry specific molecular alterations. Several mammalian target of rapamycin (mTOR) inhibitors are in preclinical or clinical trials for cancers, but the molecular basis of sensitivity or resistance to these inhibitors among patients is largely unknown. Here we have identified oncogenic variants of phosphoinositide-3-kinase, catalytic, alpha polypeptide (PIK3CA) and KRAS as determinants of response to the mTOR inhibitor everolimus. Human cancer cells carrying alterations in the PI3K pathway were responsive to everolimus, both in vitro and in vivo, except when KRAS mutations occurred concomitantly or were exogenously introduced. In human cancer cells with mutations in both PIK3CA and KRAS, genetic ablation of mutant KRAS reinstated response to the drug. Consistent with these data, PIK3CA mutant cells, but not KRAS mutant cells, displayed everolimus-sensitive translation. Importantly, in a cohort of metastatic cancer patients, the presence of oncogenic KRAS mutations was associated with lack of benefit after everolimus therapy. Thus, our results demonstrate that alterations in the KRAS and PIK3CA genes may represent biomarkers to optimize treatment of patients with mTOR inhibitors.

Anti-apoptotic and anti-inflammatory effects of hydrogen sulfide in a rat model of regional myocardial I/R.

Hydrogen sulfide (H2S) is a novel gaseous mediator produced by cystathionine-&bgr;-synthase and cystathionine-&ggr;-lyase in the cardiovascular system, including the heart. Using a rat model of regional myocardial ischemia/reperfusion, we investigated the effects of an H2S donor (sodium hydrogen sulfide [NaHS]) on the infarct size and apoptosis caused by ischemia (25 min) and reperfusion (2 h). Furthermore, we investigated the potential mechanism(s) of the cardioprotective effect(s) afforded by NaHS. Specifically, we demonstrate that NaHS (1) attenuates the increase in caspase 9 activity observed in cardiac myocytes isolated from the area at risk (AAR) of hearts subjected in vivo to regional myocardial I/R and (2) ameliorates the decrease in expression of Bcl-2 within the AAR obtained from rat hearts subjected to regional myocardial I/R. The cardioprotective effects of NaHS were abolished by 5-hydroxydeconoate, a putative mitochondrial adenosine triphosphate-sensitive potassium channel blocker. Furthermore, NaHS attenuated the increase in the I/R-induced (1) phosphorylation of p38 mitogen-activated protein kinase and Jun N-terminal kinase, (2) translocation from the cytosol to the nucleus of the p65 subunit of nuclear factor-&kgr;B, (3) intercellular adhesion molecule 1 expression, (4) polymorphonuclear leukocyte accumulation, (5) myeloperoxidase activity, (6) malondialdehyde levels, and (7) nitrotyrosine staining determined in the AAR obtained from rat hearts subjected to regional myocardial I/R. In conclusion, we demonstrate that the cardioprotective effect of NaHS is secondary to a combination of antiapoptotic and anti-inflammatory effects. The antiapoptotic effect of NaHS may be in part due to the opening of the putative mitochondrial adenosine triphosphate-sensitive potassium channels.

Generation of endogenous hydrogen sulfide by cystathionine γ-lyase limits renal ischemia/reperfusion injury and dysfunction

The generation of endogenous hydrogen sulfide may either limit or contribute to the degree of tissue injury caused by ischemia/reperfusion. A total of 74 male Wistar rats were used to investigate the effects of endogenous and exogenous hydrogen sulfide in renal ischemia/reperfusion. Administration of the irreversible cystathionine γ-lyase (CSE) inhibitor, dL-propargylglycine, prevented the recovery of renal function after 45 min ischemia and 72 h reperfusion. The hydrogen sulfide donor sodium hydrosulfide attenuated the (renal, tubular, and glomerular) dysfunction and injury caused by 45 min ischemia and 6 h reperfusion. Western blot analysis of kidneys taken at 30 min reperfusion showed that sodium hydrosulfide significantly attenuated phosphorylation of mitogen-activated protein kinases (p-38, c-JUN N-terminal protein kinase 1/2, and extracellular signal-regulated kinase 1/2) and activation of nuclear factor-κB. At 6 h reperfusion, sodium hydrosulfide significantly attenuated the histological score for acute tubular necrosis, the activation of caspase-3 and Bid, the decline in the expression of anti-apoptotic Bcl-2, and the expression of nuclear factor-κB-dependent proteins (inducible nitric oxide synthase, cyclo-oxygenase-2, and intercellular adhesion molecule-1). These findings suggest that (1) the synthesis of endogenous hydrogen sulfide by CSE is essential to protect the kidney against ischemia/reperfusion injury and dysfunction and aids in the recovery of renal function following ischemia/reperfusion, (2) hydrogen sulfide generated by sodium hydrosulfide reduces ischemia/reperfusion injury and dysfunction, and morphological changes of the kidney, and (3) the observed protective effects of hydrogen sulfide are due to both anti-apoptotic and anti-inflammatory effects.

Replacement of normal with mutant alleles in the genome of normal human cells unveils mutation-specific drug responses

Mutations in oncogenes and tumor suppressor genes are responsible for tumorigenesis and represent favored therapeutic targets in oncology. We exploited homologous recombination to knock-in individual cancer mutations in the genome of nontransformed human cells. Sequential introduction of multiple mutations was also achieved, demonstrating the potential of this strategy to construct tumor progression models. Knock-in cells displayed allele-specific activation of signaling pathways and mutation-specific phenotypes different from those obtainable by ectopic oncogene expression. Profiling of a library of pharmacological agents on the mutated cells showed striking sensitivity or resistance phenotypes to pathway-targeted drugs, often matching those of tumor cells carrying equivalent cancer mutations. Thus, knock-in of single or multiple cancer alleles provides a pharmacogenomic platform for the rational design of targeted therapies.

