DNA alterations in Cd133+ and Cd133- tumour cells enriched from intra-operative human colon tumour biopsies.

BACKGROUND: Tumour stem cells are considered important to promote disease progression, recurrence and treatment resistance following chemotherapy in colon cancer. However, genomic analyses of colorectal cancer have mainly been performed on integrated tumour tissue consisting of several different cell types in addition to differentiated tumour cells. The purpose of the present study was to compare genomic alterations in two cell fractions enriched of CD133+ and CD133-/EpCAM+ cells, respectively, obtained from fresh intraoperative human tumour biopsies. METHODS: The tumour biopsies were fractionated into CD133+ and CD133-/EpCAM+ cells by immunomagnetic separation, confirmed by immunocytochemistry and Q-PCR. DNA were extracted and used for array comparative genome hybridization (aCGH) after whole genome amplification. Frozen tumour tissue biopsies were used for DNA/RNA extraction and Q-PCR analyses to check for DNA alterations detected in the cell fractions. RESULTS: The number and size of DNA alterations were equally distributed across the cell fractions; however, large deletions were detected on chromosome 1, 7 and 19 in CD133-/EpCAM+ cells. Deletions were frequent in both cell fractions and a deletion on chromosome 19p was confirmed in 90% of the patients. CONCLUSION: Isolation of enriched cells derived from tumour tissue revealed mainly genomic deletions, which were not observed in tumour tissue DNA analyses. CD133+ cells were genetically heterogeneous among patients without any defined profile compared to CD133-/EpCAM+ cells.

Emerging drugs for cervical cancer.

INTRODUCTION: There is a shortage of therapeutical agents for invasive cervical cancer in late stages of development; however, a number of promising molecules are currently in early phases of development. AREAS COVERED: This review briefly discusses the current achievements in treating cervical cancer with an emphasis in emerging agents based on a literature search on pubmed and related sites for cervical cancer information. This is not a systematic review. EXPERT OPINION: In advanced disease, modest survival gains have been achieved with cisplatin doublets. Contrariwise, chemoradiation has increased survival rates in locally advanced disease, but there is still room for improvement. Anti-vascular endothelial growth factor and anti-epidermal growth factor receptor monoclonal antibodies are promising molecules that are at present in late-phase development, but a high number of miscellaneous agents are in early development. Strong experimental bases support that the 'Achilles' heel' of cervical cancer are the HPV-E6/E7 oncogenes. Unfortunately, agents aimed at targeting these cervical cancer-driven players are found in very early development; hence, major research efforts must be focused on developing technological strategies for their effective targeting using nucleic acid-based vehicles for safe and effective delivery to cancer cells as well as accelerating the search for small-molecule inhibitors of E6/E7 themselves or their interacting cellular proteins.

Repotrectinib (TPX-0005), effectively reduces growth of ALK driven neuroblastoma cells.

Neuroblastoma is the most commonly diagnosed extracranial tumor in the first year of life. Approximately 9% of neuroblastoma patients present germline or somatic aberrations in the gene encoding for anaplastic lymphoma kinase (ALK). This increases in high-risk neuroblastomas, which have a 14% frequency of ALK aberrations at the time of diagnosis and show increasing numbers at relapse. Abrogating ALK activity with kinase inhibitors is employed as clinical therapy in malignancies such as non-small cell lung cancer and has shown good results in pediatric inflammatory myofibroblastic tumors and anaplastic large cell lymphomas. A phase I clinical trial of the first generation ALK inhibitor, crizotinib, in neuroblastoma patients showed modest results and suggested that further investigation was needed. Continuous development of ALK inhibitors has resulted in the third generation inhibitor repotrectinib (TPX-0005), which targets the active kinase conformations of ALK, ROS1 and TRK receptors. In the present study we investigated the effects of repotrectinib in a neuroblastoma setting in vitro and in vivo. Neuroblastoma cell lines were treated with repotrectinib to investigate inhibition of ALK and to determine its effect on proliferation. PC12 cells transfected with different ALK mutant variants were used to study the efficacy of repotrectinib to block ALK activation/signaling. The in vivo effect of repotrectinib was also analyzed in a neuroblastoma xenograft model. Our results show that repotrectinib is capable of inhibiting signaling activity of a range of ALK mutant variants found in neuroblastoma patients and importantly it exhibits strong antitumor effects in a xenograft model of neuroblastoma.

Alectinib, an Anaplastic Lymphoma Kinase Inhibitor, Abolishes ALK Activity and Growth in ALK-Positive Neuroblastoma Cells.

