Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib

Non-small cell lung cancers (NSCLCs) with activating mutations in the kinase domain of the epidermal growth factor receptor (EGFR) demonstrate dramatic, but transient, responses to the reversible tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva). Some recurrent tumors have a common secondary mutation in the EGFR kinase domain, T790M, conferring drug resistance, but in other cases the mechanism underlying acquired resistance is unknown. In studying multiple sites of recurrent NSCLCs, we detected T790M in only a small percentage of tumor cells. To identify additional mechanisms of acquired resistance to gefitinib, we used NSCLC cells harboring an activating EGFR mutation to generate multiple resistant clones in vitro. These drug-resistant cells demonstrate continued dependence on EGFR and ERBB2 signaling for their viability and have not acquired secondary EGFR mutations. However, they display increased internalization of ligand-activated EGFR, consistent with altered receptor trafficking. Although gefitinib-resistant clones are cross-resistant to related anilinoquinazolines, they demonstrate sensitivity to a class of irreversible inhibitors of EGFR. These inhibitors also show effective inhibition of signaling by T790M-mutant EGFR and killing of NSCLC cells with the T790M mutation. Both mechanisms of gefitinib resistance are therefore circumvented by irreversible tyrosine kinase inhibitors. Our findings suggest that one of these, HKI-272, may prove highly effective in the treatment of EGFR-mutant NSCLCs, including tumors that have become resistant to gefitinib or erlotinib

Knocking down gene function with an RNA aptamer expressed as part of an intron

We developed a powerful expression system to produce aptamers and other types of functional RNA in yeast to examine their effects. Utilizing the intron homing process, the aptamer-coding sequences were integrated into hundreds of rRNA genes, and the aptamers were transcribed at high levels by RNA polymerase I without any additional promoter being introduced into the cell. We used this system to express an aptamer against the heat shock factor 1 (HSF1), a conserved transcription factor responsible for mobilizing specific genomic expression programs in response to stressful conditions such as elevated temperature. We observed a temperature sensitive growth retardation phenotype and specific decrease of heat shock gene expression. As HSF1 enables and promotes malignant growth and metastasis in mammals, and this aptamer binds yeast HSF1 and its mammalian ortholog with equal affinity, the results presented here attest to the potential of this aptamer as a specific and effective inhibitor of HSF1 activity

Single-step doxorubicin-selected cancer cells overexpress the ABCG2 drug transporter through epigenetic changes

Understanding the mechanisms of multidrug resistance (MDR) could improve clinical drug efficacy. Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood. We report that a single-step selection with low doses of anti-cancer agents, similar to concentrations reported in vivo, induces MDR that is mediated exclusively by ABCG2. We selected breast, ovarian and colon cancer cells (MCF-7, IGROV-1 and S-1) after exposure to 14 or 21 nM doxorubicin for only 10 days. We found that these cells overexpress ABCG2 at the mRNA and protein levels. RNA interference analysis confirmed that ABCG2 confers drug resistance. Furthermore, ABCG2 upregulation was facilitated by histone hyperacetylation due to weaker histone deacetylase 1-promoter association, indicating that these epigenetic changes elicit changes in ABCG2 gene expression. These studies indicate that the MDR phenotype arises following low-dose, single-step exposure to doxorubicin, and further suggest that ABCG2 may mediate early stages of MDR development. This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines

Synchronization of Circadian Per2 Rhythms and HSF1-BMAL1:CLOCK Interaction in Mouse Fibroblasts after Short-Term Heat Shock Pulse

Circadian rhythms are the general physiological processes of adaptation to daily environmental changes, such as the temperature cycle. A change in temperature is a resetting cue for mammalian circadian oscillators, which are possibly regulated by the heat shock (HS) pathway. The HS response (HSR) is a universal process that provides protection against stressful conditions, which promote protein-denaturation. Heat shock factor 1 (HSF1) is essential for HSR. In the study presented here, we investigated whether a short-term HS pulse can reset circadian rhythms. Circadian Per2 rhythm and HSF1-mediated gene expression were monitored by a real-time bioluminescence assay for mPer2 promoter-driven luciferase and HS element (HSE; HSF1-binding site)-driven luciferase activity, respectively. By an optimal duration HS pulse (43°C for approximately 30 minutes), circadian Per2 rhythm was observed in the whole mouse fibroblast culture, probably indicating the synchronization of the phases of each cell. This rhythm was preceded by an acute elevation in mPer2 and HSF1-mediated gene expression. Mutations in the two predicted HSE sites adjacent (one of them proximally) to the E-box in the mPer2 promoter dramatically abolished circadian mPer2 rhythm. Circadian Per2 gene/protein expression was not observed in HSF1-deficient cells. These findings demonstrate that HSF1 is essential to the synchronization of circadian rhythms by the HS pulse. Importantly, the interaction between HSF1 and BMAL1:CLOCK heterodimer, a central circadian transcription factor, was observed after the HS pulse. These findings reveal that even a short-term HS pulse can reset circadian rhythms and cause the HSF1-BMAL1:CLOCK interaction, suggesting the pivotal role of crosstalk between the mammalian circadian and HSR systems

