WISP-3 promotes angiogenesis in non-small cell lung cancer through p38/JNK-c-Jun-mediated PDGF-A upregulation.

Three well-defined laminar flow profiles were created to distinguish the influence of a gradient in shear and steady shear on platelet-derived growth factor A (PDGF-A) and monocyte chemoattractant protein-1 (MCP-1) expression in human endothelial cells. The flow profiles (16 dyne/cm2 maximum shear stress) were ramp flow (shear stress smoothly transited at flow onset), step flow (shear stress abruptly applied at flow onset), and impulse flow (shear stress abruptly applied for 3 s only). Ramp flow induced only minor expression of PDGF-A and did not increase MCP-1 expression. Step flow increased PDGF-A and MCP-1 mRNA levels 3- and 2-fold at 1.5 hours, respectively, relative to ramp flow. In contrast, impulse flow increased PDGF-A and MCP-1 expression 6- and 7-fold at 1.5 hours, and these high levels were sustained for at least 4 hours. These results indicate that a temporal gradient in shear (impulse flow and the onset of step flow) and steady shear (ramp flow and the steady component of step flow) stimulates and diminishes the expression of PDGF-A and MCP-1, respectively. NO synthase inhibitor NG-amino-L-arginine (L-NAA) was found to markedly enhance MCP-1 and PDGF-A expression induced by step flow, but decrease their expression induced by impulse flow, in a dose-dependent manner. NO donor spermine-NONOate (SPR/NO) dose-dependently reduced the MCP-1 and PDGF-A expression induced by impulse flow. Moreover, impulse flow was found to stimulate sustained (4 hours) I kappa B-alpha degradation and egr-1 mRNA induction. L-NAA prevented I kappa B-alpha degradation, whereas SPR/NO increased I kappa B-alpha resynthesis 2 hours after impulse flow. Both L-NAA and SPR/NO inhibited the impulse flow inducibility of egr-1 4 hours after the flow stimulation. The results show that both NO induced by steady shear and NO donor inhibit temporal gradient in shear-induced MCP-1 and PDGF-A expression by downregulation of their respective transcription factors NF kappa B and egr-1, whereas NO induced by impulse flow stimulates MCP-1 and PDGF-A expression by upregulation of the transcription factors. The above findings suggest distinct roles of temporal gradient in shear and steady shear in atherogenesis in vivo.

Platelet-derived growth factor A (PDGFA), the most known member of PDGF family, plays a crucial role in occurrence and progression of different tumors. However, PDGFA expression and its clinical significance in esophageal squamous cell carcinoma (ESCC) are not clear. The present study aimed to assess the expression and prognostic value of PDGFA in ESCC.The Gene Expression Omnibus databases (GSE53625, GSE23400, and GSE67269) and fresh clinical samples were employed for detecting PDGFA messenger RNA expression in ESCC. The associations of PDGFA expression with clinicopathological characteristics were evaluated by chi-square test. Kaplan–Meier analysis and Cox proportional hazard regression model were performed to determine the prognostic value of PDGFA in ESCC patients. PDGFA-related signaling pathways were defined by gene set enrichment analysis based on Gene Expression Omnibus databases.The PDGFA messenger RNA expression was upregulated in ESCC tissues compared with paired adjacent noncancerous tissues (P < .05) and was positively correlated with T stage (P < .05). Kaplan–Meier survival analysis suggested that ESCC patients with high PDGFA expression were associated with poorer overall survival compared to those with low PDGFA expression (P < .05), especially in advanced T stage (P < .05). Cox analyses showed that high expression of PDGFA was an independent predictor for poor prognosis in ESCC patients. Gene set enrichment analysis identified 3 signaling pathways (extracellular matrix receptor interaction, focal adhesion, and glycosaminoglycan biosynthesis chondroitin sulfate) that were enriched in PDGFA high expression phenotype (all P < .01).PDGFA may serve as an oncogene in ESCC and represent an independent molecular biomarker for prognosis of ESCC patients.

