A novel inhibitor of focal adhesion signaling induces caspase-independent cell death in diffuse large B-cell lymphoma.

Focal adhesion (FA) proteins have been associated with transformation, migration, metastasis, and poor outcome in many neoplasias. We previously showed that these proteins were inhibited by E7123, a new celecoxib derivative with antitumor activity, in acute myeloid leukemia. However, little is known about FAs in diffuse large B cell lymphoma (DLBCL). This paper aimed to determine whether E7123 was effective against DLBCL and whether FAs were involved in its action. We evaluated the cytotoxicity and mechanism of action of E7123 and celecoxib in DLBCL cell lines. We also assessed the E7123 in vivo activity in a DLBCL xenograft model and studied FA signaling in primary DLBCL patient samples. We found that E7123 showed higher antitumor effect than celecoxib against DLBCL cells. Its mechanism of action involved deregulation of FA, AKT, and Mcl-1 proteins, a pathway that is activated in some patient samples, apoptosis-inducing factor release and induction of caspase-independent cell death. Moreover, E7123 showed suppression of in vivo tumor growth. These findings indicate that E7123 is effective against DLBCL in vitro and in vivo, with a mechanism of action that differs from that of most current therapies for this malignancy. Our results support further preclinical evaluation of E7123.

Site-dependent E-cadherin cleavage and nuclear translocation in a metastatic colorectal cancer model.

Metastases are frequently found during colorectal cancer diagnoses and are the main determinants of clinical outcome. The lack of reliable models of metastases has precluded their mechanistic understanding and our capacity to improve outcome. We studied the effect of E-cadherin and Snail1 expression on metastagenesis in a colorectal cancer model. We microinjected SW480-ADH human colorectal cancer cells, transfected with an empty vector (Mock) or overexpressing Snail1 (Snail1(OE)) or E-cadherin (E-cadherin(OE)), in the ceca of nude mice (eight per group) and analyzed tumor growth, dissemination, and Snail1, E-cadherin, ß-catenin, and Presenilin1 (PS1) expression in local tumors and/or metastatic foci. Snail1(OE) cells disseminated only to lymph nodes, whereas Mock or E-cadherin(OE) cells spread to lymph nodes and peritoneums. Peritoneal tumor foci developed by E-cadherin(OE) cells presented an increase in E-cadherin proteolysis and nuclear translocation, and enhanced expression of proteolytically active PS1, which was linked to increased tumor growth and shortened mouse survival. Interestingly, local and lymph node tumors in mice bearing E-cadherin(OE) cells overexpressed E-cadherin, but they did not show E-cadherin proteolysis or nuclear translocation. Remarkably, E-cadherin nuclear translocation and enhanced expression of active PS1 were found in a patient with colorectal signet-ring cell carcinoma. In conclusion, we have established a colorectal cancer metastasis model in which E-cadherin proteolyis and nuclear translocation associates with aggressive foci growth only in the peritoneal microenvironment.

A celecoxib derivative inhibits focal adhesion signaling and induces caspase-8-dependent apoptosis in human acute myeloid leukemia cells.

Most acute myeloid leukemias (AMLs), including those with c-Kit or FLT3 mutations, show enhanced anchorage independent growth associated with constitutive activation of focal adhesion proteins. Moreover, these alterations increase cell survival, inhibit apoptosis and are associated with poor prognosis and resistance to chemotherapy. Therefore, the induction of apoptosis by selective inhibition of focal adhesion signaling may represent a novel anti-AML therapy. Here, we have evaluated the antitumor effect and the mechanism of action of celecoxib and E7123, a non-Cox-2 inhibitor derivative, in a panel of human AML cell lines and bone marrow mononuclear cells from AML patients. Both compounds induce cell death by inhibiting focal adhesion signaling through p130Cas, FAK and c-Src, leading to caspase-8 dependent apoptosis. This mechanism of action differs from that of classical cytotoxic drugs or of other targeted therapies, and is amenable to rational drug development. Therefore, both drugs could be developed as AML therapeutics; nevertheless, E7123 shows more activity than celecoxib against AML cells, and may not present its Cox-2 dependent cardiovascular toxicity. Finally, our results support the evaluation of celecoxib in AML patients, and the preclinical evaluation of E7123, before its possible clinical testing.

Ku70 predicts response and primary tumor recurrence after therapy in locally advanced head and neck cancer.

