Search

PTPN11 encodes the SHP2 protein tyrosine phosphatase that activates the mitogen-activated protein kinase (MAPK) pathway upstream of KRAS and MEK. PTPN11/Shp2 somatic mutations occur frequently in Juvenile myelomonocytic leukaemia (JMML); however, the role of mutated PTPN11 in lung cancer tumourigenesis and its utility as a therapeutic target has not been fully addressed. We applied mass-spectrometry-based genotyping to DNA extracted from the tumour and matched the normal tissue of 356 NSCLC patients (98 adenocarcinomas (LUAD) and 258 squamous cell carcinomas (LUSC)). Further, PTPN11 mutation cases were identified in additional cohorts, including TCGA, Broad, and MD Anderson datasets and the COSMIC database. PTPN11 constructs harbouring PTPN11 E76A, A72D and C459S mutations were stably expressed in IL-3 dependent BaF3 cells and NSCLC cell lines (NCI-H1703, NCI-H157, NCI-H1299). The MAPK and PI3K pathway activation was evaluated using Western blotting. PTPN11/Shp2 phosphatase activity was measured in whole-cell protein lysates using an Shp2 assay kit. The Shp2 inhibitor (SHPi) was assessed both in vitro and in vivo in a PTPN11-mutated cell line for improved responses to MAPK and PI3K targeting therapies. Somatic PTPN11 hotspot mutations occurred in 4/98 (4.1%) adenocarcinomas and 7/258 (2.7%) squamous cells of 356 NSCLC patients. Additional 26 PTPN11 hotspot mutations occurred in 23 and 3 adenocarcinomas and squamous cell carcinoma, respectively, across the additional cohorts. Mutant PTPN11 significantly increased the IL-3 independent survival of Ba/F3 cells compared to wildtype PTPN11 (p < 0.0001). Ba/F3, NCI-H1703, and NCI-H157 cells expressing mutant PTPN11 exhibited increased PTPN11/Shp2 phosphatase activity and phospho-ERK1/2 levels compared to cells expressing wildtype PTPN11. The transduction of the PTPN11 inactivating mutation C459S into NSCLC cell lines led to decreased phospho-ERK, as well as decreased phospho-AKT in the PTPN11-mutated NCI-H661 cell line. NCI-H661 cells (PTPN11-mutated, KRAS-wild type) were significantly more sensitive to growth inhibition by the PI3K inhibitor copanlisib (IC50: 13.9 ± 4.7 nM) compared to NCI-H1703 (PTPN11/KRAS-wild type) cells (IC50: >10,000 nM). The SHP2 inhibitor, in combination with the PI3K targeting therapy copanlisib, showed no significant difference in tumour development in vivo; however, this significantly prevented MAPK pathway induction in vitro (p < 0.0001). PTPN11/Shp2 demonstrated the in vitro features of a driver oncogene and could potentially sensitize NSCLC cells to PI3K inhibition and inhibit MAPK pathway activation following PI3K pathway targeting.

Pleural mesothelioma (PM) is an aggressive malignancy with poor prognosis. Although histology and pathologic stage are important prognostic factors, better prognostic biomarkers are needed. The ribosomal protein S6 is a downstream target of the phosphatidylinositol 3-kinase (PI3K) pathway involved in protein synthesis and cell proliferation. In previous studies, low phosphorylated S6 (pS6) immunoreactivity was significantly correlated with longer progression-free survival (PFS) and overall survival (OS) in PM patients. We aimed to correlate pS6 expression to clinical data in a large multi-centre PM cohort as part of the European Thoracic Oncology Platform (ETOP) Mesoscape project. Tissue Micro Arrays (TMAs) of PM were constructed and expression of pS6 was evaluated by a semi-quantitatively aggregate H-score. Expression results were correlated to patient characteristics as well as OS/PFS. pS6 IHC results of 364 patients from 9 centres, diagnosed between 1999 and 2017 were available. The primary histology of included tumours was epithelioid (70.3%), followed by biphasic (24.2%) and sarcomatoid (5.5%). TMAs included both treatment-naïve and tumour tissue taken after induction chemotherapy. High pS6 expression (181 patients with H-score>1.41) was significantly associated with less complete resection. In the overall cohort, OS/PFS were not significantly different between pS6-low and pS6-high patients. In a subgroup analysis non-epithelioid (biphasic and sarcomatoid) patients with high pS6 expression showed a significantly shorter OS (p

Activating mutations in KRAS are highly prevalent in solid tumours and are frequently found in 35% of lung, 45% of colorectal, and up to 90% of pancreatic cancers. Mutated KRAS is a prognostic factor for disease-free survival (DFS) and overall survival (OS) in NSCLC and is associated with a more aggressive clinical phenotype, highlighting the need for KRAS-targeted therapy. Once considered undruggable due to its smooth shallow surface, a breakthrough showed that the activated G12C-mutated KRAS isozyme can be directly inhibited via a newly identified switch II pocket. This discovery led to the development of a new class of selective small-molecule inhibitors against the KRAS G12C isoform. Sotorasib and adagrasib are approved in locally advanced or metastatic NSCLC patients who have received at least one prior systemic therapy. Currently, there are at least twelve KRAS G12C inhibitors being tested in clinical trials, either as a single agent or in combination. In this study, KRAS mutation prevalence, subtypes, rates of occurrence in treatment-resistant invasive mucinous adenocarcinomas (IMAs), and novel drug delivery options are reviewed. Additionally, the current status of KRAS inhibitors, multiple resistance mechanisms that limit efficacy, and their use in combination treatment strategies and novel multitargeted approaches in NSCLC are discussed.

Purpose: Antibody-dependent cell-mediated cytotoxicity (ADCC) is one mechanism of action of the monoclonal antibody (mAb) therapies trastuzumab and pertuzumab. Tyrosine kinase inhibitors (TKIs), like lapatinib, may have added therapeutic value in combination with mAbs through enhanced ADCC activity. Using clinical data, we examined the impact of lapatinib on HER2/EGFR expression levels and natural killer (NK) cell gene signatures. We investigated the ability of three TKIs (lapatinib, afatinib, and neratinib) to alter HER2/immune-related protein levels in preclinical models of HER2-positive (HER2+) and HER2-low breast cancer, and the subsequent effects on trastuzumab/pertuzumab-mediated ADCC. Experimental Design: Preclinical studies (proliferation assays, Western blotting, high content analysis, and flow cytometry) employed HER2+ (SKBR3 and HCC1954) and HER2-low (MCF-7, T47D, CAMA-1, and CAL-51) breast cancer cell lines. NCT00524303 provided reverse phase protein array–determined protein levels of HER2/pHER2/EGFR/pEGFR. RNA-based NK cell gene signatures (CIBERSORT/MCP-counter) post-neoadjuvant anti-HER2 therapy were assessed (NCT00769470/NCT01485926). ADCC assays utilized flow cytometry–based protocols. Results: Lapatinib significantly increased membrane HER2 levels, while afatinib and neratinib significantly decreased levels in all preclinical models. Single-agent lapatinib increased HER2 or EGFR levels in 10 of 11 (91%) tumor samples. NK cell signatures increased posttherapy (P = 0.03) and associated with trastuzumab response (P = 0.01). TKI treatment altered mAb-induced NK cell–mediated ADCC in vitro, but it did not consistently correlate with HER2 expression in HER2+ or HER2-low models. The ADCC response to trastuzumab and pertuzumab combined did not exceed either mAb alone. Conclusions: TKIs differentially alter tumor cell phenotype which can impact NK cell–mediated response to coadministered antibody therapies. mAb-induced ADCC response is relevant when rationalizing combinations for clinical investigation.