Prostate field cancerization denotes molecular alterations in histologically normal tissues adjacent to tumors. Such alterations include deregulated protein expression, as we have previously shown for the key transcription factor early growth response 1 (EGR-1) and the lipogenic enzyme fatty acid synthase (FAS). Here we add the two secreted factors macrophage inhibitory cytokine 1 (MIC-1) and platelet derived growth factor A (PDGF-A) to the growing list of protein markers of prostate field cancerization. Expression of MIC-1 and PDGF-A was measured quantitatively by immunofluorescence and comprehensively analyzed using two methods of signal capture and several groupings of data generated in human cancerous (n = 25), histologically normal adjacent (n = 22), and disease-free (n = 6) prostate tissues. A total of 208 digitized images were analyzed. MIC-1 and PDGF-A expression in tumor tissues were elevated 7.1x to 23.4x and 1.7x to 3.7x compared to disease-free tissues, respectively (p<0.0001 to p = 0.08 and p<0.01 to p = 0.23, respectively). In support of field cancerization, MIC-1 and PDGF-A expression in adjacent tissues were elevated 7.4x to 38.4x and 1.4x to 2.7x, respectively (p<0.0001 to p<0.05 and p<0.05 to p = 0.51, respectively). Also, MIC-1 and PDGF-A expression were similar in tumor and adjacent tissues (0.3x to 1.0x; p<0.001 to p = 0.98 for MIC-1; 0.9x to 2.6x; p<0.01 to p = 1.00 for PDGF-A). All analyses indicated a high level of inter- and intra-tissue heterogeneity across all types of tissues (mean coefficient of variation of 86.0%). Our data shows that MIC-1 and PDGF-A expression is elevated in both prostate tumors and structurally intact adjacent tissues when compared to disease-free specimens, defining field cancerization. These secreted factors could promote tumorigenesis in histologically normal tissues and lead to tumor multifocality. Among several clinical applications, they could also be exploited as indicators of disease in false negative biopsies, identify areas of repeat biopsy, and add molecular information to surgical margins.

BackgroundAntiangiogenetic therapy is one of the effective strategies for non-small cell lung cancer (NSCLC) treatment. Four-and-a-half LIM-domain protein 2 (FHL2) serves as a key function in cell growth and metastasis of multiple cancers, but the role of FHL2 in NSCLC angiogenesis has not been intensely examined.MethodsFHL2 expression in NSCLC tissues and cell lines and its correlation with patients prognosis were investigated by using The Cancer Genome Atlas (TCGA) database and quantitative polymerase chain reaction (qPCR). Cell Counting Kit-8 (CCK-8) assay, EdU (5-ethynyl-2'-deoxyuridine) assay, and a xenograft model were used to investigate the effects of FHL2 on NSCLC progression in vitro and in vivo. CCK-8, wound-healing, Transwell invasion, tube formation, and permeability assays were performed to determine the roles of FHL2 in angiogenesis and vascular permeability. Vascular endothelial growth factor A (VEGFA) enzyme-linked immunosorbent assay (ELISA) assay, Western blot analysis, and MK-2206 were used to investigate the specific mechanism mediated by FHL2.ResultsWe demonstrated that FHL2 was significantly upregulated in NSCLC tissues and cell lines and was associated with poor prognosis. FHL2 overexpression enhanced the cell viability of NSCLC cells, as well as the proliferation, migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs). In addition, we determined that FHL2 activated the AKT-mTOR signaling pathway in HUVECs by promoting VEGFA secretion from NSCLC cells, thereby inducing angiogenesis and vascular leakiness. We further confirmed that FHL2 also promoted NSCLC tumor growth in vivo.ConclusionsOur study revealed the role of FHL2 in NSCLC and the mechanism by which FHL2 promotes NSCLC tumorigenesis, providing novel insights into targeted therapy for NSCLC.