TREATMENT WITH THE GLYCOGEN SYNTHASE KINASE-3β INHIBITOR, TDZD-8, AFFECTS TRANSIENT CEREBRAL ISCHEMIA/REPERFUSION INJURY IN THE RAT HIPPOCAMPUS

The serine/threonine glycogen synthase kinase 3&bgr; (GSK-3&bgr;) is abundant in the central nervous system, particularly in the hippocampus, and plays a pivotal role in the pathophysiology of a number of diseases, including neurodegeneration. This study was designed to investigate the effects of GSK-3&bgr; inhibition against I/R injury in the rat hippocampus. Transient cerebral ischemia (30 min) followed by 1 h of reperfusion significantly increased generation of reactive oxygen species and modulated superoxide dismutase activity; 24 h of reperfusion evoked apoptosis (determined as mitochondrial cytochrome c release and Bcl-2 and caspase-9 expression), resulted in high plasma levels of TNF-&agr; and increased expression of cyclooxygenase-2, inducible nitric oxide synthase, and intercellular adhesion molecule-1. The selective GSK-3&bgr; inhibitor, 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), was administered before and after ischemia or during reperfusion alone to assess its potential as prophylactic or therapeutic strategy. Prophylactic or therapeutic administration of TDZD-8 caused the phosphorylation (Ser9) and hence inactivation of GSK-3&bgr;. Infarct volume and levels of S100B protein, a marker of cerebral injury, were reduced by TDZD-8. This was associated with a significant reduction in markers of oxidative stress, apoptosis, and the inflammatory response resulting from cerebral I/R. These beneficial effects were associated with a reduction of I/R-induced activation of the mitogen-activated protein kinases JNK1/2 and p38 and nuclear factor-&kgr;B. The present study demonstrates that TDZD-8 protects the brain against I/R injury by inhibiting GSK-3&bgr; activity. Collectively, our data may contribute to focus the role of GSK-3&bgr; in cerebral I/R.

Celecoxib decreases expression of the adhesion molecules ICAM‐1 and VCAM‐1 in a colon cancer cell line (HT29)

We investigated the ability of celecoxib, a selective cyclooxygenase‐2 (COX‐2) inhibitor, to modulate expression of ICAM‐1 and VCAM‐1 in the colon cancer cell line HT29.

Molecular landscape of acquired resistance to targeted therapy combinations in BRAF mutant colorectal cancer

Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1 These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer. Cancer Res; 76(15); 4504-15. ©2016 AACR.

Substance P-induced cyclooxygenase-2 expression in human umbilical vein endothelial cells

Substance P (SP) is a neuropeptide involved in neurogenic inflammation and an agonist for NK1, NK2, and NK3 receptors. SP induces prostaglandin (PG) production in various cell types, and these eicosanoids are responsible for numerous inflammatory and vascular effects. Cyclooxygenase (COX) are needed to convert arachidonic acid to PGs. The study evaluated the effect of SP on COX expression in human umbilical vein endothelial cells (HUVEC). COX‐2 protein expression was upregulated by SP with a peak at 100 nM and at 20 h; in the same experimental conditions COX‐1 protein expression was unchanged. A correlation between COX‐2 expression and PGI2 and PGE2 release was detected. Dexamethasone (DEX) inhibited SP‐mediated COX‐2 expression. Mitogen‐activated protein kinases (MAPK) p38 and p42/44 were activated by SP, whereas SB202190 and PD98059, inhibitors of these kinases, blocked COX‐2 expression. 5,5‐dimethyl‐3‐(3‐fluorophenyl)‐4‐(4‐methylsulphonyl)phenyl‐2(5H)‐furanone (DFU), an experimental selective COX‐2 inhibitor, blocked SP‐induced PG release. By RT–PCR and Western blot analysis, we demonstrated that NK1 and NK2 but not NK3 receptors are present on HUVEC. Selective NK1 and NK2 agonists, namely [Sar9, Met(O2)11]SP and [β‐Ala8] NKA(4–10), upregulated COX‐2 protein expression and PG production, whereas senktide (Suc–Asp–Phe–MePhe–Gly–Leu–Met–NH2), a selective NK3 agonist, was ineffective in this respect. The NK1 selective antagonist L703,606 ((cis)‐2‐(diphenylmethyl)‐N‐((2‐iodophenyl)‐methyl)‐1‐azabicyclo(2.2.2)octan‐3‐amine) and the NK2 selective antagonist SR 48,968 ((S)‐N‐methyl‐N‐(4‐(4‐acetylamino‐4‐phenylpiperidino)‐2‐(3,4 dichlorophenyl)butyl) benzamide) competitively antagonised SP‐induced effects. The study shows HUVEC to possess functional NK1 and NK2 receptors, which mediate the ability of SP to induce expression of COX‐2 in HUVEC, thus showing a previously‐undetected effect of SP on endothelial cells.

Celecoxib modulates adhesion of HT29 colon cancer cells to vascular endothelial cells by inhibiting ICAM-1 and VCAM-1 expression

Cyclooxygenase‐2 (COX‐2) is highly expressed during inflammation and can promote the progression of colorectal cancer. Interactions between cancer cells and vascular endothelial cells are key events in this process. Recently, the selective COX‐2 inhibitor, celecoxib, was shown to inhibit expression of the adhesion molecules, ICAM‐1 and VCAM‐1, in the human colon cancer cell line HT29 and to inhibit adhesion of HT29 cells to FCS‐coated plastic wells. Here, we evaluated the effects of celecoxib on adhesion of HT29 cells to human umbilical vein endothelial cells (HUVEC), mediated by ICAM‐1 and VCAM‐1, to assess further the potential protective effects of celecoxib on cancer development.