PIM kinases have been shown to play a role in prostate cancer development and progression, as well as in some of the hallmarks of cancer, especially proliferation and apoptosis. Their upregulation in prostate cancer has been correlated with decreased patient overall survival and therapy resistance. Initial efforts to inhibit PIM with monotherapies have been hampered by compensatory upregulation of other pathways and drug toxicity, and as such, it has been suggested that co-targeting PIM with other treatment approaches may permit lower doses and be a more viable option in the clinic. Here, we present the rationale and basis for co-targeting PIM with inhibitors of PI3K/mTOR/AKT, JAK/STAT, MYC, stemness, and RNA Polymerase I transcription, along with other therapies, including androgen deprivation, radiotherapy, chemotherapy, and immunotherapy. Such combined approaches could potentially be used as neoadjuvant therapies, limiting the development of resistance to treatments or sensitizing cells to other therapeutics. To determine which drugs should be combined with PIM inhibitors for each patient, it will be key to develop companion diagnostics that predict response to each co-targeted option, hopefully providing a personalized medicine pathway for subsets of prostate cancer patients in the future.

Objectives The majority of patients with non-small cell lung cancer (NSCLC) present with advanced stage disease, at which time chemotherapy is usually the most common treatment option. While somewhat effective, patients treated with platinum-based regimens will eventually develop resistance, with others presenting with intrinsic resistance. Multiple pathways have been implicated in chemo-resistance, however the critical underlying mechanisms have yet to be elucidated. The aim of this project was to determine the role of inflammatory mediators in cisplatin-resistance in NSCLC. Materials and methods Inflammatory mediator, NF-κB, and its associated pathways were investigated in an isogenic model of cisplatin-resistant NSCLC using age-matched parental (PT) and corresponding cisplatin-resistant (CisR) sublines. Pathways were assessed using mass spectrometry, western blot analysis and qRT-PCR. The cisplatin sensitizing potential of an NF-κB small molecule inhibitor, DHMEQ, was also assessed by means of viability assays and western blot analysis. Results Proteomic analysis identified dysregulated NF-κB responsive targets in CisR cells when compared to PT cells, with increased NF-κB expression identified in four out of the five NSCLC sub-types examined (CisR versus PT). DHMEQ treatment resulted in reduced NF-κB expression in the presence of cisplatin, and re-sensitized CisR cells to the cytotoxic effects of the drug. Conclusion This study identified NF-ĸB as a potential therapeutic target in cisplatin-resistant NSCLC. Furthermore, inhibition of NF-ĸB using DHMEQ re-sensitized chemo-resistant cells to cisplatin treatment.

Simple Summary PIM kinases interact with major oncogenic players, including the PI3K/Akt pathway, and provide an escape mechanism leading to drug resistance. This study examined PIM kinase expression in NSCLC and the potential of PIM1 as a prognostic marker. The effect on cell signaling of novel preclinical PI3K/mTOR/PIM kinase inhibitor IBL-301 was compared to PI3K/mTOR inhibition in vitro and ex vivo. PI3K-mTOR inhibitor sensitive (H1975P) and resistant (H1975GR) cells were compared for altered IL6/STAT3 pathway expression and sensitivity to IBL-301. All three PIM kinases are expressed in NSCLC and PIM1 is a marker of poor prognosis. IBL-301 inhibited c-Myc, the PI3K-Akt and JAK/STAT pathways in vitro and in NSCLC tumor tissue explants. IBL-301 also inhibited secreted pro-inflammatory cytokine MCP-1. PIM kinases were activated in H1975GR cells which were more sensitive to IBL-301 than H1975P cells. A miRNA signature of PI3K-mTOR resistance was validated. Co-targeting PIM kinase and PI3K-mTOR warrants further clinical investigation. Abstract PIM kinases are constitutively active proto-oncogenic serine/threonine kinases that play a role in cell cycle progression, metabolism, inflammation and drug resistance. PIM kinases interact with and stabilize p53, c-Myc and parallel signaling pathway PI3K/Akt. This study evaluated PIM kinase expression in NSCLC and in response to PI3K/mTOR inhibition. It investigated a novel preclinical PI3K/mTOR/PIM inhibitor (IBL-301) in vitro and in patient-derived NSCLC tumor tissues. Western blot analysis confirmed PIM1, PIM2 and PIM3 are expressed in NSCLC cell lines and PIM1 is a marker of poor prognosis in patients with NSCLC. IBL-301 decreased PIM1, c-Myc, pBAD and p4EBP1 (Thr37/46) and peIF4B (S406) protein levels in-vitro and MAP kinase, PI3K-Akt and JAK/STAT pathways in tumor tissue explants. IBL-301 significantly decreased secreted pro-inflammatory cytokine MCP-1. Altered mRNA expression, including activated PIM kinase and c-Myc, was identified in Apitolisib resistant cells (H1975GR) by an IL-6/STAT3 pathway array and validated by Western blot. H1975GR cells were more sensitive to IBL-301 than parent cells. A miRNA array identified a dysregulated miRNA signature of PI3K/mTOR drug resistance consisting of regulators of PIM kinase and c-Myc (miR17-5p, miR19b-3p, miR20a-5p, miR15b-5p, miR203a, miR-206). Our data provides a rationale for co-targeting PIM kinase and PI3K-mTOR to improve therapeutic response in NSCLC.

PIM and PI3K/mTOR pathways are often dysregulated in prostate cancer, and may lead to decreased survival, increased metastasis and invasion. The pathways are heavily interconnected and act on a variety of common effectors that can lead to the development of resistance to drug inhibitors. Most current treatments exhibit issues with toxicity and resistance. We investigated the novel multikinase PIM/PI3K/mTOR inhibitor, AUM302, versus a combination of the PIM inhibitor, AZD-1208, and the PI3K/mTOR inhibitor BEZ235 (Dactolisib) to determine their impact on mRNA and phosphoprotein expression, as well as their functional efficacy. We have determined that around 20% of prostate cancer patients overexpress the direct targets of these drugs, and this cohort are more likely to have a high Gleason grade tumour (≥ Gleason 8). A co-targeted inhibition approach offered broader inhibition of genes and phosphoproteins in the PI3K/mTOR pathway, when compared to single kinase inhibition. The preclinical inhibitor AUM302, used at a lower dose, elicited a comparable or superior functional outcome compared with combined AZD-1208 + BEZ235, which have been investigated in clinical trials, and could help to reduce treatment toxicity in future trials. We believe that a co-targeting approach is a viable therapeutic strategy that should be developed further in pre-clinical studies.

Objectives Lung cancer is the most common cause of cancer mortality worldwide and, while tobacco smoke remains the primary cause, there is increasing concern that vaping and E-cigarette use may also increase lung cancer risk. This review concentrates on the current data, scholarship and active foci of research regarding potential cancer risk and oncogenic mechanisms of vaping and lung cancer. Materials and methods We performed a literature review of current and historical publications on lung cancer oncogenesis, vaping device/e-liquid contents and daughter products, molecular oncogenic mechanisms and the fundamental, potentially oncogenic, effects of electronic cigarette smoke/e-liquid products. Results E-cigarette devices and vaping fluids demonstrably contain a series of both definite and probable oncogens including nicotine derivatives (e.g. nitrosnornicotine, nitrosamine ketone), polycyclic aromatic hydrocarbons, heavy metals (including organometal compounds) and aldehydes/other complex organic compounds. These arise both as constituents of the e-liquid (with many aldehydes and other complex organics used as flavourings) and as a result of pyrolysis/complex organic reactions in the electronic cigarette device (including unequivocal carcinogens such as formaldehyde – formed from pyrolysis of glycerol). Various studies demonstrate in vitro transforming and cytotoxic activity of these derivatives. E-cigarette device use has been significantly increasing – particularly amongst the younger cohort and non-smokers; thus, this is an area of significant concern for the future. Conclusion Although research remains somewhat equivocal, there is clear reason for concern regarding the potential oncogenicity of E-Cigarettes/E-Liquids with a strong basic and molecular science basis. Given lag times (extrapolating from tobacco smoke data) of perhaps 20 years, this may have significant future public health implications. Thus, the authors feel further study in this field is strongly warranted and consideration should be made for tighter control and regulation of these products.