Platelet-derived growth factor A (PDGFA) has been shown to be upregulated in several tumors, contributing to their malignant phenotypes. However, its expression and function in head and neck squamous cell carcinoma (HNSC) are not clearly understood. Thus, we aimed to evaluate this issue using bioinformatic analyses and primary experimental validation. The expression of PDGFA was analyzed using popular bio-databases and further validated by RT-PCR and immunohistochemical staining. Survival analyses were then performed. The association between PDGFA expression levels and immune cell infiltration in the immune microenvironment was assessed. PDGFA has been found to be significantly upregulated in a variety of cancers, including HNSC, and increased PDGFA expression may be an independent prognostic factor associated with immune cell infiltration in HNSC. Overexpression of PDGFA in HNSC is significantly associated with poor prognosis and immune cell infiltration in the tumor microenvironment (TME). PDGFA has potential as a molecular indicator for diagnosis, prognosis, and immune processes in HNSC.

Dependence of fibroblast infiltration in tumor stroma on type IV collagen-initiated integrin signal through induction of platelet-derived growth factor

Pancreatic Ductal Adenocarcinoma (PDA) has one of the worst prognoses of all cancers. Mucin 1 (MUC1), a transmembrane mucin glycoprotein, is a key modulator of several signaling pathways that affect oncogenesis, motility, and metastasis. Its expression is known to be associated with poor prognosis in patients. However, the precise mechanism remains elusive. We report a novel association of MUC1 with Platelet-Derived Growth Factor-A (PDGFA). PDGFA is one of the many drivers of tumor growth, angiogenesis, and metastasis in PDA. Using mouse PDA models as well as human samples, we show clear evidence that MUC1 regulates the expression and secretion of PDGFA. This, in turn, influences proliferation and invasion of pancreatic cancer cells leading to higher tumor burden in vivo. In addition, we reveal that MUC1 over expressing cells are heavily dependent on PDGFA both for proliferation and invasion while MUC1-null cells are not. Moreover, PDGFA and MUC1 are critical for translocation of βcatenin to the nucleus for oncogenesis to ensue. Finally, we elucidate the underlying mechanism by which MUC1 regulates PDGFA expression and secretion in pancreatic cancer cells. We show that MUC1 associates with Hif1-α, a known transcription factor involved in controlling PDGFA expression. Furthermore, MUC1 facilitates Hif1-α translocation to the nucleus. In summary, we have demonstrated that MUC1-induced invasion and proliferation occurs via increased exogenous production of PDGFA. Thus, impeding MUC1 regulation of PDGFA signaling may be therapeutically beneficial for patients with PDA.

Pancreatic cancer remains one of the most deadly cancers with a grave prognosis. Despite continuous efforts to improve remedial values, limited progress has been made. We have reported that dual specificity phosphatase 28 (DUSP28) has a critical role of chemo-resistance and migration in pancreatic cancers. However, its mechanism remains unclear. Here, we further clarify the function of DUSP28 in pancreatic cancers. Analysis using a public microarray database and in vitro assay indicated a critical role of platelet derived growth factor A (PDGF-A) in pancreatic cancer malignancy. PDGF-A was positively regulated by DUSP28 expression at the mRNA and protein levels. Enhanced DUSP28 sensitized pancreatic cancer cells to exogenous PDGF-A treatment in migration, invasion, and proliferation. Transfection with siRNA targeting DUSP28 blunted the influence of administered PDGF-A by inhibition of phosphorylation of FAK, ERK1/2, and p38 signalling pathways. In addition, DUSP28 and PDGF-A formed an acquired autonomous autocrine-signaling pathway. Furthermore, targeting DUSP28 inhibited the tumor growth and migratory features through the blockade of PDGF-A expression and intracellular signaling in vivo. Our results establish novel insight into DUSP28 and PDGF-A related autonomous signaling pathway in pancreatic cancer.

