Comparison of EMT mediated tyrosine kinase inhibitor resistance in NSCLC.

In the United States, lung cancer is the second most common cancer in men and women. In 2017, 222,500 new cases and 155,870 deaths from lung cancer are estimated to have occurred. A tyrosine kinase receptor, epidermal growth factor receptor (EGFR), is over expressed or mutated in non-small cell lung cancer (NSCLC) resulting in increased cell proliferation and survival. Tyrosine kinase inhibitors (TKIs) are currently being used as therapy for NSCLC patients, however, they have limited efficacy in NSCLC patients due to acquisition of resistance. This study investigates the role of epithelial-mesenchymal transition (EMT) in the development of resistance against TKIs in NSCLC. Currently, the role of p120-catenin, Kaiso factor and PRMT-1 in reversal of EMT in T790M mutated and TKI-resistant NSCLC cells is a new line of study. In this investigation we found upregulation of cytoplasmic p120-catenin, which was co-localized with Kaiso factor. In the nucleus, binding of p120-catenin to Kaiso factor initiates transcription by activating EMT-transcription factors such as Snail, Slug, Twist, and ZEB1. PRMT-1 was also found to be upregulated, which induces methylation of Twist and repression of E-cadherin activity, thus promoting EMT. We confirmed that TKI-resistant cells have mesenchymal cell type characteristics based on their cell morphology and gene or protein expression of EMT related proteins. EMT proteins, Vimentin and N-cadherin, displayed increased expression, whereas E-cadherin expression was downregulated. Finally, we found that the knockdown of p120-catenin and PRMT-1 by siRNA or use of a PRMT-1 inhibitor Furamidine increased Erlotinib sensitivity and could reverse EMT to overcome TKI resistance.

Oligonucleotides Targeting Telomeres and Telomerase in Cancer.

Telomeres and telomerase have become attractive targets for the development of anticancer therapeutics due to their involvement in cancer cell immortality. Currently, several therapeutics have been developed that directly target telomerase and telomeres, such as telomerase inhibitors and G-quadruplex stabilizing ligands. Telomere-specific oligonucleotides that reduce telomerase activity and disrupt telomere architecture are also in development as novel anticancer therapeutics. Specifically, GRN163L and T-oligos have demonstrated promising anticancer activity in multiple cancers types via induction of potent DNA damage responses. Currently, several miRNAs have been implicated in the regulation of telomerase activity and may prove to be valuable targets in the development of novel therapies by reducing expression of telomerase subunits. Targeting miRNAs that are known to increase expression of telomerase subunits may be another strategy to reduce carcinogenesis. This review aims to provide a comprehensive understanding of current oligonucleotide-based anticancer therapies that target telomeres and telomerase. These studies may help design novel therapeutic approaches to overcome the challenges of oligonucleotide therapy in a clinical setting.

Mechanism of c-Met and EGFR tyrosine kinase inhibitor resistance through epithelial mesenchymal transition in non-small cell lung cancer.

According to currently available estimates from Cancer Research UK, 14.1 million new lung cancer cases were diagnosed and a staggering 8.2 million people worldwide died from lung cancer in 2012. EGFR and c-Met are two tyrosine kinase receptors most commonly overexpressed or mutated in Non-small Cell Lung Cancer (NSCLC) resulting in increased proliferation and survival of lung cancer cells. Tyrosine kinase inhibitors (TKIs), such as erlotinib, approved by the FDA as first/second line therapy for NSCLC patients have limited clinical efficacy due to acquired resistance. In this manuscript, we investigate and discuss the role of epithelial mesenchymal transition (EMT) in the development of resistance against EGFR and c-Met TKIs in NSCLC. Our findings show that Zeb-1, a transcriptional repressor of E-Cadherin, is upregulated in TKI-resistant cells causing EMT. We observed that TKI-resistant cells have increased gene and protein expression of EMT related proteins such as Vimentin, N-Cadherin, ß-Catenin and Zeb-1, while expression of E-Cadherin, an important cell adhesion molecule, was suppressed. We also confirmed that TKI-resistant cells display mesenchymal cell type morphology, and have upregulation of ß-Catenin which may regulate expression of Zeb-1, a transcriptional repressor of E-Cadherin in TKI-resistant NSCLC cells. Finally, we show that down-regulating Zeb-1 by inducing miR-200a or ß-Catenin siRNA can increase drug sensitivity of TKI-resistant cells.