5-Fluorouracil and cisplatin-based induction chemotherapy (IC) is commonly used to treat locally advanced head and neck squamous cell carcinoma (HNSCC). The role of nonhomologous end joining (NHEJ) genes (Ku70, Ku80 and DNA-PKcs) in double-strand break (DSB) repair, genomic instability and apoptosis suggest a possible impact on tumor response to radiotherapy, 5-fluorouracil or cisplatin, as these agents are direct or indirect inductors of DSBs. We evaluated the relationship between Ku80, Ku70 or DNA PKcs mRNA expression in pretreatment tumor biopsies, and tumor response to IC or local recurrence, in 50 patients with HNSCC. Additionally, in an independent cohort of 75 patients with HNSCC, we evaluated the relationship between tumor Ku70 protein expression and the same clinical outcomes or patient survival. Tumors in the responder group had significantly higher mRNA levels for Ku70, Ku80 and DNA-PKcs than those in the nonresponder group. Ku70 mRNA was the marker most significantly associated with response to IC. Moreover, high tumor Ku70 mRNA expression was associated with significantly longer local recurrence-free survival (LRFS). Ku70 protein expression was also significantly related to response, and patients with higher percentage of tumor cells expressing Ku70 had longer LRFS. In addition, the percentage of Ku70 positive cells, tumor localization and node involvement were significantly associated with overall survival of patient. Therefore, Ku70 expression is a candidate predictive marker that could distinguish patients who are likely to benefit from chemoradiotherapy or radiotherapy after the induction chemotherapy treatment, suggesting a contribution of the NHEJ system in HNSCC clinical outcome.

Novel triiodophenol derivatives induce caspase-independent mitochondrial cell death in leukemia cells inhibited by Myc.

2,4,6-Triiodophenol (Bobel-24, AM-24) was originally described as a nonsteroid antiinflammatory molecule. We have synthesized three derivatives of Bobel-24 (Bobel-4, Bobel-16, and Bobel-30) and tested their activities as putative antileukemic agents. We have found that Bobel-24 and Bobel-16 were dual inhibitors of cyclooxygenase and 5-lipoxygenase, whereas Bobel-4 and Bobel-30 were selective against 5-lipoxygenase. We have tested the antiproliferative activity of these compounds on a panel of cell lines derived from myeloid and lymphoid leukemias (K562, Raji, HL-60, and Molt4). The cytotoxic IC(50) in these cell lines ranged between 14 and 50 micromol/L, but it was higher for nontransformed cells such as 32D, NIH3T3, or human leukocytes. All compounds showed cytotoxic activity on all tested cell lines, accompanied by DNA synthesis inhibition and arrest in the G(0)/G(1) phase. Bobel-16, Bobel-4, and Bobel-24 induced a caspase-independent cell death in K562 and Raji cells, accompanied by chromatin condensation, cytochrome c release, and dissipation of mitochondrial membrane potential in a concentration-dependent manner and production of reactive oxygen species. As the proto-oncogene MYC is involved in mitochondrial biogenesis and survival of leukemia cells, we tested its effect on bobel activity. Bobel-24 induced down-regulation of MYC in K562 and, consistently, ectopic expression of MYC results in partial protection towards the cytotoxic effect of Bobel-24. In conclusion, Bobel derivatives induce a caspase- and Bcl-2-independent cell death in which mitochondrial permeabilization and MYC down-regulation are involved. Bobels may serve as prototypes for the development of new agents for the therapy of leukemia.

CKMT1 and NCOA1 expression as a predictor of clinical outcome in patients with advanced-stage head and neck squamous cell carcinoma.

BACKGROUND: We studied the association between the expression of a subset of previously identified genes and clinical outcome in patients with head and neck cancer. METHODS: We analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) the expression of 89 genes in tumor biopsies from stage III to IVa/b chemotherapy treated patients (n = 46). Two additional cohorts analyzed by RNAseq (The Cancer Genome Atlas [TCGA] project; n = 371) or immunohistochemistry (IHC; n = 73) were used to validate results. RESULTS: Thirty genes were associated with local-recurrence or progression-free survival. The best multi-gene decision-tree model to predict local recurrence included nuclear receptor coactivator 1 (NCOA1) and serum-amyloid A2 (SAA2) expression, whereas the best model to predict disease recurrence included creatine kinase mitochondrial 1 (CKMT1) and metal-regulatory transcription factor 1 (MTF1). Both models were associated with cancer-specific survival. Results were confirmed analyzing the RNAseq data included in the TCGA project. CKMT1 and NCOA1 were identified as independent risk factors for survival in an independent cohort analyzed by immunohistochemistry. CONCLUSION: CKMT1 and NCOA1 expression has prognostic significance in advanced-stage head and neck carcinoma. © 2015 Wiley Periodicals, Inc. Head Neck 38: E1392-E1403, 2016.

Expression of IL-1α correlates with distant metastasis in patients with head and neck squamous cell carcinoma.