Background and Objectives A previous study of ours indicated that platelet-derived growth factor-A (PDGF-A) mRNA expression in biopsy specimens can identify a subgroup of high-risk gastric carcinoma patients, while clinicopathologic studies have shown that lymph node involvement is an important risk factor for predicting overall survival. To identify gastric carcinoma patients at high risk for recurrence, we assessed a two-step evaluation consisting of mRNA expression of tumor growth–related factors and the histopathologic findings. Methods The reverse transcriptase–polymerase chain reaction (RT-PCR) was used to assay the gene expression of PDGF-A and transforming growth factor-β1 (TGF-β1) in 69 gastric carcinoma endoscopic biopsy specimens (prospective cohort). The corresponding gastric carcinoma surgical specimens were classified histologically. Finally, the patients' survival curves were calculated. The relationships among the mRNA expression, histopathologic findings, and survival period were analyzed statistically. Results Nodal involvement correlated with PDGF-A and TGF-β1 mRNA expression in early and advanced carcinomas, respectively. Both PDGF-A mRNA and TGF-β1 mRNA expression were independent preoperative prognostic indicators in advanced cases. The ratio of involved nodes (n1) to total perigastric lymph nodes dissected (percentage of involved nodes) was the most independent postoperative prognostic indicator in advanced cases. Early carcinomas were divided preoperatively into two types. Advanced carcinomas were divided preoperatively into three. These were divided again postoperatively according to the percentage of involved nodes into high- and low-malignacy groups. Conclusions A two-step evaluation of the malignant potential of gastric carcinoma by a combination of preoperative evaluation for PDGF-A and TGF-β1 expression and postoperative pathologic examination would yield a more accurate prognosis for patients with gastric carcinoma. J. Surg. Oncol. 1999;72:142–149. © 1999 Wiley-Liss, Inc.

A previous study of ours indicated that platelet-derived growth factor-A (PDGF-A) mRNA expression in biopsy specimens can identify a subgroup of high-risk gastric carcinoma patients, while clinicopathologic studies have shown that lymph node involvement is an important risk factor for predicting overall survival. To identify gastric carcinoma patients at high risk for recurrence, we assessed a two-step evaluation consisting of mRNA expression of tumor growth-related factors and the histopathologic findings. The reverse transcriptase-polymerase chain reaction (RT-PCR) was used to assay the gene expression of PDGF-A and transforming growth factor-beta1 (TGF-beta1) in 69 gastric carcinoma endoscopic biopsy specimens (prospective cohort). The corresponding gastric carcinoma surgical specimens were classified histologically. Finally, the patients' survival curves were calculated. The relationships among the mRNA expression, histopathologic findings, and survival period were analyzed statistically. Nodal involvement correlated with PDGF-A and TGF-beta1 mRNA expression in early and advanced carcinomas, respectively. Both PDGF-A mRNA and TGF-beta1 mRNA expression were independent preoperative prognostic indicators in advanced cases. The ratio of involved nodes (n1) to total perigastric lymph nodes dissected (percentage of involved nodes) was the most independent postoperative prognostic indicator in advanced cases. Early carcinomas were divided preoperatively into two types. Advanced carcinomas were divided preoperatively into three. These were divided again postoperatively according to the percentage of involved nodes into high- and low-malignacy groups. A two-step evaluation of the malignant potential of gastric carcinoma by a combination of preoperative evaluation for PDGF-A and TGF-beta1 expression and postoperative pathologic examination would yield a more accurate prognosis for patients with gastric carcinoma.