Modulation of Heat Shock Transcription Factor 1 as a Therapeutic Target for Small Molecule Intervention in Neurodegenerative Disease

A yeast-based small molecule screen identifies a novel activator of human HSF1 and protein chaperone expression and which appears to alleviate the toxicity of protein misfolding diseases

Cytosolic phospholipase A2-α expression in breast cancer is associated with EGFR expression and correlates with an adverse prognosis in luminal tumours

BACKGROUND: The eicosanoid signalling pathway promotes the progression of malignancies through the production of proliferative prostaglandins (PGs). Cytosolic phospholipase A(2)α (cPLA(2)α) activity provides the substrate for cyclooxygenase-dependent PG release, and we have previously found that cPLA(2)α expression correlated with EGFR/HER2 over-expression in a small number of breast cancer cell lines. METHODS: The importance of differential cPLA(2)α activity in clinical breast cancer was established by relating the expression of cPLA(2)α in tissue samples from breast cancer patients, and two microarray-based gene expression datasets to different clinicopathological and therapeutic parameters. RESULTS: High cPLA(2)α mRNA expression correlated with clinical parameters of poor prognosis, which are characteristic of highly invasive tumours of the HER2-positive and basal-like subtype, including low oestrogen receptor expression and high EGFR expression. High cPLA(2)α expression decreased overall survival in patients with luminal cancers, and correlated with a reduced effect of tamoxifen treatment. The cPLA(2)α expression was an independent predictive parameter of poor response to endocrine therapy in the first 5 years of follow-up. CONCLUSION: This study shows a role of cPLA(2)α in luminal breast cancer progression, in which the enzyme could represent a novel therapeutic target and a predictive marker

Novel therapies in breast cancer: what is new from ASCO 2008

<p>Abstract</p> <p>Introduction</p> <p>Breast cancer is the most common female cancer and the second most common cause of female cancer-related deaths in the United States. World-wide, more than one million women will be diagnosed with breast cancer annually. In 2007, more than 175,000 women were diagnosed with breast cancer in the United States. However, deaths due to breast cancer have decreased in the recent years in part because of improved screening techniques, surgical interventions, understanding of the pathogenesis of the disease, and utilization of traditional chemotherapies in a more efficacious manner. One of the more exciting areas of improvement in the treatment of breast cancer is the entrance of novel therapies now available to oncologists. In the field of cancer therapeutics, the area of targeted and biologic therapies has been progressing at a rapid rate, particularly in the treatment of breast cancer.</p> <p>Since the advent of imatinib for the successful treatment of chronic myelogenous leukemia in the 2001, clinicians have been searching for comparable therapies that could be as efficacious and as tolerable. In order for targeted therapies to be effective, the agent must be able to inhibit critical regulatory pathways which promote tumor cell growth and proliferation. The targets must be identifiable, quantifiable and capable of being interrupted.</p> <p>In the field of breast cancer, two advances in targeted therapy have led to great strides in the understanding and treatment of breast cancer, namely hormonal therapy for estrogen positive receptor breast cancer and antibodies directed towards the inhibition of human epidermal growth factor receptor (HER)2. These advances have revolutionized the understanding and the treatment strategies for breast cancer. Building upon these successes, a host of novel agents are currently being investigated and used in clinical trials that will hopefully prove to be as fruitful. This review will focus on novel therapies in the field of breast cancer with a focus on metastatic breast cancer (MBC) and updates from the recent annual ASCO meeting and contains a summary of the results.</p

HER2-family signalling mechanisms, clinical implications and targeting in breast cancer.

Approximately 20 % of human breast cancers (BC) overexpress HER2 protein, and HER2-positivity is associated with a worse prognosis. Although HER2-targeted therapies have significantly improved outcomes for HER2-positive BC patients, resistance to trastuzumab-based therapy remains a clinical problem. In order to better understand resistance to HER2-targeted therapies in HER2-positive BC, it is necessary to examine HER family signalling as a whole. An extensive literature search was carried out to critically assess the current knowledge of HER family signalling in HER2-positive BC and response to HER2-targeted therapy. Known mechanisms of trastuzumab resistance include reduced receptor-antibody binding (MUC4, p95HER2), increased signalling through alternative HER family receptor tyrosine kinases (RTK), altered intracellular signalling involving loss of PTEN, reduced p27kip1, or increased PI3K/AKT activity and altered signalling via non-HER family RTKs such as IGF1R. Emerging strategies to circumvent resistance to HER2-targeted therapies in HER2-positive BC include co-targeting HER2/PI3K, pan-HER family inhibition, and novel therapies such as T-DM1. There is evidence that immunity plays a key role in the efficacy of HER-targeted therapy, and efforts are being made to exploit the immune system in order to improve the efficacy of current anti-HER therapies. With our rapidly expanding understanding of HER2 signalling mechanisms along with the repertoire of HER family and other targeted therapies, it is likely that the near future holds further dramatic improvements to the prognosis of women with HER2-positive BC