Currently there are five EGFR tyrosine kinase inhibitors (TKIs) available for the treatment of EGFR-mutated non-small cell lung cancer (NSCLC). However drug resistance is inevitable for virtually all patients and disease progression occurs within 1 to 2 years of starting a TKI. Third-generation agents, such as osimertinib, show improved response rates and extended median overall survival (OS), with potential to overcome previously untreatable resistance mechanisms. However acquired resistance mutations and activation of bypass RTK signalling mechanisms can mediate primary and secondary resistance to all EGFR TKIs. We have identfied activated STAT3 pathway signalling in an erlotinib resistant cell line model HCC827ER compared to the erlotinib sensitive parent cells HCC827P. pSTAT3 and PIM-1 kinase expression is activated in the total resistant cell population (HCC827ER) as well as the EMT-like HCC827ER subclone 10 cells compared to HCC827P cells. MET amplified HCC827ER subclone 3 cells show elevated PIM-1 expression. Exposure to pan-PIM inhibitor (AZD1208) alone and in combination with erlotinib resulted in a decrease in protein kinase phosphorylation in Clone 3 but had little effect on Clone 10 cells. Here, we examine the efficacy of STAT3 pathway inhibitor BBI608 alone and in combination with erlotinib in the erlotinib sensitive and resistant cells. BBI608 is a small molecule STAT3 inhibitor known to suppress cancer relapse, progression and metastasis. We hypothesise that co-targeting STAT3 and EGFR may provide a more durable response to treatment and overcome EGFR TKI resistance.

PIM and PI3K/mTOR pathways are often dysregulated in prostate cancer, and may lead to decreased survival, increased metastasis and invasion. The pathways are heavily interconnected and act on a variety of common effectors that can lead to the development of resistance to drug inhibitors. Most current treatments exhibit issues with toxicity and resistance. We investigated the novel multikinase PIM/PI3K/mTOR inhibitor, AUM302, versus a combination of the PIM inhibitor, AZD-1208, and the PI3K/mTOR inhibitor BEZ235 (Dactolisib) to determine their impact on mRNA and phosphoprotein expression, as well as their functional efficacy. We have determined that around 20% of prostate cancer patients overexpress the direct targets of these drugs, and this cohort are more likely to have a high Gleason grade tumour (≥ Gleason 8). A co-targeted inhibition approach offered broader inhibition of genes and phosphoproteins in the PI3K/mTOR pathway, when compared to single kinase inhibition. The preclinical inhibitor AUM302, used at a lower dose, elicited a comparable or superior functional outcome compared with combined AZD-1208 + BEZ235, which have been investigated in clinical trials, and could help to reduce treatment toxicity in future trials. We believe that a co-targeting approach is a viable therapeutic strategy that should be developed further in pre-clinical studies.

PD-L1, as assessed by immunohistochemistry, is a predictive biomarker for immuno-oncology treatment in lung cancer. Different scoring methods have been used to assess its status, resulting in a wide range of positivity rates. We use the European Thoracic Oncology Platform Lungscape non-small cell lung carcinoma cohort to explore this issue. PD-L1 expression was assessed via immunohistochemistry on tissue microarrays (up to four cores per case), using the DAKO 28-8 immunohistochemistry assay, following a two-round external quality assessment procedure. All samples were analyzed under the same protocol. Cross-validation of scoring between tissue microarray and whole sections was performed in 10% randomly selected samples. Cutoff points considered: ≥1, 50 (primarily), and 25%. At the two external quality assessment rounds, tissue microarray scoring agreement rates between pathologists were: 73% and 81%. There were 2008 cases with valid immunohistochemistry tissue microarray results (50% all cores evaluable). Concordant cases at 1, 25, and 50% were: 85, 91, and 93%. Tissue microarray core results were identical for 70% of cases. Sensitivity of the tissue microarray method for 1, 25, and 50% was: 80, 78, and 79% (specificity: 90, 95, 98%). Complete agreement between tissue microarrays and whole sections was achieved for 60% of the cases. Highest sensitivity rates for 1% and 50% cutoffs were detected for higher number of cores. Underestimation of PD-L1 expression on small samples is more common than overestimation. We demonstrated that classification of PD-L1 on small biopsy samples does not represent the overall expression of PD-L1 in all non-small cell cancer carcinoma cases, although the majority of cases are ‘correctly’ classified. In future studies, sampling more and larger biopsies, recording the biopsy size and tumor load may permit further refinement, increasing predictive accuracy.

Background An increasing number of anti-cancer therapeutic agents target specific mutant proteins that are expressed by many different tumor types. Successful use of these therapies is dependent on the presence or absence of somatic mutations within the patient’s tumor that can confer clinical efficacy or drug resistance. Methods The aim of our study was to determine the type, frequency, overlap and functional proteomic effects of potentially targetable recurrent somatic hotspot mutations in 47 cancer-related genes in multiple disease sites that could be potential therapeutic targets using currently available agents or agents in clinical development. Results Using MassArray technology, of the 1300 patient tumors analysed 571 (43.9%) had at least one somatic mutation. Mutations were identified in 30 different genes. KRAS (16.5%), PIK3CA (13.6%) and BRAF (3.8%) were the most frequently mutated genes. Prostate (10.8%) had the lowest number of somatic mutations identified, while no mutations were identified in sarcoma. Ocular melanoma (90.6%), endometrial (72.4%) and colorectal (66.4%) tumors had the highest number of mutations. We noted high concordance between mutations in different parts of the tumor (94%) and matched primary and metastatic samples (90%). KRAS and BRAF mutations were mutually exclusive. Mutation co-occurrence involved mainly PIK3CA and PTPN11, and PTPN11 and APC. Reverse Phase Protein Array (RPPA) analysis demonstrated that PI3K and MAPK signalling pathways were more altered in tumors with mutations compared to wild type tumors. Conclusions Hotspot mutational profiling is a sensitive, high-throughput approach for identifying mutations of clinical relevance to molecular based therapeutics for treatment of cancer, and could potentially be of use in identifying novel opportunities for genotype-driven clinical trials.

[EN] Background Reported prevalence of driver gene mutations in non-small-cell lung cancer (NSCLC) is highly variable and clinical correlations are emerging. Using NSCLC biomaterial and clinical data from the European Thoracic Oncology Platform Lungscape iBiobank, we explore the epidemiology of mutations and association to clinicopathologic features and patient outcome (relapse-free survival, time-to-relapse, overall survival). Methods Clinically annotated, resected stage I¿III NSCLC FFPE tissue was assessed for gene mutation using a microfluidics-based multiplex PCR platform. Mutant-allele detection sensitivity is¿>1% for most of the ~150 (13 genes) mutations covered in the multiplex test. Results Multiplex testing has been carried out in 2063 (76.2%) of the 2709 Lungscape cases (median follow-up 4.8¿years). FFPE samples mostly date from 2005 to 2008, yet recently extracted DNA quality and quantity was generally good. Average DNA yield/case was 2.63¿µg; 38 cases (1.4%) failed QC and were excluded from study; 95.1% of included cases allowed the complete panel of mutations to be tested. Most common were KRAS, MET, EGFR and PIK3CA mutations with overall prevalence of 23.0%, 6.8%, 5.4% and 4.9%, respectively. KRAS and EGFR mutations were significantly more frequent in adenocarcinomas: PIK3CA in squamous cell carcinomas. MET mutation prevalence did not differ between histology groups. EGFR mutations were found predominantly in never smokers; KRAS in current/former smokers. For all the above mutations, there was no difference in outcome between mutated and non-mutated cases. Conclusion Archival FFPE NSCLC material is adequate for multiplex mutation analysis. In this large, predominantly European, clinically annotated stage I¿III NSCLC cohort, none of the mutations characterized showed prognostic significance.