It has been demonstrated that exosomes derived from HPV-16 E7-over-expressiong non-small cell lung cancer (NSCLC) cells (E7 Exo) trigger increased levels of epidermal growth factor receptor (EGFR) and miR-381-3p. The purpose of this investigation was to examine the role of E7 Exo in NSCLC angiogenesis, and to analyze the contribution of exosomal EGFR and miR-381-3p to it. The influence of E7 Exo on the proliferation and migration of human umbilical vein endothelial cells (HUVECs) was assessed using colony formation and transwell migration assays. Experiments on both cells and animal models were conducted to evaluate the angiogenic effect of E7 Exo treatment. The involvement of exosomal EGFR and miR-381-3p in NSCLC angiogenesis was further investigated through suppressing exosome release or EGFR activation, or by over-expressing miR-381-3p. Treatment with E7 Exo increased the proliferation, migration, and tube formation capacities of HUVECs, as well as angiogenesis in animal models. The suppression of exosome release or EGFR activation in NSCLC cells decreased the E7-induced enhancements in HUVEC migration and tube formation, and notably reduced vascular endothelial growth factor A (VEGFA) and Ang-1 levels. HUVECs that combined miR-381-3p mimic transfection and E7 Exo treatment exhibited a more significant tube-forming capacity than E7 Exo-treated HUVECs alone, but were reversed by the miR-381-3p inhibitor. The angiogenesis induced by HPV-16 E7 in NSCLC is mediated through exosomal EGFR and miR-381-3p.

Non-small cell lung cancer (NSCLC) has markedly increased morbidity and mortality rates worldwide. Circular RNAs were shown to regulate NSCLC progression. But the underlying pathways of the circRPPH1-mediated regulation of NSCLC still need further exploration. We evaluated circRPPH1 levels in NSCLC tissues and cell lines via qRT-PCR. Moreover, using ectopic plasmid incorporation and siRNA assays, we analysed the circRPPH1-mediated regulation of cell proliferation (CP), cell migration (CM) and cell invasion (CI) in NSCLC cell lines (H1975 and A549 cells), using CCK-8, colony forming, scratch wound and transwell assays, respectively. CircRPPH1 levels were remarkably high in the NSCLC tissues and cell lines. The transfection experiments showed that circRPPH1 overexpression was able to promote CP, CM and CI of NSCLC cells, while CP, CM and CI were significantly restrained by the knockdown of circRPPH1. We also displayed that circRPPH1 knockdown suppressed the cell progression via inactivating the PI3K/AKT and JAK2/STAT3 signalling axes. Subsequently, in vivo experiment in nude mice was demonstrated that the inhibition of circRPPH1 could reduce the tumour growth of NSCLC. circRPPH1 may accelerate the growth and metastasis of NSCLC, in culture conditions and in animal models, by stimulating the PI3K/AKT and JAK2/STAT3 signalling axes, thus promoting the development of NSCLC.

A small, international, closed-door conference on Novel Agents in the Treatment of Lung Cancer, held in Cambridge, Massachusetts, October 17–18, 2003, was convened to present and discuss findings from recent and ongoing trials of investigational drugs for the treatment of lung cancer. Invited participants from the Netherlands, Italy, Spain, and the United States presented new data regarding the role of molecularly targeted agents in the treatment of advanced lung cancer and discussed their significance for clinical care. The conference format combined brief presentations with extended periods of open discussion. The conclusions reached over the course of the 2-day conference are summarized briefly below and presented at greater length in the individual papers and accompanying discussions that comprise the conference proceedings.Over the past decade we have witnessed a dramatic increase in our understanding of lung cancer biology. This greater knowledge has led to the identification of new therapeutic targets as well as the development of innovative preclinical model systems. Over the past year we have seen the fruit of this effort in the discovery of pemetrexed, gefitinib, and erlotinib as active agents in lung cancer. For the most part, these agents have modest activity when used as single agents in patients with previously treated lung cancer. Preclinical modeling of these agents and drugs such as the antisense molecule to protein kinase C (Affinitac) predicted that they would have synergy with chemotherapy. A major disappointment has been the failure of these novel agents to improve survival when added to standard chemotherapy, calling into question the validity of the preclinical model systems.Much discussion focused on understanding the reasons why so many compounds that appeared promising in preclinical and early-phase clinical studies did not fulfill that promise when taken to large-scale randomized trials. Participants identified several key issues that will need to be addressed in the investigation of other novel compounds still in early development, primarily concerning the selection, interpretation, and reporting of preclinical studies and the design and interpretation of Phase I/II studies. Fundamentally, participants felt that industry has moved