Oncogenic receptor tyrosine kinases including anaplastic lymphoma kinase (ALK) are implicated in numerous solid and hematologic cancers. ALK mutations are reported in an estimated 9% of neuroblastoma and recent reports indicate that the percentage of ALK-positive cases increases in the relapsed patient population. Initial clinical trial results have shown that it is difficult to inhibit growth of ALK positive neuroblastoma with crizotinib, motivating investigation of next generation ALK inhibitors with higher affinity for ALK. Here, alectinib, a potent next generation ALK inhibitor with antitumor activity was investigated in ALK-driven neuroblastoma models. Employing neuroblastoma cell lines and mouse xenografts we show a clear and efficient inhibition of ALK activity by alectinib. Inhibition of ALK activity was observed in vitro employing a set of different constitutively active ALK variants in biochemical assays. The results suggest that alectinib is an effective inhibitor of ALK kinase activity in ALK addicted neuroblastoma and should be considered as a potential future therapeutic option for ALK-positive neuroblastoma patients alone or in combination with other treatments.

Phosphoproteome and gene expression profiling of ALK inhibition in neuroblastoma cell lines reveals conserved oncogenic pathways.

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that is a clinical target of major interest in cancer. Mutations and rearrangements in ALK trigger the activation of the encoded receptor and its downstream signaling pathways. ALK mutations have been identified in both familial and sporadic neuroblastoma cases as well as in 30 to 40% of relapses, which makes ALK a bona fide target in neuroblastoma therapy. Tyrosine kinase inhibitors (TKIs) that target ALK are currently in clinical use for the treatment of patients with ALK-positive non-small cell lung cancer. However, monotherapy with the ALK inhibitor crizotinib has been less encouraging in neuroblastoma patients with ALK alterations, raising the question of whether combinatorial therapy would be more effective. In this study, we established both phosphoproteomic and gene expression profiles of ALK activity in neuroblastoma cells exposed to first- and third-generation ALK TKIs, to identify the underlying molecular mechanisms and identify relevant biomarkers, signaling networks, and new therapeutic targets. This analysis has unveiled various important leads for novel combinatorial treatment strategies for patients with neuroblastoma and an increased understanding of ALK signaling involved in this disease.

FosB transcription factor regulates COX-2 expression in colorectal cancer cells without affecting PGE2 expression.

The expression levels of cyclooxygenase (COX)-2 and the prostaglandin E2 (PGE2) content have been associated with poor prognosis in patients with colorectal cancer (CRC). There is a strong correlation between COX-2 expression and PGE2 production in tissues from CRC patients, suggesting an important role for COX-2 on the regulation of PGE2 production. Previous studies by the present authors, where CRC patients were divided into high- or low-COX-2 expressing tumors, displayed important differences in the expression levels of several transcription factors involved in carcinogenesis. Among them, FBJ murine osteosarcoma viral oncogene homolog B (FosB), which is a member of the activator protein-1 complex, was the highest upregulated transcription factor in patients with high expression levels of COX-2. The present study aimed to investigate the role of FosB on the COX-2/PGE2 axis in CRC cells with high COX-2 expression levels. Interference RNA technology was used to knockdown FosB expression in HCA-7 cells, and 72 h later the messenger (m)RNA expression levels of COX-1 and COX-2, as well as the PGE2 content, were measured. The results indicated that FosB knockdown decreased the expression levels of COX-2 but did not affect the PGE2 content or the mRNA expression levels of COX-1. The present findings suggest an important role for FosB on the regulation of COX-2 expression, but no effect on the regulation of the PGE2 levels. In addition, the present results imply independent regulatory mechanisms for COX-2 expression and PGE2 content.

MEK inhibitor trametinib does not prevent the growth of anaplastic lymphoma kinase (ALK)-addicted neuroblastomas.

Activation of the RAS-RAF-MEK-ERK signaling pathway is implicated in driving the initiation and progression of multiple cancers. Several inhibitors targeting the RAS-MAPK pathway are clinically approved as single- or polyagent therapies for patients with specific types of cancer. One example is the MEK inhibitor trametinib, which is included as a rational polytherapy strategy for treating EML4-ALK-positive, EGFR-activated, or KRAS-mutant lung cancers and neuroblastomas that also contain activating mutations in the RAS-MAPK pathway. In addition, in neuroblastoma, a heterogeneous disease, relapse cases display an increased rate of mutations in ALK, NRAS, and NF1, leading to increased activation of RAS-MAPK signaling. Co-targeting ALK and the RAS-MAPK pathway is an attractive option, because monotherapies have not yet produced effective results in ALK-addicted neuroblastoma patients. We evaluated the response of neuroblastoma cell lines to MEK-ERK pathway inhibition by trametinib. In contrast to RAS-MAPK pathway-mutated neuroblastoma cell lines, ALK-addicted neuroblastoma cells treated with trametinib showed increased activation (inferred by phosphorylation) of the kinases AKT and ERK5. This feedback response was mediated by the mammalian target of rapamycin complex 2-associated protein SIN1, resulting in increased survival and proliferation that depended on AKT signaling. In xenografts in mice, trametinib inhibited the growth of EML4-ALK-positive non-small cell lung cancer and RAS-mutant neuroblastoma but not ALK-addicted neuroblastoma. Thus, our results advise against the seemingly rational option of using MEK inhibitors to treat ALK-addicted neuroblastoma.