T-oligo as an anticancer agent in colorectal cancer.

In the United States, there will be an estimated 96,830 new cases of colorectal cancer (CRC) and 50,310 deaths in 2014. CRC is often detected at late stages of the disease, at which point there is no effective chemotherapy. Thus, there is an urgent need for effective novel therapies that have minimal effects on normal cells. T-oligo, an oligonucleotide homologous to the 3'-telomere overhang, induces potent DNA damage responses in multiple malignant cell types, however, its efficacy in CRC has not been studied. This is the first investigation demonstrating T-oligo-induced anticancer effects in two CRC cell lines, HT-29 and LoVo, which are highly resistant to conventional chemotherapies. In this investigation, we show that T-oligo may mediate its DNA damage responses through the p53/p73 pathway, thereby inhibiting cellular proliferation and inducing apoptosis or senescence. Additionally, upregulation of downstream DNA damage response proteins, including E2F1, p53 or p73, was observed. In LoVo cells, T-oligo induced senescence, decreased clonogenicity, and increased expression of senescence associated proteins p21, p27, and p53. In addition, downregulation of POT1 and TRF2, two components of the shelterin protein complex which protects telomeric ends, was observed. Moreover, we studied the antiproliferative effects of T-oligo in combination with an EGFR tyrosine kinase inhibitor, Gefitinib, which resulted in an additive inhibitory effect on cellular proliferation. Collectively, these data provide evidence that T-oligo alone, or in combination with other molecularly targeted therapies, has potential as an anti-cancer agent in CRC.

The Effects of Smoking on Telomere Length, Induction of Oncogenic Stress, and Chronic Inflammatory Responses Leading to Aging.

Telomeres, potential biomarkers of aging, are known to shorten with continued cigarette smoke exposure. In order to further investigate this process and its impact on cellular stress and inflammation, we used an in vitro model with cigarette smoke extract (CSE) and observed the downregulation of telomere stabilizing TRF2 and POT1 genes after CSE treatment. hTERT is a subunit of telomerase and a well-known oncogenic marker, which is overexpressed in over 85% of cancers and may contribute to lung cancer development in smokers. We also observed an increase in hTERT and ISG15 expression levels after CSE treatment, as well as increased protein levels revealed by immunohistochemical staining in smokers' lung tissue samples compared to non-smokers. The effects of ISG15 overexpression were further studied by quantifying IFN-γ, an inflammatory protein induced by ISG15, which showed greater upregulation in smokers compared to non-smokers. Similar changes in gene expression patterns for TRF2, POT1, hTERT, and ISG15 were observed in blood and buccal swab samples from smokers compared to non-smokers. The results from this study provide insight into the mechanisms behind smoking causing telomere shortening and how this may contribute to the induction of inflammation and/or tumorigenesis, which may lead to comorbidities in smokers.

PRMT-1 and p120-Catenin as EMT Mediators in Osimertinib Resistance in NSCLC.

Osimertinib, an irreversible tyrosine kinase inhibitor, is a first-line therapy in EGFR-mutant NSCLC patients. Prolonged treatment with Osimertinib leads to resistance due to an acquired C797S mutation in the EGFR domain and other mechanisms, such as epithelial-mesenchymal transition (EMT). In this study, we investigated the role of PRMT-1 and p120-catenin in mediating Osimertinib resistance (OR) through EMT. These studies found upregulation of gene and protein expression of PRMT-1, p120-catenin and Kaiso factor. Knockdown of p120-catenin using siRNA increased OR efficacy by 45% as compared to cells treated with mock siRNA and OR. After 24 h of transfection, the percentage wound closure in cells transfected with p120-catenin siRNA was 26.2%. However, in mock siRNA-treated cells the wound closure was 7.4%, showing its involvement in EMT. We also found high levels of p120-catenin expressed in 30% of smokers as compared to 5.5% and 0% of non-smokers and quit-smokers (respectively) suggesting that smoking may influence p120-catenin expression in NSCLC patients. These results suggest that biomarkers such as PRMT-1 may mediate EMT by methylating Twist-1 and increasing p120-catenin expression, which causes transcriptional activation of genes associated with Kaiso factor to promote EMT in Osimertinib-resistant cells.