The presence of IL-1 in human cancers is associated with aggressive tumor biology but its prognostic value is unknown. We studied whether IL-1α expression is a prognostic marker of distant metastasis in patients with head and neck squamous cell carcinoma (HNSCC). IL-1α mRNA and protein levels were determined in tumor samples and cancer cell lines using RT-PCR and ELISA. The effects of constitutive IL-1α expression by tumor lines were characterized. IL-1α mRNA and protein secretion were higher in tumor samples from patients who later developed distant metastasis than in patients who did not. By using distant metastasis as a dependent variable, patients were classified into two categories of IL-1α transcript-levels. The high-IL-1α group had a significantly lower five-year distant metastasis-free survival than the low-IL-1α group [70.0% (CI 95%: 55.9-84.1%) vs 94.7% (CI 95%:90.2-99.2%)]. When IL-1α transcript-levels were combined with clinical factors related to tumor metastasis, the predictive power of the model increased significantly. Additionally, transcript levels of IL-1α correlated significantly with those of the IL-1 family genes and genes related to the metastatic process. IL-1 treatment of microvascular endothelial cells increased adhesion of HNSCC cells but no differences were found based on constitutive IL-1α expression by tumor cells. Nevertheless, IL-1α produced by tumor cells effectively increased their transmigration across the endothelium. We found a significant relationship between IL-1α expression and development of distant metastasis in HNSCC patients. IL-1α expression could help to define a subset of patients at high risk of distant metastasis who could benefit from adjuvant treatment.

Bobel-24 and derivatives induce caspase-independent death in pancreatic cancer regardless of apoptotic resistance.

The poor prognosis of pancreatic cancer and poor sensitivity to current therapeutics, associated with resistance to apoptosis, urge the search for new drugs. We previously described the induction of caspase-independent mithochondrial death in leukemia cells by Bobel-24 (AM-24) and derivatives. Here, we explored whether these compounds induce a similar cytotoxicity in human pancreatic carcinoma cell lines (NP18, NP9, NP31, and NP29). Bobel-24 or Bobel-16 induced cytotoxicity and DNA synthesis inhibition in all cell lines and apoptosis in all lines, except for NP9. Caspase and/or poly(ADP-ribose) polymerase-1 (PARP-1) activity inhibition experiments showed that cytotoxicity was mainly induced through apoptosis in NP18 and through a caspase-independent process in NP9. Moreover, in NP29 or NP31 cell lines, both caspase-dependent and caspase-independent cell death mechanisms coexisted. Cell death was associated with reactive oxygen species (ROS) production, mitochondrial depolarization, cytochrome c and apoptosis-inducing factor (AIF) release, AIF nuclear translocation, and lysosomal cathepsin release. Inhibition of ROS production, mitochondrial pore permeability, PARP-1, or phospholipase A2 partially prevented cell death. Moreover, cathepsin B inhibition or down-regulation by small interfering RNA partially blocked cell death. In conclusion, Bobel-24 and derivatives trigger caspase-independent lysosomal and mitochondrial death in all tested human pancreatic cancer lines, irrespective of their degree of apoptotic sensitivity, becoming the only active cytotoxic mechanism in the apoptosis-resistant NP9 line. This mechanism may overcome the resistance to apoptosis observed in pancreatic carcinoma when treated with current genotoxic drugs.

Celecoxib induces anoikis in human colon carcinoma cells associated with the deregulation of focal adhesions and nuclear translocation of p130Cas.

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, is effective as chemopreventive against colon cancer and it is the only nonsteoroidal antiinflammatory drug approved by the FDA for adjuvant therapy in patients with familial adenomatous polyposis. It is also being evaluated, within Phase II and III clinical trials, in combination with standard chemotherapy to treat sporadic colorectal cancer. Nevertheless, its antitumor mechanism of action is still not fully understood. In this study, we have evaluated the in vitro growth inhibitory effect of celecoxib in colon carcinoma cells and analyzed its mechanism of action. We report that the deregulation of the focal adhesion assembly protein Crk-associated substrate 130 kDa (p130Cas) by celecoxib plays a relevant role in the cytotoxic effect of this drug. Thus, celecoxib induces the proteolysis of p130Cas and the nuclear translocation of the 31 kDa generated fragment leading to apoptosis. Furthermore, overexpression of wild-type p130Cas reverts, in part, the growth inhibitory effect of celecoxib. In contrast, FAK and AKT do not appear to be involved in this activity. Our data suggest, for the first time, that the antitumor mechanism of action of celecoxib includes the induction of anoikis, an effect that is not related to COX-2 inhibition. Besides providing new insights into the antitumor effect of celecoxib, this novel mechanism of action holds potential relevance in drug development. Indeed, our results open the possibility to develop new celecoxib derivatives that induce anoikis without COX-2 inhibition so as to avoid the cardiovascular toxicity recently described for the COX-2 inhibitors.

K-ras Asp12 mutant neither interacts with Raf, nor signals through Erk and is less tumorigenic than K-ras Val12.