too precipitously to bring novel compounds into Phase III clinical trials in a competitive push to be the first with a new class of agents. It is understandable that we are impatient with advanced lung cancer. In its advanced stages, this is a disease that is uniformly fatal with a median survival of 9 months. This accelerated advance to Phase III clinical studies without better understanding of who may benefit from a given novel agent has had the support of regulatory authorities and academic researchers anxious to find effective regimens. A major concern raised by participants in the conference was that the recent pattern of negative Phase III trials threatens the future of the field, if companies and other investors in research decide to reallocate resources to disease conditions with a higher likelihood of successful outcomes.The success rate in proceeding from the preclinical model to the patient in the clinic is markedly lower for oncology compared with other indications. The predictive value of preclinical studies needs to be improved. One issue identified by conference participants is the importance of defining the target for the novel molecular agents and demonstrating (a) that the target is relevant and (b) that the agent in fact has a clinically meaningful impact on the target. Cell lines and xenografts will continue to provide necessary but not sufficient data. At the conference the potential role of genetically engineered mouse models was explored, and great hope was expressed that in the future these might be able to better predict efficacy of novel treatments.In discussing the generally poor predictive value of the preclinical models, conference participants concluded that there is an obvious need not only for better models but also for better utilization of the existing models. Although not optimal, the available cell line and xenograft models provide useful information if they are rationally used and rigorously interpreted. In the past, positive results in selected cell lines have been presented without reporting whether other cell lines were also run and found negative. Many felt strongly that multiple cell lines should be looked at and the full data set should be used to influence the go/no go decision in clinical development. Positive results should be confirmed in different cell lines or different models before proceeding to clinical trial, and all data should be reported with SDs given.In addition to validating target, preclinical models should be used to explore schedule and dose questions. Early use and more thorough interpretation of preclinical pharmacokinetic and pharmacodynamic data might help to design Phase II trials that are more likely to be representative. Conference participants called for more complete and consistent reporting of preclinical data, with an end to the common practice of selective presentation of preclinical data to justify planned clinical trials. The standard for data reporting should be as stringent for preclinical as for clinical studies, with all responses and nonresponses reported.As with the preclinical studies, early-stage clinical trials should include more thorough analysis of clinical data, such as distinguishing the subsets of patients who did or did not respond to the investigational agent. While recognizing the inherent difficulty in obtaining tissue from lung cancer patients, the group felt that, wherever possible, study designs should include at least baseline and posttreatment biopsies to assess or confirm the agent’s efficacy against the molecular target and the tumor itself. Even if the potential targets are not well “flushed out” at the time of the study, tumor tissue should be archived so that it can be looked at in the future because newer technology might clarify potential mechanisms of response or resistance.A number of novel agents that appeared promising in Phase II studies, even those with single-agent activity, have proved disappointing when taken to larger randomized trials. To avoid expending resources in unproductive large trials, statistical models should be used to determine what type of early-phase data (response rate, time to progression, or other surrogate end points), with what magnitude of apparent effect, would be predictive of positive outcomes in larger randomized Phase III trials. Because the typical Phase II trial only enrolls up to 40–50 patients, the duration of follow-up and the number of events must be adequate to allow interpretation. Both Phase I and Phase II trials need to be better powered. Phase I trials should include a pharmacodynamic marker whenever possible to verify that the drug (and the dosing regimen) is in fact hitting the putative target.Clinical studies evaluating novel agents have been designed primarily to gain regulatory approval for an agent. Two study designs have predominated: (a) using the investigational agent as monotherapy and comparing this with standard chemotherapy; or (b) combining the novel agent with a standard chemotherapy regimen to produce a doublet or triplet. Whereas these are certainly reasonable approaches, new designs for Phase II trials should be considered, including adjuvant and