Outpatient satisfaction and quality of health care in North Indian medical institute.

PURPOSE: Outpatient departments (OPDs) need to monitor the quality of care and patient satisfaction for continuous quality improvement. Additionally, there is a need for an increase in focused literature on patient satisfaction and quality of health care at a tertiary care level. The purpose of this paper is to attempt to fulfil this need. DESIGN/METHODOLOGY/APPROACH: A cross-sectional hospital-based study among OPD patients was undertaken, where investigators conducted interviews with 120 patients at entry (registration), 120 patients at the OPD clinic (60 doctor-patient interactions and 60 exit interviews), and a further 120 patients at investigation facilities. Patient satisfaction, client convenience facilities, prescription quality, doctor-patient interaction and other quality elements as described in the study were given score of 0 or 1. FINDINGS: At exit, 52 (86.6 percent) patients were satisfied with the OPD care. The mean total quality score was 80.9 percent of the total scores. It was above 90 percent of the total score for patient convenience facilities and for doctor-patient interaction, 76 percent for the prescription quality of the doctors and 43.3 percent for signage display. The mean score for patient-doctor interaction was found to be significantly lower (3.6/5) among dissatisfied patients compared to the satisfied patients (4.7/5). Satisfied patients reported a significantly higher consultation time (12.4 minutes) with a doctor compared to dissatisfied patients (8.5 minutes) (p = 0.04). RESEARCH LIMITATIONS/IMPLICATIONS: Not using a Likert scale to measure patient satisfaction could be considered a limitation However, the authors also arrived at similar conclusions with their tools as with the use of Likert scales in other studies. Furthermore, findings are limited to medicine and surgery general OPDs in a tertiary care setting. Any interpretation beyond this frame may be done with caution. PRACTICAL IMPLICATIONS: Hospitals should encourage good patient-doctor interaction as it has emerged as the key factor associated with patient satisfaction. SOCIAL IMPLICATIONS: Quality improvements in public sector health institutes can lead to better utilization of health care by the poor and compromised sections of society and can lead to a reduction in the inequity associated with health care. ORIGINALITY/VALUE: This paper fulfils the need to evaluate quality of hospital care in public sector hospitals at the tertiary care level. The methods and tools used are simple and extensive enough to capture information at multiple service points.

Mediating EGFR-TKI Resistance by VEGF/VEGFR Autocrine Pathway in Non-Small Cell Lung Cancer.

NSCLC treatment includes targeting of EGFR with tyrosine kinase inhibitors (TKIs) such as Erlotinib; however, resistance to TKIs is commonly acquired through T790M EGFR mutations or overexpression of vascular endothelial growth factor receptor-2 (VEGFR-2). We investigated the mechanisms of EGFR-TKI resistance in NSCLC cell lines with EGFR mutations or acquired resistance to Erlotinib. These studies showed upregulated gene and protein expression of VEGF, VEGFR-2, and a VEGF co-receptor neuropilin-1 (NP-1) in Erlotinib-resistant (1.4-5.3-fold) and EGFR double-mutant (L858R and T790M; 4.1-8.3-fold) NSCLC cells compared to parental and EGFR single-mutant (L858R) NSCLC cell lines, respectively. Immunofluorescence and FACS analysis revealed increased expression of VEGFR-2 and NP-1 in EGFR-TKI-resistant cell lines compared to TKI-sensitive cell lines. Cell proliferation assays showed that treatment with a VEGFR-2 inhibitor combined with Erlotinib lowered cell survival in EGFR double-mutant NSCLC cells to 9% compared to 72% after treatment with Erlotinib alone. Furthermore, Kaplan-Meier analysis revealed shorter median survival in late-stage NSCLC patients with high vs. low VEGFR-2 expression (14 mos vs. 21 mos). The results indicate that VEGFR-2 may play a key role in EGFR-TKI resistance and that combined treatment of Erlotinib with a VEGFR-2 inhibitor may serve as an effective therapy in NSCLC patients with EGFR mutations.