INTRODUCTION NSCLC is the leading cause for cancer-related deaths worldwide. New therapeutic targets are needed, as development of resistance to current treatment, such as platinum-based chemotherapy, is inevitable. The purpose of this study was to determine the functional relevance and therapeutic potential of cell division cycle associated 3 protein (CDCA3) in NSCLC. METHODS The expression of CDCA3 in squamous and nonsquamous NSCLC was investigated by using bioinformatics, Western blot analysis of matched tumor and normal tissue, and immunohistochemistry of a tissue microarray. The function of CDCA3 in NSCLC was determined by using several in vitro assays with small interfering RNA depleting CDCA3 in a panel of three immortalized human bronchial epithelial cell (HBEC) lines and seven NSCLC cell lines. RESULTS In this study, cell division cycle associated 3 gene (CDCA3) transcripts were identified as highly increased in NSCLC versus in nonmalignant tissue, with high levels of CDCA3 being associated with poor patient prognosis. CDCA3 protein was also increased in NSCLC tissue and expression was limited to tumor cells. CDCA3 expression was similarly increased in a panel of NSCLC cell lines compared with in three HBEC lines. Although depletion of CDCA3 in the HBEC lines did not affect cellular proliferation, depletion of CDCA3 expression markedly reduced the proliferation of all NSCLC cell lines. CDCA3 depletion caused a defective G2/M-phase cell cycle progression, upregulation of p21 independent of p53, and induction of cellular senescence. CONCLUSIONS Our findings highlight CDCA3 as a prognostic factor and potential novel therapeutic target in NSCLC through inhibition of tumor growth and promotion of tumor senescence.

PIM kinases are a class of serine/threonine kinases that play a role in several of the hallmarks of cancer including cell cycle progression, metabolism, inflammation and immune evasion. Their constitutively active nature and unique catalytic structure has led them to be an attractive anticancer target through the use of small molecule inhibitors. This review highlights the enhanced activity of PIM kinases in cancer that can be driven by hypoxia in the tumour microenvironment and the important role that aberrant PIM kinase activity plays in resistance mechanisms to chemotherapy, radiotherapy, anti-angiogenic therapies and targeted therapies. We highlight an interaction of PIM kinases with numerous major oncogenic players, including but not limited to, stabilisation of p53, synergism with c-Myc, and notable parallel signalling with PI3K/Akt. We provide a comprehensive overview of PIM kinase's role as an escape mechanism to targeted therapies including PI3K/mTOR inhibitors, MET inhibitors, anti-HER2/EGFR treatments and the immunosuppressant rapamycin, providing a rationale for co-targeting treatment strategies for a more durable patient response. The current status of PIM kinase inhibitors and their use as a combination therapy with other targeted agents, in addition to the development of novel multi-molecularly targeted single therapeutic agents containing a PIM kinase targeting moiety are discussed.

In pre-clinical studies, triple-negative breast cancer (TNBC) cells have demonstrated sensitivity to the multi-targeted kinase inhibitor dasatinib, however, clinical trials with single-agent dasatinib showed limited efficacy in unselected populations of breast cancer, including TNBC. To study potential mechanisms of resistance to dasatinib in TNBC, we established a cell line model of acquired dasatinib resistance (231-DasB). Following an approximately three-month exposure to incrementally increasing concentrations of dasatinib (200 nM to 500 nM) dasatinib, 231-DasB cells were resistant to the agent with a dasatinib IC50 value greater than 5 &mu, M compared to 0.04 &plusmn, 0.001 &micro, M in the parental MDA-MB-231 cells. 231-DasB cells also showed resistance (2.2-fold) to the Src kinase inhibitor PD180970. Treatment of 231-DasB cells with dasatinib did not inhibit phosphorylation of Src kinase. The 231-DasB cells also had significantly increased levels of p-Met compared to the parental MDA-MB-231 cells, as measured by luminex, and resistant cells demonstrated a significant increase in sensitivity to the c-Met inhibitor, CpdA, with an IC50 value of 1.4 &plusmn, 0.5 &micro, M compared to an IC50 of 6.8 &plusmn, 0.2 &micro, M in the parental MDA-MB-231 cells. Treatment with CpdA decreased p-Met and p-Src in both 231-DasB and MDA-MB-231 cells. Combined treatment with dasatinib and CpdA significantly inhibited the growth of MDA-MB-231 parental cells and prevented the emergence of dasatinib resistance. If these in vitro findings can be extrapolated to human cancer treatment, combined treatment with dasatinib and a c-Met inhibitor may block the development of acquired resistance and improve response rates to dasatinib treatment in TNBC.

// Susan Heavey 1 , Sinead Cuffe 1 , Stephen Finn 1 , Vincent Young 2 , Ronan Ryan 2 , Siobhan Nicholson 3 , Niamh Leonard 3 , Niall McVeigh 1 , Martin Barr 1 , Kenneth O’Byrne 4 , Kathy Gately 1 1 Department of Clinical Medicine, Trinity College Dublin, Dublin, Ireland 2 Department of Cardiothoracic Surgery, St. James’s Hospital, Dublin, Ireland 3 Department of Histopathology, St James Hospital, Dublin, Ireland 4 Cancer and Ageing Research Program, Institute of Health and Biomedical Innovation at the Translational Research Institute (TRI), Queensland University of Technology, Brisbane, Australia Correspondence to: Susan Heavey, email: heaveys@tcd.ie Keywords: PI3K, MEK, NSCLC, lung, co-target Received: September 29, 2015 Accepted: September 12, 2016 Published: October 19, 2016 ABSTRACT Clinical PI3K inhibition has been somewhat disappointing, due to both inadequate patient stratification and compensatory cell signalling through bypass mechanisms. As such, investigation of PI3K-MEK co-targeted inhibition has been recommended. With high mortality rates and a clear need for new therapeutic intervention strategies, non-small cell lung cancer (NSCLC) is an important setting to investigate the effectiveness of this approach. Here, 174 NSCLC tumours were screened for 150 mutations by Fluidigm technology, with 15 patients being profiled for phosphoprotein expression. The effects of GDC-0941 (a pan PI3K inhibitor), GDC-0980 (a dual PI3K/mTOR inhibitor) and GDC-0973 (a MEK inhibitor) alone and in combination were assessed in 3 NSCLC cell lines. PIK3CA was mutated in 5.17% of NSCLC patients. GDC-0941 and GDC-0980 treatment induced anti-proliferative and pro-apoptotic responses across all NSCLC cell lines, while GDC-0973 treatment induced only anti-proliferative responses. GDC-0980 and GDC-0973 combined treatment led to significant increases in apoptosis and synergistic reductions in proliferation across the panel of cell lines. This study found that the PI3K/MEK co-targeted inhibition strategy is synergistic in all 3 molecular subtypes of NSCLC investigated. Consequently, we would advocate clinical trials for NSCLC patients combining GDC-0980 and GDC-0973, each of which are separately under clinical investigation currently.