Feasibility and antitumor efficacy in vivo, of simultaneously targeting glycolysis, glutaminolysis and fatty acid synthesis using lonidamine, 6-diazo-5-oxo-L-norleucine and orlistat in colon cancer.

The aim of the present study was to investigate in vivo the feasibility and efficacy of the combination of lonidamine (LND), 6-diazo-5-oxo-L-norleucine (DON) and orlistat to simultaneously target glycolysis, glutaminolysis and de novo synthesis of fatty acids, respectively. The doses of LND and DON used in humans were translated to mouse doses (77.7 mg/kg and 145.5 mg/kg, respectively) and orlistat was used at 240 mg/kg. Three schedules of LND, DON and orlistat at different doses were administered by intraperitoneal injection to BALB/c mice in a 21-day cycle (schedule 1: LND, 0.5 mg/day; DON, 0.25 mg/day 1, 5 and 9; orlistat, 240 mg/kg/day; schedule 2: LND, 0.1 mg/day; DON, 0.5 mg/day 1, 5 and 9; orlistat, 240 mg/kg/day; schedule 3: LND, 0.5 mg/day; DON, 0.08 mg/day 1, 5 and 9; orlistat, 360 mg/kg/day) to assess tolerability. To determine the antitumor efficacy, a syngeneic tumor model in BALB/c mice was created using colon cancer CT26.WT cells, and a xenogeneic tumor model was created in nude mice using the human colon cancer SW480 cell line. Mice were treated with schedule 1. Animals were weighed, clinically inspected during the experiment and the tumor volume was measured at day 21. The 3 schedules assessed in the tolerability experiments were well tolerated, as mice maintained their weight and no evident clinical signs of toxicity were observed. Combination treatment with schedule 1 significantly decreased tumor growth in each mouse model. No evident signs of toxicity were observed and mice maintained their weight during treatment. The triple metabolic blockade of the malignant phenotype appears feasible and promising for cancer therapy.

Antitumor effects of a drug combination targeting glycolysis, glutaminolysis and de novo synthesis of fatty acids.

There is a strong rationale for targeting the metabolic alterations of cancer cells. The most studied of these are the higher rates of glycolysis, glutaminolysis and de novo synthesis of fatty acids (FAs). Despite the availability of pharmacological inhibitors of these pathways, no preclinical studies targeting them simultaneously have been performed. In the present study it was determined whether three key enzymes for glycolysis, glutaminolysis and de novo synthesis of FAs, hexokinase-2, glutaminase and fatty acid synthase, respectively, were overexpressed as compared to primary fibroblasts. In addition, we showed that at clinically relevant concentrations lonidamine, 6-diazo-5-oxo-L-norleucine and orlistat, known inhibitors of the mentioned enzymes, exerted a cell viability inhibitory effect. Genetic downregulation of the three enzymes also reduced cell viability. The three drugs were highly synergistic when administered as a triple combination. Of note, the cytotoxicity of the triple combination was low in primary fibroblasts and was well tolerated when administered into healthy BALB/c mice. The results suggest the feasibility and potential clinical utility of the triple metabolic targeting which merits to be further studied by using either repositioned old drugs or newer, more selective inhibitors.

Reviving Lonidamine and 6-Diazo-5-oxo-L-norleucine to Be Used in Combination for Metabolic Cancer Therapy.

Abnormal metabolism is another cancer hallmark. The two most characterized altered metabolic pathways are high rates of glycolysis and glutaminolysis, which are natural targets for cancer therapy. Currently, a number of newer compounds to block glycolysis and glutaminolysis are being developed; nevertheless, lonidamine and 6-diazo-5-oxo-L-norleucine (DON) are two old drugs well characterized as inhibitors of glycolysis and glutaminolysis, respectively, whose clinical development was abandoned years ago when the importance of cancer metabolism was not fully appreciated and clinical trial methodology was less developed. In this review, a PubMed search using the words lonidamine and 6-diazo-5-oxo-L-norleucine (DON) was undertaken to analyse existing information on the preclinical and clinical studies of these drugs for cancer treatment. Data show that they exhibit antitumor effects; besides there is also the suggestion that they are synergistic. We conclude that lonidamine and DON are safe and potentially effective drugs that need to be reevaluated in combination as metabolic therapy of cancer.