Treatment of Metastatic Melanoma with a Combination of Immunotherapies and Molecularly Targeted Therapies.

Melanoma possesses invasive metastatic growth patterns and is one of the most aggressive types of skin cancer. In 2021, it is estimated that 7180 deaths were attributed to melanoma in the United States alone. Once melanoma metastasizes, traditional therapies are no longer effective. Instead, immunotherapies, such as ipilimumab, pembrolizumab, and nivolumab, are the treatment options for malignant melanoma. Several biomarkers involved in tumorigenesis have been identified as potential targets for molecularly targeted melanoma therapy, such as tyrosine kinase inhibitors (TKIs). Unfortunately, melanoma quickly acquires resistance to these molecularly targeted therapies. To bypass resistance, combination treatment with immunotherapies and single or multiple TKIs have been employed and have been shown to improve the prognosis of melanoma patients compared to monotherapy. This review discusses several combination therapies that target melanoma biomarkers, such as BRAF, MEK, RAS, c-KIT, VEGFR, c-MET and PI3K. Several of these regimens are already FDA-approved for treating metastatic melanoma, while others are still in clinical trials. Continued research into the causes of resistance and factors influencing the efficacy of these combination treatments, such as specific mutations in oncogenic proteins, may further improve the effectiveness of combination therapies, providing a better prognosis for melanoma patients.

Resistance to Molecularly Targeted Therapies in Melanoma.

Malignant melanoma is the most aggressive type of skin cancer with invasive growth patterns. In 2021, 106,110 patients are projected to be diagnosed with melanoma, out of which 7180 are expected to die. Traditional methods like surgery, radiation therapy, and chemotherapy are not effective in the treatment of metastatic and advanced melanoma. Recent approaches to treat melanoma have focused on biomarkers that play significant roles in cell growth, proliferation, migration, and survival. Several FDA-approved molecular targeted therapies such as tyrosine kinase inhibitors (TKIs) have been developed against genetic biomarkers whose overexpression is implicated in tumorigenesis. The use of targeted therapies as an alternative or supplement to immunotherapy has revolutionized the management of metastatic melanoma. Although this treatment strategy is more efficacious and less toxic in comparison to traditional therapies, targeted therapies are less effective after prolonged treatment due to acquired resistance caused by mutations and activation of alternative mechanisms in melanoma tumors. Recent studies focus on understanding the mechanisms of acquired resistance to these current therapies. Further research is needed for the development of better approaches to improve prognosis in melanoma patients. In this article, various melanoma biomarkers including BRAF, MEK, RAS, c-KIT, VEGFR, c-MET and PI3K are described, and their potential mechanisms for drug resistance are discussed.

Oligonucleotides and microRNAs Targeting Telomerase Subunits in Cancer Therapy.

Telomerase provides cancer cells with replicative immortality, and its overexpression serves as a near-universal marker of cancer. Anti-cancer therapeutics targeting telomerase have garnered interest as possible alternatives to chemotherapy and radiotherapy. Oligonucleotide-based therapies that inhibit telomerase through direct or indirect modulation of its subunits, human telomerase reverse transcriptase (hTERT) and human telomerase RNA gene (hTERC), are a unique and diverse subclass of telomerase inhibitors which hold clinical promise. MicroRNAs that play a role in the upregulation or downregulation of hTERT and respective progression or attenuation of cancer development have been effectively targeted to reduce telomerase activity in various cancer types. Tumor suppressor miRNAs, such as miRNA-512-5p, miRNA-138, and miRNA-128, and oncogenic miRNAs, such as miRNA-19b, miRNA-346, and miRNA-21, have displayed preclinical promise as potential hTERT-based therapeutic targets. Antisense oligonucleotides like GRN163L and T-oligos have also been shown to uniquely target the telomerase subunits and have become popular in the design of novel cancer therapies. Finally, studies suggest that G-quadruplex stabilizers, such as Telomestatin, preserve telomeric oligonucleotide architecture, thus inhibiting hTERC binding to the telomere. This review aims to provide an adept understanding of the conceptual foundation and current state of therapeutics utilizing oligonucleotides to target the telomerase subunits, including the advantages and drawbacks of each of these approaches.

