Modulation of the Ribonucleotide Reductase-Antimetabolite Drug Interaction in Cancer Cell Lines

RRM1 is a determinant of gemcitabine efficacy in cancer patients. However, the precision of predicting tumor response based on RRM1 levels is not optimal. We used gene-specific overexpression and RNA interference to assess RRM1's impact on different classes of cytotoxic agents, on drug-drug interactions, and the modulating impact of other molecular and cellular parameters. RRM1 was the dominant determinant of gemcitabine efficacy in various cancer cell lines. RRM1 also impacted the efficacy of other antimetabolite agents. It did not disrupt the interaction of two cytotoxic agents when combined. Cell lines with truncation, deletion, and null status of p53 were resistant to gemcitabine without apparent relationship to RRM1 levels. Pemetrexed and carboplatin sensitivity did not appear to be related to p53 mutation status. The impact of p53 mutations in patients treated with gemcitabine should be studied in prospective clinical trials to develop a model with improved precision of predicting drug efficacy

Ubiquitin-Specific Peptidase 10 (USP10) Deubiquitinates and Stabilizes MutS Homolog 2 (MSH2) to Regulate Cellular Sensitivity to DNA Damage

MSH2 is a key DNA mismatch repair protein, which plays an important role in genomic stability. In addition to its DNA repair function, MSH2 serves as a sensor for DNA base analogs-provoked DNA replication errors and binds to various DNA damage-induced adducts to trigger cell cycle arrest or apoptosis. Loss or depletion of MSH2 from cells renders resistance to certain DNA-damaging agents. Therefore, the level of MSH2 determines DNA damage response. Previous studies showed that the level of MSH2 protein is modulated by the ubiquitin-proteasome pathway, and histone deacetylase 6 (HDAC6) serves as an ubiquitin E3 ligase. However, the deubiquitinating enzymes, which regulate MSH2 remain unknown. Here we report that ubiquitin-specific peptidase 10 (USP10) interacts with and stabilizes MSH2. USP10 deubiquitinates MSH2 in vitro and in vivo. Moreover, the protein level of MSH2 is positively correlated with the USP10 protein level in a panel of lung cancer cell lines. Knockdown of USP10 in lung cancer cells exhibits increased cell survival and decreased apoptosis upon the treatment of DNA-methylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and antimetabolite 6-thioguanine (6-TG). The above phenotypes can be rescued by ectopic expression of MSH2. In addition, knockdown of MSH2 decreases the cellular mismatch repair activity. Overall, our results suggest a novel USP10-MSH2 pathway regulating DNA damage response and DNA mismatch repair

Clinical Efficacy and Predictive Molecular Markers of Neoadjuvant Gemcitabine and Pemetrexed in Resectable Non-small Cell Lung Cancer

BackgroundA trial of neoadjuvant gemcitabine and pemetrexed (GP) chemotherapy in patients with resectable non-small cell lung cancer was conducted. The goal was to achieve a disease response rate of 50% and to determine if the expression levels of genes associated with GP metabolism are predictive of response.MethodsPatients had staging with a computed tomography scan, whole body F-18 fluorodeoxyglucose positron emission tomography, and mediastinoscopy. Four biweekly cycles of GP were given. Patients were restaged, and those with resectable stage IB-III disease had thoracotomy. Fresh frozen tumor specimens were collected before and after chemotherapy and the mRNA levels of 14 target genes determined by real-time reverse transcription polymerase chain reaction.ResultsFifty-two patients started therapy. The radiographic disease response rate was 35% (95% confidence interval 21.7-49.6%), and the progression rate was 6%. Forty-six patients had a thoracotomy. The complete tumor resection rate was 77% (40/52). There were no perioperative deaths or deaths related to chemotherapy. Tumor response to chemotherapy was inversely correlated with the level of expression of RRM1 (p < 0.001; regulatory subunit of ribonucleotide reductase) and TS (p = 0.006; thymidylate synthase); i.e., the reduction in tumor size was greater in those with low levels of expression.ConclusionsNeoadjuvant GP is well tolerated and produces an objective response rate of 35%. Tumoral RRM1 and TS mRNA levels are predictive of disease response and should be considered as parameters for treatment selection in future trials with this regimen

Ribonucleotide reductase is not limiting for mitochondrial DNA copy number in mice

Peer reviewe

Cancer Cachexia: Traditional Therapies and Novel Molecular Mechanism-Based Approaches to Treatment

The complex syndrome of cancer cachexia (CC) that occurs in 50% to 80% cancer patients has been identified as an independent predictor of shorter survival and increased risk of treatment failure and toxicity, contributing to the mortality and morbidity in this population. CC is a pathological state including a symptom cluster of loss of muscle (skeletal and visceral) and fat, manifested in the cardinal feature of emaciation, weakness affecting functional status, impaired immune system, and metabolic dysfunction. The most prominent feature of CC is its non-responsiveness to traditional treatment approaches; randomized clinical trials with appetite stimulants, 5-HT3 antagonists, nutrient supplementation, and Cox-2 inhibitors all have failed to demonstrate success in reversing the metabolic abnormalities seen in CC. Interventions based on a clear understanding of the mechanism of CC, using validated markers relevant to the underlying metabolic abnormalities implicated in CC are much needed. Although the etiopathogenesis of CC is poorly understood, studies have proposed that NFkB is upregulated in CC, modulating immune and inflammatory responses induce the cellular breakdown of muscle, resulting in sarcopenia. Several recent laboratory studies have shown that n-3 fatty acid may attenuate protein degradation, potentially by preventing NFkB accumulation in the nucleus, preventing the degradation of muscle proteins. However, clinical trials to date have produced mixed results potentially attributed to timing of interventions (end stage) and utilizing outcome markers such as weight which is confounded by hydration, cytotoxic therapies, and serum cytokines. We propose that selective targeting of proteasome activity with a standardized dose of omega-3-acid ethyl esters, administered to cancer patients diagnosed with early stage CC, in addition to a standard intervention with nutritionally adequate diet and appetite stimulants, will alter metabolic abnormalities by downregulating NFkB, preventing the breakdown of myofibrillar proteins and resulting in increasing serum protein markers, lean body mass, and functional status