Different mutant amino acids in the Ras proteins lead to distinct transforming capacities and different aggressiveness in human tumors. K-Ras Asp12 (K12D) is more prevalent in benign than in malignant human colorectal tumors, whereas K-Ras Val12 (K12V) associates with more advanced and metastatic carcinomas, higher recurrence and decreased survival. Here, we tested, in a nude mouse xenograft model, whether different human K-Ras oncogenes mutated at codon 12 to Val, Asp or Cys would confer NIH3T3 fibroblasts distinct oncogenic phenotypes. We studied tumor histology and growth, apoptotic and mitotic rates, activation of signal transduction pathways downstream of Ras and regulation of the cell cycle and apoptotic proteins in tumors derived from the implanted transformants. We found that the K12V oncogene induces a more aggressive tumorigenic phenotype than the K12D oncogene, whereas K12C does not induce tumors in this model. Thus, K12V mutant tumors proliferate about seven times faster, and have higher cellularity and mitotic rates than the K12D mutant tumors. A molecular analysis of the induced tumors shows that the K12V mutant protein interacts with Raf-1 and transduces signals mainly through the Erk pathway. Unexpectedly, in tumors induced by the K12D oncogene, the K-Ras mutant protein does not interact with Raf-1 nor activates the Erk canonical pathway. Instead, it transduces signals through the PI3K/Akt, JNK, p38 and FAK pathways. Finally, the higher growth rate of the K12V tumors associates with enhanced Rb phosphorylation, and PCNA and cyclin B upregulation, consistent with faster G1/S and G2/M transitions, without alteration of apoptotic regulation.

G1 cyclin/cyclin-dependent kinase-coordinated phosphorylation of endogenous pocket proteins differentially regulates their interactions with E2F4 and E2F1 and gene expression.

Mitogenic stimulation leads to activation of G(1) cyclin-dependent kinases (CDKs), which phosphorylate pocket proteins and trigger progression through the G(0)/G(1) and G(1)/S transitions of the cell cycle. However, the individual role of G(1) cyclin-CDK complexes in the coordinated regulation of pocket proteins and their interaction with E2F family members is not fully understood. Here we report that individually or in concert cyclin D1-CDK and cyclin E-CDK complexes induce distinct and coordinated phosphorylation of endogenous pocket proteins, which also has distinct consequences in the regulation of pocket protein interactions with E2F4 and the expression of p107 and E2F1, both E2F-regulated genes. The up-regulation of these two proteins and the release of p130 and pRB from E2F4 complexes allows formation of E2F1 complexes not only with pRB but also with p130 and p107 as well as the formation of p107-E2F4 complexes. The formation of these complexes occurs in the presence of active cyclin D1-CDK and cyclin E-CDK complexes, indicating that whereas phosphorylation plays a role in the abrogation of certain pocket protein/E2F interactions, these same activities induce the formation of other complexes in the context of a cell expressing endogenous levels of pocket and E2F proteins. Of note, phosphorylated p130 "form 3," which does not interact with E2F4, readily interacts with E2F1. Our data also demonstrate that ectopic overexpression of either cyclin is sufficient to induce mitogen-independent growth in human T98G and Rat-1 cells, although the effects of cyclin D1 require downstream activation of cyclin E-CDK2 activity. Interestingly, in T98G cells, cyclin D1 induces cell cycle progression more potently than cyclin E. This suggests that cyclin D1 activates pathways independently of cyclin E that ensure timely progression through the cell cycle.

Short amino acid stretches can mediate amyloid formation in globular proteins: the Src homology 3 (SH3) case.

Protein misfolding and deposition underlie an increasing number of debilitating human disorders. We have shown that model proteins unrelated to disease, such as the Src homology 3 (SH3) domain of the p58alpha subunit of bovine phosphatidyl-inositol-3'-kinase (PI3-SH3), can be converted in vitro into assemblies with structural and cytotoxic properties similar to those of pathological aggregates. By contrast, homologous proteins, such as alpha-spectrin-SH3, lack the capability of forming amyloid fibrils at a measurable rate under any of the conditions we have so far examined. However, transplanting a small sequence stretch (6 aa) from PI3-SH3 to alpha-spectrin-SH3, comprising residues of the diverging turn and adjacent RT loop, creates an amyloidogenic protein closely similar in its behavior to the original PI3-SH3. Analysis of specific PI3-SH3 mutants further confirms the involvement of this region in conferring amyloidogenic properties to this domain. Moreover, the inclusion in this stretch of two consensus residues favored in SH3 sequences substantially inhibits aggregation. These findings show that short specific amino acid stretches can act as mediators or facilitators in the incorporation of globular proteins into amyloid structures, and they support the suggestion that natural protein sequences have evolved in part to code for structural characteristics other than those included in the native fold, such as avoidance of aggregation.