Potential Telomere-Related Pharmacological Targets.

Telomeres function as protective caps at the terminal portion of chromosomes, containing non-coding nucleotide sequence repeats. As part of their protective function, telomeres preserve genomic integrity and minimize chromosomal exposure, thus limiting DNA damage responses. With continued mitotic divisions in normal cells, telomeres progressively shorten until they reach a threshold at a point where they activate senescence or cell death pathways. However, the presence of the enzyme telomerase can provide functional immortality to the cells that have reached or progressed past senescence. In senescent cells that amass several oncogenic mutations, cancer formation can occur due to genomic instability and the induction of telomerase activity. Telomerase has been found to be expressed in over 85% of human tumors and is labeled as a near-universal marker for cancer. Due to this feature being present in a majority of tumors but absent in most somatic cells, telomerase and telomeres have become promising targets for the development of new and effective anticancer therapeutics. In this review, we evaluate novel anticancer targets in development which aim to alter telomerase or telomere function. Additionally, we analyze the progress that has been made, including preclinical studies and clinical trials, with therapeutics directed at telomere-related targets. Furthermore, we review the potential telomere-related therapeutics that are used in combination therapy with more traditional cancer treatments. Throughout the review, topics related to medicinal chemistry are discussed, including drug bioavailability and delivery, chemical structure-activity relationships of select therapies, and the development of a unique telomere assay to analyze compounds affecting telomere elongation.

EGFR/c-Met and mTOR signaling are predictors of survival in non-small cell lung cancer.

BACKGROUND: EGFR/c-Met activation/amplification and co-expression, mTOR upregulation/activation, and Akt/Wnt signaling upregulation have been individually associated with more aggressive disease and characterized as potential prognostic markers for lung cancer patients. METHODS: Tumors obtained from 109 participants with stage I-IV non-small cell lung cancer (NSCLC) were studied for EGFR/c-Met co-localization as well as for total and active forms of EGFR, c-Met, mTOR, S6K, beta-catenin, and Axin2. Slides were graded by two independent blinded pathologists using a validated scoring system. Protein expression profile correlations were assessed using Pearson correlation and Spearman's rho. Prognosis was assessed using Kaplan-Meier analysis. RESULTS: Protein expression profile analysis revealed significant correlations between EGFR/p-EGFR (p = 0.0412) and p-mTOR/S6K (p = 0.0044). Co-localization of p-EGFR/p-c-Met was associated with increased p-mTOR (p = 0.0006), S6K (p = 0.0018), and p-S6K (p < 0.0001) expression. In contrast, active beta-catenin was not positively correlated with EGFR/c-Met nor any activated proteins. Axin2, a negative regulator of the Wnt pathway, was correlated with EGFR, p-EGFR, p-mTOR, p-S6K, EGFR/c-Met co-localization, and p-EGFR/p-c-Met co-localization (all p-values <0.03). Kaplan-Meier analysis revealed shorter median survival in participants with high expression of Axin2, total beta-catenin, total/p-S6K, total/p-mTOR, EGFR, and EGFR/c-Met co-localization compared with low expression. After controlling for stage of disease at diagnosis, subjects with late-stage disease demonstrated shorter median survival when exhibiting high co-expression of EGFR/c-Met (8.1 month versus 22.3 month, p = 0.050), mTOR (6.7 month versus 22.3 month, p = 0.002), and p-mTOR (8.1 month versus 25.4 month, p = 0.004) compared with low levels. CONCLUSIONS: These findings suggest that increased EGFR/c-Met signaling is correlated with upregulated mTOR/S6K signaling, which may in turn be associated with shorter median survival in late-stage NSCLC.