Introduction: The PI3K pathway is often dysregulated in lung cancer, making it an ideal therapeutic target. Pan- and isoform-specific inhibitors of the PI3K pathway are currently being evaluated in clinical trials. However, all patients treated thus far develop progressive disease due to the emergence of bypass signaling mechanisms. We developed a panel of lung cancer preclinical models to elucidate potential mechanisms of resistance to PI3K-mTOR inhibitor GDC-0980 (apitolisib). In-depth characterization of the PIK3CA mutant positive H1975 sensitive cells (H1975P) versus apitolisib-resistant cells (H1975GR) identified activation of MACC1 as an early marker of drug resistance and the subsequent activation of all three PIM kinases. Inhibition of MACC1 and PIM kinase by siRNAs and the effect of IBL-301 (a novel PIM/PI3K/mTOR inhibitor) were examined for effects on cell viability/proliferation and downstream signaling pathways in vitro. Methods: Profiling of H1975P versus H1975GR cells was undertaken using a mRNA/lncRNA array (Arraystar Inc) to quantify the expression of 20,730 mRNAs and 40,173 lncRNAs. Concomitant changes were assessed in the IL6/STAT3 signaling pathway by a QPCR array, immunofluorescence, Western blot and miRNA arrays. The effect of MACC1 and PIM kinase inhibition by siRNAs and targeted agents were examined by Western blot and cell viability assays. Results: MACC1 mRNA was upregulated 77 fold in H1975GR cells after 1-month exposure to apitolisib, and its regulated proteins HGF and MET were upregulated (10.06 and 1.65 fold, respectively). At 4 months MACC1 mRNA was increased by 44 fold, HGF 2 fold and MET 6.6 fold while several members of the IL-6/STAT3 pathway including c-Myc and PIM1, PIM2 and PIM3 kinases were also significantly activated (25.5, 19.8, 3.3 and 31.9 fold respectively). Results were also validated by Western blot. A miRNA signature of PI3K/mTOR inhibitor resistance was validated that can be used to monitor patients on treatment. Inhibition of PIM kinases by siRNA and targeted therapy restored sensitivity to PI3K-mTOR inhibitors. Novel triple-targeted therapy IBL-301 decreased PIM kinase, c-Myc and MACC1 expression. IC50 values of IBL-301 were significantly less for H1975GR cells (0.02μM) compared to H1975P cells (0.75μM), highlighting activated PIM kinase as a mechanism of resistance to PI3K-mTOR inhibition. Conclusion: Activated MACC1, PIM kinases and their interacting partners play a role in resistance to PI3K-mTOR inhibitors in PIK3CA mutant NSCLC. Our data provide a rationale for combined or sequential targeting of PI3K-mTOR and PIM kinases to significantly delay resistance and provide durable treatment responses in patients with PIK3CA mutant lung tumors. Citation Format: Gillian Moore, Samira Elbai, Susan Heavey, Stephen Finn, Michael O'Neill, Kathy Gately. Activation of the MACC1/PIM/cMyc axis confers resistance to PI3K/mTOR inhibition in PIK3CA mutant NSCLC [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr B14.

The PI3K-mTOR pathway is involved in regulating all hallmarks of cancer, and is often dysregulated in NSCLC, making it an attractive therapeutic target in this setting. Acquired resistance to PI3K-mTOR inhibition is a major hurdle to overcome in the success of PI3K-mTOR targeted agents. H460, A549, and H1975 resistant cells were generated by prolonged treatment in culture with Apitolisib (GDC-0980), a dual PI3K-mTOR inhibitor over a period of several months, from age-matched parent cells. Resistance was deemed to have developed when a log fold difference in IC50 had been achieved. Resistant cell lines also exhibited resistance to another widely investigated PI3K-mTOR dual inhibitor; Dactolisib (BEZ235). Cell lines were characterised at the level of mRNA (expression array profiling expression of >150 genes), miRNA (expression array profiling of 2100 miRNAs), protein (bottoms-up label-free mass spectrometry) and phosphoprotein (expression array profiling of 84 phospho/total proteins). Key alterations were validated by qPCR and Western blot. H1975 cells were initially most sensitive to Apitolisib (GDC-0980), but developed resistance more quickly than the other cell lines, perhaps due to increased selective pressure from the impressive initial effect. In-depth molecular profiling suggested epithelial-mesenchymal transition (EMT) may play a role in resistance to PI3K-mTOR dual inhibition in NSCLC.

The impact of host immunity on outcome in nonsmall cell lung cancer (NSCLC) is controversial. We examined the relationship between lymphoid infiltration patterns in NSCLC and prognosis.Tumour- and stroma-infiltrating CD3+, CD8+ and forkhead box P3 (Foxp3)+ T-lymphocytes were identified using immunohistochemistry and a novel image analysis algorithm to assess total, cytotoxic and regulatory T-lymphocyte counts, respectively, in 196 NSCLC cases. The median cell count was selected as a cut-point to define patient subgroups and the ratio of the corresponding tumour islet:stroma (TI/S) counts was determined.There was a positive association between overall survival and increased CD8+ TI/S ratio (hazard ratio (HR) for death 0.44, p+ TI/S ratio and overall survival (HR 4.86, p+ islet (HR 0.48, p+ stromal (HR 0.23, p+ and CD8+ stromal counts and high Foxp3+ islet infiltration conferred a worse survival (HR 1.55, 2.19 and 3.14, respectively). By multivariate analysis, a high CD8+ TI/S ratio conferred an improved survival (HR 0.48, p=0.002) but a high Foxp3+ TI/S ratio was associated with worse survival (HR 3.91, pMicrolocalisation of infiltrating T-lymphocytes is a powerful predictor of outcome in resected NSCLC.

The insulin-like growth factor 1 receptor (IGF1R) pathway plays an important role in the pathogenesis of non-small cell lung cancer (NSCLC) and also provides a mechanism of resistance to targeted therapies. IGF1R is therefore an ideal therapeutic target and several inhibitors have entered clinical trials. However, thus far the response to these inhibitors has been poor, highlighting the importance of predictive biomarkers to identify patient cohorts who will benefit from these targeted agents. It is well-documented that mutations and/or deletions in the epidermal growth factor receptor (EGFR) tyrosine kinase (TK) domain predict sensitivity of NSCLC patients to EGFR TK inhibitors. Single-nucleotide polymorphisms (SNPs) in the IGF pathway have been associated with disease, including breast and prostate cancer. The aim of the present study was to elucidate whether the IGF1R TK domain harbours SNPs, somatic mutations or deletions in NSCLC patients and correlates the mutation status to patient clinicopathological data and prognosis. Initially 100 NSCLC patients were screened for mutations/deletions in the IGF1R TK domain (exons 16–21) by sequencing analysis. Following the identification of SNP rs2229765, a further 98 NSCLC patients and 866 healthy disease-free control patients were genotyped using an SNP assay. The synonymous SNP (rs2229765) was the only aberrant base change identified in the IGF1R TK domain of 100 NSCLC patients initially analysed. SNP rs2229765 was detected in exon 16 and was found to have no significant association between IGF1R expression and survival. The GA genotype was identified in 53.5 and 49.4% of NSCLC patients and control individuals, respectively. No significant difference was found in the genotype (P=0.5487) or allele (P=0.9082) frequencies between the case and control group. The present findings indicate that in contrast to the EGFR TK domain, the IGF1R TK domain is not frequently mutated in NSCLC patients. The synonymous SNP (rs2229765) had no significant association between IGF1R expression and survival in the cohort of NSCLC patients.

Lung cancer is the second most common type of cancer in the world and is the most common cause of cancer-related death in both men and women. Research into causes, prevention and treatment of lung cancer is ongoing and much progress has been made recently in these areas, however survival rates have not significantly improved. Therefore, it is essential to develop biomarkers for early diagnosis of lung cancer, prediction of metastasis and evaluation of treatment efficiency, as well as using these molecules to provide some understanding about tumour biology and translate highly promising findings in basic science research to clinical application. In this investigation, two-dimensional difference gel electrophoresis and mass spectrometry were initially used to analyse conditioned media from a panel of lung cancer and normal bronchial epithelial cell lines. Significant proteins were identified with heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1), pyruvate kinase M2 isoform (PKM2), Hsc-70 interacting protein and lactate dehydrogenase A (LDHA) selected for analysis in serum from healthy individuals and lung cancer patients. hnRNPA2B1, PKM2 and LDHA were found to be statistically significant in all comparisons. Tissue analysis and knockdown of hnRNPA2B1 using siRNA subsequently demonstrated both the overexpression and potential role for this molecule in lung tumorigenesis. The data presented highlights a number of in vitro derived candidate biomarkers subsequently verified in patient samples and also provides some insight into their roles in the complex intracellular mechanisms associated with tumour progression.