Spontaneous changes in intermediate filament protein expression patterns in lung cancer cell lines

The usefulness of cell lines in the study and prediction of the clinical behaviour of lung cancer is still a matter of debate. However, lung tumour cell cultures have been of value in investigations concerning molecular and cell biological aspects of these neoplasms. Especially in the examination of characteristics specific for the main types of differentiation (squamous cell carcinoma, adenocarcinoma, small cell carcinoma), in vitro studies have been most important. Twenty eight lung cancer cell lines were cultured for up to four years, and were examined at regular intervals for their intermediate filament protein (IFP) expression patterns using a panel of cytokeratin (CK) and neurofilament (NF) antibodies. These studies showed that the classic type of small cell lung cancer (SCLC) cell lines contain CKs 8, 18, and occasionally CK 19, while the variant-type SCLC cell lines generally express no CKs but can contain NFs. Non-SCLC cell lines, such as squamous cell carcinoma and adenocarcinoma cell lines, contain CKs 7 (in most cases), 8, 18 and 19. In one variant SCLC cell line and in one adenocarcinoma cell line CKs 4, 10 and 13, characteristic of squamous cell differentiation, were found. Although most cell lines have remained stable with respect to growth characteristics and IFP expression patterns, five lung cancer cultures exhibited a transition from one cell type to another, paralleled by changes in IFP expression. Progressions from classic to variant SCLC cell lines have been observed, next to conversions from variant SCLC to cell lines re-expressing cytokeratins. In some cases this resulted in a coexpression of CKs and NFs within a cell line and even within individual tumour cells. These results strongly support the earlier finding that CK expression in SCLC cell lines is a reliable marker for the classic type of differentiation, while the absence of CKs and the presence of NFs marks the variant type of differentiation. Our results are discussed in view of previous histological findings

Identification of actionable targets for breast cancer intervention using a diversity outbred mouse model

HER2-targeted therapy has improved breast cancer survival, but treatment resistance and disease prevention remain major challenges. Genes that enable HER2/Neu oncogenesis are the next intervention targets. A bioinformatics discovery platform of HER2/Neu-expressing Diversity Outbred (DO) F1 Mice was established to identify cancer-enabling genes. Quantitative Trait Loci (QTL) associated with onset ages and growth rates of spontaneous mammary tumors were sought. Twenty-six genes in 3 QTL contain sequence variations unique to the genetic backgrounds that are linked to aggressive tumors and 21 genes are associated with human breast cancer survival. Concurrent identification of TSC22D3, a transcription factor, and its target gene LILRB4, a myeloid cell checkpoint receptor, suggests an immune axis for regulation, or intervention, of disease. We also investigated TIEG1 gene that impedes tumor immunity but suppresses tumor growth. Although not an actionable target, TIEG1 study revealed genetic regulation of tumor progression, forming the basis of the genetics-based discovery platform

Extracellular Signal-regulated Kinase (ERK) Phosphorylates Histone Deacetylase 6 (HDAC6) at Serine 1035 to Stimulate Cell Migration

Histone deacetylase 6 (HDAC6) is well known for its ability to promote cell migration through deacetylation of its cytoplasmic substrates such as α-tubulin. However, how HDAC6 itself is regulated to control cell motility remains elusive. Previous studies have shown that one third of extracellular signal-regulated kinase (ERK) is associated with the microtubule cytoskeleton in cells. Yet, no connection between HDAC6 and ERK has been discovered. Here, for the first time, we reveal that ERK binds to and phosphorylates HDAC6 to promote cell migration via deacetylation of α-tubulin. We have identified two novel ERK-mediated phosphorylation sites: threonine 1031 and serine 1035 in HDAC6. Both sites were phosphorylated by ERK1 in vitro, whereas Ser-1035 was phosphorylated in response to the activation of EGFR-Ras-Raf-MEK-ERK signaling pathway in vivo. HDAC6-null mouse embryonic fibroblasts rescued by the nonphosphorylation mimicking mutant displayed significantly reduced cell migration compared with those rescued by the wild type. Consistently, the nonphosphorylation mimicking mutant exerted lower tubulin deacetylase activity in vivo compared with the wild type. These data indicate that ERK/HDAC6-mediated cell motility is through deacetylation of α-tubulin. Overall, our results suggest that HDAC6-mediated cell migration could be governed by EGFR-Ras-Raf-MEK-ERK signaling

Multi-Level Targeting of the Phosphatidylinositol-3-Kinase Pathway in Non-Small Cell Lung Cancer Cells

Introduction: We assessed expression of p85 and p110a PI3K subunits in non-small cell lung cancer (NSCLC) specimens and the association with mTOR expression, and studied effects of targeting the PI3K/AKT/mTOR pathway in NSCLC cell lines. Methods: Using Automated Quantitative Analysis we quantified expression of PI3K subunits in two cohorts of 190 and 168 NSCLC specimens and correlated it with mTOR expression. We studied effects of two PI3K inhibitors, LY294002 and NVP-BKM120, alone and in combination with rapamycin in 6 NSCLC cell lines. We assessed activity of a dual PI3K/mTOR inhibitor