A selective small molecule inhibitor of c-Met, PHA665752, inhibits tumorigenicity and angiogenesis in mouse lung cancer xenografts.

The c-Met receptor tyrosine kinase is emerging as a novel target in many solid tumors, including lung cancer. PHA-665752 was identified as a small molecule, ATP competitive inhibitor of the catalytic activity of the c-Met kinase. Here, we show that treatment with PHA665752 reduced NCI-H69 (small cell lung cancer) and NCI-H441 (non-small cell lung cancer) tumorigenicity in mouse xenografts by 99% and 75%, respectively. Reduction in tumor size was also observed by magnetic resonance imaging of tumors in mice. PHA665752 inhibited c-Met phosphorylation at the autophosphorylation and c-Cbl binding sites in mouse xenografts derived from non-small cell lung cancer cell lines (NCI-H441 and A549) and small cell lung cancer cell line (NCI-H69). PHA665752 also inhibited angiogenesis by >85% in all the abovementioned cell lines and caused an angiogenic switch which resulted in a decreased production of vascular endothelial growth factor and an increase in the production of the angiogenesis inhibitor thrombospondin-1. These studies show the feasibility of selectively targeting c-Met with ATP competitive small molecule inhibitors and suggest that PHA665752 may provide a novel therapeutic approach to lung cancer.

Synergism of EGFR and c-Met pathways, cross-talk and inhibition, in non-small cell lung cancer.

BACKGROUND: c-Met and EGFR receptors are widely expressed on cancer cells; they are implicated in the development and progression of cancer through a plethora of effects on cell cycle progression, apoptosis, motility and metastasis and are potential targets for combination therapy. EGFR receptor tyrosine kinases are currently being targeted in a number of malignancies. METHODS: Apoptosis was studied by FACS analysis using propidium iodide. EGF and HGF signaling intermediates were studied by western blotting. Cell proliferation was determined by MTT assays. Cell motility was done by time lapse confocal microscopy. RESULTS: c-Met and EGFR were both expressed in A549, H1838, H2170, SW900, SW1573, H358, SKLU-1, and H1993 non small cell lung cancer (NSCLC) cell lines. Both EGF and HGF at 100 ng/ml in medium showed a synergistic effect on cell proliferation at 48-72 h as seen by a proliferation assay in A549, H1838, and SKMES cells. In A549 and H1838 cell lines, HGF (40 ng/ml) and EGF (5 ng/ml) induced synergistic phosphorylation on c-Met (Tyr 1003/1230/1234/1235). Additionally, synergistic phosphorylation of Akt (Ser-473) and phospho-ERK1+ERK2 (Thr202/Tyr204) was also seen indicating that EGF and HGF could induce synergistic phosphorylation of important signaling intermediates. Treatment with EGF and HGF at 100 ng/ml for 2 h also leads to an additive effect in inducing cell motility (especially membrane ruffling) in H1993 cells. A novel c-Met small molecule tyrosine kinase inhibitor SU11274 and EGFR tyrosine kinase inhibitors Tyrphostin AG1478 and gefitinib (Iressa) were tested to study their effect in combination on proliferation and apoptosis in lung cancer cells. Interestingly, a synergistic effect on inhibition of cell proliferation was seen in the presence of SU11274 and Tyrphostin AG1478. 0.5 microM Tyrphostin AG1478 and 2 microM SU11274 inhibited growth by 21% and 25%, respectively; a combination of both tyrosine kinase inhibitors inhibited growth by 65%. Interestingly, EGFR inhibitor (gefitinib, Iressa) and c-Met inhibitor (SU11274) also had a synergistic effect on apoptosis in H358 cells. CONCLUSION: There was a synergistic effect of EGF and HGF on proliferation, downstream activation of signal transduction and an additive effect seen on motility. These studies show that a combination of HGF and EGF tyrosine kinase inhibitors on NSCLC, could potentially be targeted in a synergistic fashion.

c-Met is a potentially new therapeutic target for treatment of human melanoma.