In this study, we developed an image analysis algorithm for quantification of two potential apoptotic biomarkers in non-small-cell lung cancer (NSCLC): FLIP and procaspase-8. Immunohistochemical expression of FLIP and procaspase-8 in 184 NSCLC tumors were assessed. Individual patient cores were segmented and classified as tumor and stroma using the Definiens Tissue Studio. Subsequently, chromogenic expression of each biomarker was measured separately in the nucleus and cytoplasm and reported as a quantitative histological score. The software package pROC was applied to define biomarker thresholds. Cox proportional hazards analysis was applied to generate hazard ratios (HRs) and associated 95% CI for survival. High cytoplasmic expression of tumoral (but not stromal) FLIP was associated with a 2.5-fold increased risk of death in lung adenocarcinoma patients, even when adjusted for known confounders (HR 2.47, 95% CI 1.14–5.35). Neither nuclear nor cytoplasmic tumoral procaspase-8 expression was associated with overall survival in lung adenocarcinoma patients; however, there was a significant trend (P for trend=0.03) for patients with adenocarcinomas with both high cytoplasmic FLIP and high cytoplasmic procaspase-8 to have a multiplicative increased risk of death. Notably, high stromal nuclear procaspase-8 expression was associated with a reduced risk of death in lung adenocarcinoma patients (adjusted HR 0.31, 95% CI 0.15–0.66). On further examination, the cells with high nuclear procaspase-8 were found to be of lymphoid origin, suggesting that the better prognosis of patients with tumors with high stromal nuclear procaspase-8 is related to immune infiltration, a known favorable prognostic factor. No significant associations were detected in analysis of lung squamous cell carcinoma patients. Our results suggest that cytoplasmic expression of FLIP in the tumor and nuclear expression of procaspase-8 in the stroma are prognostically relevant in non-small-cell adenocarcinomas but not in squamous cell carcinomas of the lung.

Background Trastuzumab is an anti-HER2 monoclonal antibody (mAb) therapy capable of antibody-dependent cell-mediated cytotoxicity (ADCC) and used in the treatment of HER2+ breast cancer. Through interactions with FcƴR+ immune cell subsets, trastuzumab functions as a passive immunotherapy. The EGFR/HER2-targeting tyrosine kinase inhibitor (TKI) lapatinib and the next generation TKIs afatinib and neratinib, can alter HER2 levels, potentially modulating the ADCC response to trastuzumab. Using LDH-release assays, we investigated the impact of antigen modulation, assay duration and peripheral blood mononuclear cell (PBMC) activity on trastuzumab-mediated ADCC in breast cancer models of maximal (SKBR3) and minimal (MCF-7) target antigen expression to determine if modulating the ADCC response to trastuzumab using TKIs may be a viable approach for enhancing tumor immune reactivity. Methods HER2 levels were determined in lapatinib, afatinib and neratinib-treated SKBR3 and MCF-7 using high content analysis (HCA). Trastuzumab-mediated ADCC was assessed following treatment with TKIs utilising a colorimetric LDH release-based protocol at 4 and 12 h timepoints. PBMC activity was assessed against non-MHC-restricted K562 cells. A flow cytometry-based method (CFSE/7-AAD) was also used to measure trastuzumab-mediated ADCC in medium-treated SKBR3 and MCF-7. Results HER2 antigen levels were significantly altered by the three TKIs in both cell line models. The TKIs significantly reduced LDH levels directly in SKBR3 cells but not MCF-7. Lapatinib and neratinib augment trastuzumab-related ADCC in SKBR3 but the effect was not consistent with antigen expression levels and was dependent on volunteer PBMC activity (vs. K562). A 12 h assay timepoint produced more consistent results. Trastuzumab-mediated ADCC (PBMC:target cell ratio of 10:1) was measured at 7.6 ± 4.7% (T12) by LDH assay and 19 ± 3.2 % (T12) using the flow cytometry-based method in the antigen-low model MCF-7. Conclusions In the presence of effector cells with high cytotoxic capacity, TKIs have the ability to augment the passive immunotherapeutic potential of trastuzumab in SKBR3, a model of HER2+ breast cancer. ADCC levels detected by LDH release assays are extremely low in MCF-7; the flow cytometry-based CFSE/7-AAD method is more sensitive and consistent for the determination of ADCC in HER2-low models.

Evaluation of protein and metabolite expression patterns in blood using mass spectrometry and high-throughput antibody-based screening platforms has potential for the discovery of new biomarkers for managing breast cancer patient treatment. Previously identified blood-based breast cancer biomarkers, including cancer antigen 15.3 (CA15-3) are useful in combination with imaging (computed tomography scans, magnetic resonance imaging, X-rays) and physical examination for monitoring tumour burden in advanced breast cancer patients. However, these biomarkers suffer from insufficient levels of accuracy and with new therapies available for the treatment of breast cancer, there is an urgent need for reliable, non-invasive biomarkers that measure tumour burden with high sensitivity and specificity so as to provide early warning of the need to switch to an alternative treatment. The aim of this study was to identify a biomarker signature of tumour burden using cancer and non-cancer (healthy controls/non-malignant breast disease) patient samples. Results demonstrate that combinations of three candidate biomarkers from Glutamate, 12-Hydroxyeicosatetraenoic acid, Beta-hydroxybutyrate, Factor V and Matrix metalloproteinase-1 with CA15-3, an established biomarker for breast cancer, were found to mirror tumour burden, with AUC values ranging from 0.71 to 0.98 when comparing non-malignant breast disease to the different stages of breast cancer. Further validation of these biomarker panels could potentially facilitate the management of breast cancer patients, especially to assess changes in tumour burden in combination with imaging and physical examination.

Chemoresistance is a major therapeutic challenge to overcome in NSCLC, in order to improve the current survival rates of

Background: Thromboxane synthase (TXS) metabolizes prostaglandin H2 into thromboxanes, which are biologically active on cancer cells. TXS over-expression has been reported in a range of cancers, and associated with angiogenesis and poor outcome. TXS has been identified as a potential therapeutic target in NSCLC. This study examines a link between TXS expression, angiogenesis, and survival in NSCLC. Methods: TXS and VEGF metabolite levels were measured in NSCLC serum samples (n=46) by EIA. TXB2 levels were correlated with VEGF. A 204-patient TMA was stained for TXS, VEGF, and CD-31 expression. Expression was correlated with a range of clinical parameters, including overall survival. TXS expression was correlated with VEGF and CD-31. Stable TXS clones were generated and the effect of overexpression on tumor growth and angiogenesis markers was examined in-vitro and in-vivo (xenograft mouse model). Results: Serum TXB2 levels were correlated with VEGF (p

Research studies aimed at advancing cancer prevention, diagnosis, and treatment depend on a number of key resources, including a ready supply of high-quality annotated biospecimens from diverse ethnic populations that can be used to test new drugs, assess the validity of prognostic biomarkers, and develop tailor-made therapies. In November 2011, KHCCBIO was established at the King Hussein Cancer Center (KHCC) with the support of Seventh Framework Programme (FP7) funding from the European Union (khccbio.khcc.jo). KHCCBIO was developed for the purpose of achieving an ISO accredited cancer biobank through the collection, processing, and preservation of high-quality, clinically annotated biospecimens from consenting cancer patients, making it the first cancer biobank of its kind in Jordan. The establishment of a state-of-the-art, standardized biospecimen repository of matched normal and lung tumor tissue, in addition to blood components such as serum, plasma, and white blood cells, was achieved through the support and experience of its European partners, Trinity College Dublin, Biostór Ireland, and accelopment AG. To date, KHCCBIO along with its partners, have worked closely in establishing an ISO Quality Management System (QMS) under which the biobank will operate. A Quality Policy Manual, Validation, and Training plan have been developed in addition to the development of standard operating procedures (SOPs) for consenting policies on ethical issues, data privacy, confidentiality, and biobanking bylaws. SOPs have also been drafted according to best international practices and implemented for the donation, procurement, processing, testing, preservation, storage, and distribution of tissues and blood samples from lung cancer patients, which will form the basis for the procurement of other cancer types. KHCCBIO will be the first ISO accredited cancer biobank from a diverse ethnic Middle Eastern and North African population. It will provide a unique and valuable resource of high-quality human biospecimens and anonymized clinicopathological data to the cancer research communities world-wide.