preoperative studies, where tumor samples taken from a small number of patients before and after a brief neoadjuvant course of treatment may aid in determining whether the agent has a detectable impact on the tumor. In this setting, multicenter trials that give priority to the collection and analysis of surgical samples may be of service in developing a better understanding of what is happening at a molecular level and in correlating therapy response with population subgroups.There was much discussion about the merits of the randomized Phase II trial. Several conference participants argued in favor of randomized Phase II studies with an active control arm, whereas others cautioned against the risks of overinterpreting the data because the patient numbers are generally too low to permit confident interpretation of the results. Multiple arm early-phase trials were viewed as expending resources that should be reserved for Phase III trials of agents that have established their potential. Participants agreed that there should be more emphasis on multicenter Phase II studies rather than single-institution studies, in which the patient population may not be representative. Conference participants also agreed that an investigational agent should demonstrate antitumor activity as monotherapy in Phase II before it is further evaluated as combination therapy in large Phase III trials. However, exceptions may be possible when novel agents are working primarily as sensitizers of chemotherapy or radiotherapy, and their major role is expected only in combination.Ultimately, better surrogate markers of antitumor activity are needed than clinical response rate or time to progression (an end point that is dependent on the frequency of follow-up). Early-phase trials should include efforts to validate both biomarkers of optimal drug dosing and surrogate markers of drug efficacy. Too many agents have gone on to Phase II/III trials without resolving these issues, and agents that failed in large trials in the general population might have shown efficacy if the dosing and the proper surrogate markers had been determined in the preliminary trials. In this regard, a biomarker that measures pharmacokinetics should not be substituted for a surrogate marker. The latter must be validated as correlating with both target-related changes within the tumor and clinical outcomes.In vivo imaging in humans may prove to be a powerful tool in elucidating the effects of the targeted therapies, and incorporation of imaging studies such as positron emission tomography or dynamic contrast magnetic resonance imaging in early-phase trials may also prove of utility in determining which agents should move forward to randomized trials. One recently developed study design treats all patients for a predetermined time, at which point all patients are imaged to identify response or lack of response, permitting an early determination of whether the drug has an effect on time to progression.Obtaining biopsy tissue is a difficult goal in lung cancer trials. Patients with metastatic lung cancer often have a diagnosis made by just a fine-needle aspiration. Often there is no archival tissue. In breast cancer, by contrast, the majority of patients have had the removal of a primary tumor, and paraffin samples are available. Only with great commitment will it be possible to obtain this tissue. Surrogate tissue is also a possibility. In early-phase trials it is feasible to biopsy surrogate tissue to look for markers of response. This will be much harder to coordinate in larger Phase III studies involving multiple sites of care.Participants were asked to determine the relevant benchmarks that should serve as standards by which to compare new treatments for stage IV non-small cell lung cancer (NSCLC). The group agreed that two distinct standards had evolved for interpreting a Phase III study as positive. For industry-sponsored multi-institution studies, the benchmark was a 10-month median survival with a 35% response rate and a 4.5-month median time to progression. In contrast, for cooperative group studies, the benchmark has been an 8-month median survival, 25% response rate, and a 4-month median time to progression. These differences may largely be explained by different patient characteristics and, in particular, by the entry of many stage III patients in drug company-sponsored studies as compared with cooperative group studies, in which the patient population is often limited to patients with mainly stage IV disease.In designing trials of the novel targeted agents, there is the issue of whether enrollment should be restricted to patients whose tumor expresses the target. In principle, the answer to this question should be yes. However, often we do not appreciate the relevant target. In a disease like chronic myelogenous leukemia, the presence of the bcr-abl is pathognomonic for the disease. In most solid tumors the genetic changes that produce malignancy are complex and incompletely understood. An agent that is highly effective for a small subset of patients may go unappreciated if that subset is not included in the clinical study. This strategy becomes problematic with agents such as