PURPOSE: c-Met is a receptor tyrosine kinase involved in cell growth, invasion, metastases, and angiogenesis. In this study, we investigated the role of c-Met in melanoma biology using a novel small-molecule tyrosine kinase inhibitor SU11274 and small interfering (si) RNA against the receptor. EXPERIMENTAL DESIGN: The effects of SU11274 and c-Met siRNA were studied on proliferation, apoptosis, differentiation, reactive oxygen species, and intracellular signaling. c-Met mutations were examined, and the expression of c-Met and activated c-Met was studied in nevi, primary, and metastatic melanoma. RESULTS: c-Met was expressed in 6:7 melanoma cell lines by immunoblotting. SU11274 inhibited cell growth in all melanoma cell lines by 85% to 98% with an IC(50) between 1 and 2.5 mumol/L and caused apoptosis (12-58%) in five out of six cell lines. siRNA against c-Met inhibited proliferation of melanoma cells by 60%. This is the first study that shows that SU11274 and siRNA induced microphthalmia-associated transcription factor (MITF) and several other melanoma differentiation proteins and a morphologically differentiated phenotype. SU11274 also inhibited reactive oxygen species formation and phosphorylation of c-Met receptor, AKT and S-6 kinase by the hepatocyte growth factor. A new missense c-Met mutation N948S was identified in cell lines and R988C in tumor tissue in the juxtamembrane domain of c-Met. It was found that c-Met was expressed in 88% of melanomas and 15% of nevi, and that c-Met (pY1003) was activated in 21% of human melanomas. CONCLUSION: These results support the role of c-Met in proliferation, apoptosis, differentiation, and tumor progression of melanoma. SU11274 could be used in the therapeutic inhibition of melanoma.

Current Molecular-Targeted Therapies in NSCLC and Their Mechanism of Resistance.

Lung cancer is treated with many conventional therapies, such as surgery, radiation, and chemotherapy. However, these therapies have multiple undesirable side effects. To bypass the side effects elicited by these conventional treatments, molecularly-targeted therapies are currently in use or under development. Current molecularly-targeted therapies effectively target specific biomarkers, which are commonly overexpressed in lung cancers and can cause increased tumorigenicity. Unfortunately, several molecularly-targeted therapies are associated with initial dramatic responses followed by acquired resistance due to spontaneous mutations or activation of signaling pathways. Acquired resistance to molecularly targeted therapies presents a major clinical challenge in the treatment of lung cancer. Therefore, to address this clinical challenge and to improve lung cancer patient prognosis, we need to understand the mechanism of acquired resistance to current therapies and develop additional novel therapies. This review concentrates on various lung cancer biomarkers, including EGFR, ALK, and BRAF, as well as their potential mechanisms of drug resistance.

Mechanism of Action of G-Quadruplex-Forming Oligonucleotide Homologous to the Telomere Overhang in Melanoma.

T-oligo, a guanine-rich oligonucleotide homologous to the 3'-telomeric overhang of telomeres, elicits potent DNA-damage responses in melanoma cells; however, its mechanism of action is largely unknown. Guanine-rich oligonucleotides can form G-quadruplexes (G4), which are stabilized by the hydrogen bonding of guanine residues. In this study, we confirmed the G4-forming capabilities of T-oligo using nondenaturing PAGE, nuclear magnetic resonance, and immunofluorescence. Using an anti-G-quadruplex antibody, we showed that T-oligo can form G4 in the nuclei of melanoma cells. Furthermore, using DNase I in a nuclease degradation assay, G4-T-oligo was found to be more stable than single-stranded T-oligo. G4-T-oligo had decreased antiproliferative effects compared with single-stranded T-oligo. However, G4-T-oligo has similar cellular uptake as single-stranded T-oligo, as shown by FACS analysis. Inhibition of JNK, which causes DNA damage-induced apoptosis, partially reversed the antiproliferative activity of T-oligo. T-oligo also inhibited mRNA expression of human telomerase reverse transcriptase, a catalytic subunit of telomerase that was reversed by JNK inhibition. Furthermore, two shelterin complex proteins TRF2/POT1 were found to be up-regulated and bound by T-oligo, suggesting that T-oligo may mediate dissociation of these proteins from the telomere overhang. These studies show that T-oligo can form a G-quadruplex and that the antitumor effects of T-oligo may be mediated through POT1/TRF2 and via human telomerase reverse transcriptase inhibition through JNK activation.