Recent experimental and biomarker evidence indicates that the epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor 1 (IGF1R) interact in the pathogenesis of malignant epithelial tumors, including lung cancer. This study examines the expression of both receptors and their prognostic significance in surgically resected non-small-cell lung cancer (NSCLC).EGFR and IGF1R expression were evaluated in 184 patients with NSCLC (83 squamous cell carcinomas [SCCs], 83 adenocarcinomas [ADCs], and 18 other types) using immunohistochemical (IHC) analysis. Expression of both receptors was examined in matched fresh frozen normal and tumor tissues from 40 patients with NSCLC (20 SCCs and 20 ADCs) by Western blot analysis.High EGFR expression was detected in 51% of patients, and SCCs had higher EGFR expression than did non-SCCs (57.4% vs. 42.5%; P = .028). High IGF1R expression was observed in 53.8% of patients, with SCC having higher expression than non-SCC (62.6% vs. 37.3%; P = .0004). A significant association was shown between EGFR and IGF1R protein overexpression (P.005). Patients with high expression of both receptors had a poorer overall survival (OS) (P = .04). Higher EGFR and IGF1R expression was detected in resected tumors relative to matched normal tissues (P = .0004 and P = .0009), with SCC having higher expression levels than ADC.Our findings indicate a close interrelationship between EGFR and IGF1R. Coexpression of both receptors correlates with poor survival. This subset of patients may benefit from treatments cotargeting EGFR and IGF1R.

Introduction Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality globally, having a 5 year survival rate of less than 15%. PI3K-mTOR signalling has been implicated in various hallmarks of cancer and is frequently dysregulated in NSCLC. Efforts to therapeutically target the PI3K-mTOR pathway have been hampered by the inevitable emergence of drug resistance inhibiting a durable response to treatment. Our group developed NSCLC cell line models of acquired resistance to PI3K-mTOR inhibitor GDC-0980. Resistant cells (H1975GR) were also less sensitive to PI3K-mTOR dual targeting inhibitor, BEZ235 compared to matched parent cells (H1975P) making them an ideal model to identify and interrogate drug resistance mechanisims. Material and methods The sensitivity of GDC-0980 resistant cells (H1975GR) versus age-matched parent cells (H1975P) to BEZ235 following a 72 hour treatment was compared using a Cell Titre Blue cell viability assay (n=3). Alterations to the mRNA expression profile of H1975GR versus H1975P were examined using an IL-6/STAT3 signalling-specific RT2 gene profiler array (n=1). Selected genes from the array were validated by SYBR-based qPCR, immunofluorescence (IF) and western blot analysis (n=3–4). 11 miRNAs (regulated by or regulators of c-Myc/PIM) plus housekeeping control miRNAs were quantified in the H1975P/GR model by QPCR. IC50 values of pan-PIM kinase inhibitors AZD1208, IBL101 and novel PI3K/mTOR/PIM inhibitor IBL301 inhibiting growth in H1975P versus H1975GR were compared using the BrdU assay. The effect of these drugs on RTK and c-Myc expression were examined in H1975GR by IF and western blot analysis. Results and discussions In-depth characterisation of the H1975GR cells identified activation of several receptor tyrosine kinases (RTKs) including EGFR, IGF1R, EPHA2 and members of the IL-6/STAT3 pathway including c-Myc and PIM1, PIM2 and PIM3 kinases. IL-6/STAT3 overexpression in cancer stimulates angiogenesis, migration, invasiveness, cytokine signalling and notably drug resistance. A miRNA signature of PI3K/mTOR inhibitor resistance was validated which can be used to monitor patients on treatment. IC50 values of IBL301 were significantly less for H1975GR cells (0.02 uM) compared to H1975P cells (0.75 uM) highlighting activated PIM kinase as a mechanism of resistance to PI3K-mTOR inhibition. Targeting PIM kinase in H1975GR cells inhibits activated c-Myc and RTKs. Conclusion Dual targeting of both PIM and PI3K-mTOR may provide a more durable response to treatment for patients with NSCLC.

Introduction: Despite advances in NSCLC therapeutics, including small molecule and antibody based targeted therapies, the majority of patients with advanced disease will ultimately relapse. Lung cancer is characterized by genomic instability leading to tissue invasion, metastasis and resistance to systemic treatments. hSSB1 has a key role in the detection and repair of DNA damage (double- strand breaks, replication fork arrest and oxidative stress). Recently we demonstrated that hSSB1 is phosphorylated by DNA-PK in response to replication stress to promote cellular survival. Here we establish that hSSB1 is a prognostic factor for NSCLC a potential novel target for therapy. Methods: We analyzed the prognostic significance of hSSB1 mRNA expression from public on line databases and from protein expression in an NSCLC tissue macro-array (TMA) through hSSB1 immunohistochemistry. hSSB1 mRNA levels were analyzed in matched normal, tumour adenocarcinoma and squamous cell tumour samples. We explored the impact of hSSB1 expression on NSCLC cell line proliferation by depleting hSSB1 with specific small interfering (si)RNA. We also measured the impact of hSSB1 inhibition on NSCLC cells proliferation with the use of a DNA-PK inhibitor and a novel agent, DKLS02. Results: hSSB1 expression was associated with poor prognosis for lung cancer, high levels of mRNA and protein expression correlating with a worse overall survival. We also observed that hSSB1 depletion leads to increased lung cancer cell death. The same result was observed when inhibiting hSSB1 by treating lung cancer cells with a DNA-PK inhibitor. Targeting hSSB1 with DKLS02 induces cell death in vitro and inhibits tumour growth in vivo in platinum sensitive and resistant tumours. Conclusion: Our results indicate that hSSB1 is a prognostic factor in NSCLC. Moreover, targeting hSSB1 may prove to be an effective therapeutic strategy for the treatment of chemosenstive and resistant NSCLC in the future. Disclosure: All authors have declared no conflicts of interest.

Introduction: Lung cancer is the leading cause of all cancer death worldwide. It is characterized by genomic instability leading to tissue invasion, metastasis and resistance to chemotherapy, notably cisplatin. Being the protein guardian of the genome, hSSB1 has a key role in the detection and repair of DNA damage (double-strand breaks, replication fork arrest and oxidative stress). Recently we have shown that hSSB1 is phosphorylated by DNA-PK at serine residue 134 in response to replication stress to promote cellular survival. We wanted to check is hSSB1 could be involved in the mechanism of non-small cell lung cancer (NSCLC) proliferation, establishing hSSB1 not only as a novel prognostic factor for NSCLC but also as a potential target for a new therapy strategy. Methods: We analyzed the prognostic significance of hSSB1 mRNA expression from public on line database and from protein expression in an NSCLC tissue macro-array (TMA) through hSSB1 immunohistochemistry staining. hSSB1 mRNA levels were analyzed in matched normal, tumour adenocarcinoma and squamous cell tumour samples. We explored the impact of hSSB1 expression on NSCLC cell lines proliferation by depleting hSSB1 with specific small interfering (si)RNA. We also measured the impact of hSSB1 inhibition on NSCLC cells proliferation with the use of a DNA-PK inhibitor. Results: hSSB1 expression was associated with poor prognosis for lung cancer, high levels of mRNA and protein expression correlating with a worse overall survival. We also observed that hSSB1 depletion leads to increased lung cancer cell death. The same result was observed when inhibiting hSSB1 by treating lung cancer cells with a DNA-PK inhibitor. Conclusion: Our results set hSSB1 as a prognostic factor in non-small cell lung cancer. Moreover, targeting hSSB1 proved to be effective to stop lung cancer cells proliferation, showing the potential as a new treatment for NSCLC. Disclosure: All authors have declared no conflicts of interest.

* Article is free to read on publisher website. Abstract Background Ras-related nuclear protein (Ran) is required for cancer cell survival in vitro and human cancer progression, but the molecular mechanisms are largely unknown. Methods We investigated the effect of the v-myc myelocytomatosis viral oncogene homolog (Myc) on Ran expression by Western blot, chromatin immunoprecipitation, and luciferase reporter assays and the effects of Myc and Ran expression in cancer cells by soft-agar, cell adhesion, and invasion assays. The correlation between Myc and Ran and the association with patient survival were investigated in 14 independent patient cohorts (n = 2430) and analyzed with Spearman's rank correlation and Kaplan-Meier plots coupled with Wilcoxon-Gehan tests, respectively. All statistical tests were two-sided. Results Myc binds to the upstream sequence of Ran and transactivates Ran promoter activity. Overexpression of Myc upregulates Ran expression, whereas knockdown of Myc downregulates Ran expression. Myc or Ran overexpression in breast cancer cells is associated with cancer progression and metastasis. Knockdown of Ran reverses the effect induced by Myc overexpression in breast cancer cells. In clinical data, a positive association between Myc and Ran expression was revealed in 288 breast cancer and 102 lung cancer specimens. Moreover, Ran expression levels differentiate better or poorer survival in Myc overexpressing breast (χ2 = 24.1; relative risk [RR] = 9.1, 95% confidence interval [CI] = 3.3 to 24.7, P

1 ISSN 1758-1966 10.2217/LMT.12.51 © 2013 Future Medicine Ltd Lung Cancer Manage. (2013) 2(1), 1–4 Lung cancer, in particular non-smallcell lung cancer (NSCLC), is the most common cause of cancer-related death in the world [1]. Rates of lung cancer will continue to rise owing to high levels of cigarette consumption in Asia and the developing world, and aging populations. Despite increased surgical resection rates, optimization of chemoradiotherapy and the introduction of targeted therapies, the treatment outcome for NSCLC remains poor [1]. The current standard of care for the majority of patients suitable for therapy involves treatment with a platinum-based chemotherapy regimen [1]. However, only a proportion of patients benefit and the remainder develop resistance to platinum-based therapy. Ongoing research aims to understand both the molecular mechanisms underpinning this resistance and identify agents or therapies that could sensitize or even resensitize patients to cisplatin-based therapeutic regimens. Targeting epigenetic mechanisms may represent one such approach. At its simplest, epigenetics can be described as stable and heritable changes in gene expression that are not due to changes in the primary DNA sequence, and is most commonly used to describe chromatinbased regulation of gene expression [1,2]. The main mechanisms used involve the laying down of modifications to DNA and histones, that are then utilized by various chromatin-modifying enzymes which can be categorized as ‘readers’, ‘writers’ and ‘erasers’ [2]. More recently, miRNAs, a form of noncoding RNA, have also been shown to affect epigenetic regulation by directly targeting the epigenetic machinery, leading them to be described as ‘epi-miRNAs’ [1]. Aberrant epigenetic regulation is now well established as a frequent event in NSCLC, with all known forms of epigenetic regulation playing a role [1]. This ‘epigenetic landscape’ can potentially be targeted and may have important implications for the management and treatment of NSCLC [1,2]. Critically, an emerging body of evidence now links some of these epigenetic events with cisplatin resistance.

Background Baicalein is a widely used Chinese herbal medicine derived from Scutellaria baicalenesis, which has been traditionally used as anti-inflammatory and anti-cancer therapy. In this study we examined the anti-tumour pathways activated following baicalein treatment in non-small cell lung cancer (NSCLC), both in-vitro and in-vivo. Methods The effect of baicalein treatment on H-460 cells in-vitro was assessed using both BrdU assay (cell proliferation) and High Content Screening (multi-parameter apoptosis assay). A xenograft nude mouse model was subsequently established using these cells and the effect of baicalein on tumour growth and survival assessed in-vivo. Tumours were harvested from these mice and histological tissue analysis carried out. VEGF, 12-lipoxygenase and microvessel density (CD-31) were assessed by immunohistochemistry (IHC), while H and E staining was carried out to assess mitotic index. Gene expression profiling was carried out on corresponding RNA samples using Human Cancer Pathway Finder Arrays and qRT-PCR, with further gene expression analysis carried out using qRT-PCR. Results Baicalein significantly decreased lung cancer proliferation in H-460 cells in a dose dependent manner. At the functional level, a dose-dependent induction in apoptosis associated with decreased cellular f-actin content, an increase in nuclear condensation and an increase in mitochondrial mass potential was observed. Orthotopic treatment of experimental H-460 tumours in athymic nude mice with baicalein significantly (p

Apoptosis is a highly regulated cellular process that functions to remove undesired cells from multicellular organisms. This pathway is often disrupted in cancer, providing tumours with a mechanism to avoid cell death and promote growth and survival. The putative tumour suppressor, SASH1 (SAM and SH3 domain containing protein 1), has been previously implicated in the regulation of apoptosis; however, the molecular role of SASH1 in this process is still unclear. In this study, we demonstrate that SASH1 is cleaved by caspase-3 following UVC-induced apoptosis. Proteolysis of SASH1 enables the C-terminal fragment to translocate from the cytoplasm to the nucleus where it associates with chromatin. The overexpression of wild-type SASH1 or a cleaved form of SASH1 representing amino acids 231–1247 leads to an increase in apoptosis. Conversely, mutation of the SASH1 cleavage site inhibits nuclear translocation and prevents the initiation of apoptosis. SASH1 cleavage is also required for the efficient translocation of the transcription factor nuclear factor-κB (NF-κB) to the nucleus. The use of the NF-κB inhibitor DHMEQ demonstrated that the effect of SASH1 on apoptosis was dependent on NF-κB, indicating a codependence between SASH1 and NF-κB for this process.

Few patients diagnosed with lung cancer are still alive 5 years after diagnosis. The aim of the current study was to conduct a 10-year review of a consecutive series of patients undergoing curative-intent surgical resection at the largest tertiary referral centre to identify prognostic factors.Case records of all patients operated on for lung cancer between 1998 and 2008 were reviewed. The clinical features and outcomes of all patients with non-small cell lung cancer (NSCLC) stage I-IV were recorded.A total of 654 patients underwent surgical resection with curative intent during the study period. Median overall survival for the entire cohort was 37 months. The median age at operation was 66 years, with males accounting for 62.7 %. Squamous cell type was the most common histological subtype, and lobectomies were performed in 76.5 % of surgical resections. Pneumonectomy rates decreased significantly in the latter half of the study (25 vs. 16.3 %), while sub-anatomical resection more than doubled (2 vs. 5 %) (p0.005). Clinico-pathological characteristics associated with improved survival by univariate analysis include younger age, female sex, smaller tumour size, smoking status, lobectomy, lower T and N status and less advanced pathological stage. Age, gender, smoking status and tumour size, as well as T and N descriptors have emerged as independent prognostic factors by multivariate analysis.We identified several factors that predicted outcome for NSCLC patients undergoing curative-intent surgical resection. Survival rates in our series are comparable to those reported from other thoracic surgery centres.