Treating Cancer by Targeting Telomeres and Telomerase.

Telomerase is expressed in more than 85% of cancer cells. Tumor cells with metastatic potential may have a high telomerase activity, allowing cells to escape from the inhibition of cell proliferation due to shortened telomeres. Human telomerase primarily consists of two main components: hTERT, a catalytic subunit, and hTR, an RNA template whose sequence is complimentary to the telomeric 5'-dTTAGGG-3' repeat. In humans, telomerase activity is typically restricted to renewing tissues, such as germ cells and stem cells, and is generally absent in normal cells. While hTR is constitutively expressed in most tissue types, hTERT expression levels are low enough that telomere length cannot be maintained, which sets a proliferative lifespan on normal cells. However, in the majority of cancers, telomerase maintains stable telomere length, thereby conferring cell immortality. Levels of hTERT mRNA are directly related to telomerase activity, thereby making it a more suitable therapeutic target than hTR. Recent data suggests that stabilization of telomeric G-quadruplexes may act to indirectly inhibit telomerase action by blocking hTR binding. Telomeric DNA has the propensity to spontaneously form intramolecular G-quadruplexes, four-stranded DNA secondary structures that are stabilized by the stacking of guanine residues in a planar arrangement. The functional roles of telomeric G-quadruplexes are not completely understood, but recent evidence suggests that they can stall the replication fork during DNA synthesis and inhibit telomere replication by preventing telomerase and related proteins from binding to the telomere. Long-term treatment with G-quadruplex stabilizers induces a gradual reduction in the length of the G-rich 3' end of the telomere without a reduction of the total telomere length, suggesting that telomerase activity is inhibited. However, inhibition of telomerase, either directly or indirectly, has shown only moderate success in cancer patients. Another promising approach of targeting the telomere is the use of guanine-rich oligonucleotides (GROs) homologous to the 3' telomere overhang sequence (T-oligos). T-oligos, particularly a specific 11-base oligonucleotide (5'-dGTTAGGGTTAG-3') called T11, have been shown to induce DNA damage responses (DDRs) such as senescence, apoptosis, and cell cycle arrest in numerous cancer cell types with minimal or no cytostatic effects in normal, non-transformed cells. As a result, T-oligos and other GROs are being investigated as prospective anticancer therapeutics. Interestingly, the DDRs induced by T-oligos in cancer cells are similar to the effects seen after progressive telomere degradation in normal cells. The loss of telomeres is an important tumor suppressor mechanism that is commonly absent in transformed malignant cells, and hence, T-oligos have garnered significant interest as a novel strategy to combat cancer. However, little is known about their mechanism of action. In this review, we discuss the current understanding of how T-oligos exert their antiproliferative effects in cancer cells and their role in inhibition of telomerase. We also discuss the current understanding of telomerase in cancer and various therapeutic targets related to the telomeres and telomerase.

Induction of a p95/Nbs1-mediated S phase checkpoint by telomere 3' overhang specific DNA.

Telomere shortening induces a nonproliferative senescent phenotype, believed to reduce cancer risk, and telomeres are involved in a poorly understood manner in responses to DNA damage. Although telomere disruption induces p53 and triggers apoptosis or cell cycle arrest, the features of the disrupted telomere that trigger this response and the precise mechanism involved are poorly understood. Using human cells, we show that DNA oligonucleotides homologous to the telomere 3' overhang sequence specifically induce and activate p53 and activate an S phase checkpoint by modifying the Nijmegen breakage syndrome protein, known to mediate the S phase checkpoint after DNA damage. These responses are mediated, at least in part, by the ATM kinase and are not attributable to disruption of cellular telomeres. Based on these and earlier data, we propose that these oligonucleotides mimic a physiological signal, exposure of the telomere 3' overhang due to opening of the normal telomere loop structure, and hence evoke these protective antiproliferative responses in the absence of DNA damage or telomere disruption.