the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, for which the mechanism of action is, at best, poorly understood. In this example, clinical efficacy has not correlated with expression of the presumed target. Ultimately, there is no simple answer to this question. When it is clear what the target of interest is (which is the exception in lung cancer), it makes sense to enrich the population for such patients. When it is not known, it makes more sense to enroll broadly, collect tumor tissue, and ask correlative questions in a post hoc manner.Novel targeted agents are not the only drugs for which we need to improve our ability to predict response. Platinum-based combination therapy only helps a minority of patients with advanced NSCLC. Understanding chemotherapy sensitivity and resistance is a major goal as well. The mRNA expression levels of several genes (ERCC1, RRM1 and XPD) in the nucleotide excision repair pathway are implicated in cisplatin resistance, and RRM1appears to be a marker for gemcitabine/cisplatin resistance. Trials to further evaluate the prognostic significance of RRM1mRNA should be done to test the hypothesis that patients with low levels of RRM1 will benefit from gemcitabine/platinum chemotherapy.Genomic polymorphisms are another potential predictive marker of response and toxicity to chemotherapy. Pharmacodynamics is a potentially powerful tool for determining the effects of therapy and the tumor characteristics of those likely to respond. Predictive markers of therapeutic response need to be carefully distinguished from prognostic factors. For example, in untreated patients, expression of some poor prognosis markers, such as cyclooxygenase (COX)-2, may increase in response to therapy and serve as a valid target.For a marker to be predictive of response, a given drug has to have activity. In lung cancer, erlotinib and gefitinib showed activity in recent studies, but predictive markers of response have not yet been identified, although several leads have been reported. In terms of predictive markers, mitogen-activated protein kinase has potential and should be more carefully studied. It needs to be demonstrated that phospho-specific antibodies accurately detect phosphorylated receptor if there is a delay in processing the tissue specimen. More uniform methods of collecting and analyzing tissue for predictive markers should be developed before the validity of this and other markers can be confirmed. Another potential mechanism, PTEN loss and resultant phosphatidylinositol 3′-kinase/Akt pathway constitutive activation, may be important in mediating resistance to gefitinib. Given the redundancy of the pathways, it is likely that several markers, rather than one, may need to be elucidated.The participants were in agreement with regard to the current status of chemotherapy for metastatic NSCLC. All agreed that in metastatic disease, platinum-based combination chemotherapy provides a modest survival benefit compared with best supportive care. Two-drug regimens offer improved response and survival rates compared with one drug, whereas three-drug regimens do not improve survival in advanced NSCLC. Studies attempting to determine optimal therapy duration have found that prolonging treatment beyond three to four cycles increases toxicity with no increase in response or survival.During the 1990s, at least five “new” chemotherapeutic agents that had activity in lung cancer (paclitaxel, docetaxel, vinorelbine, gemcitabine, and irinotecan) have shown single-agent activity in advanced NSCLC. These agents are usually used in combination with a platinum compound, either cisplatin or carboplatin. Nonplatinum doublets have not been found to offer an advantage over platinum doublets, and decreased survival has been observed in two trials in the nonplatinum arms. In combination with a platinum agent, the newer agents appear to offer a slight improvement in outcome compared with older regimens. The randomized trials that have evaluated these modern platinum-based doublets have not shown that any specific combination is superior when survival is the primary measure of outcome. Some studies have suggested that cisplatin doublets are superior to carboplatin doublets, but such a difference has not been consistently observed. More research is needed to determine the optimal regimen for performance status 2 patients and for the elderly, a heterogeneous and understudied group of patients.Docetaxel remains the standard of care for second-line therapy for NSCLC. This agent has been shown to prolong survival compared with best supportive care. Recently, pemetrexed has been shown to have activity similar to that of and is with and of is also a to The participants felt that second-line treatment for advanced is an that is for potential therapeutic Both and pemetrexed are with which to novel may a prognosis in lung cancer, but there are many any effect is In lung cancer has been shown to and and tumor cell and are This is the for as an important target in lung cancer. Although was found to be expressed in over of the the level of expression did not with clinical response to tyrosine kinase in the studies of the trials, the tyrosine kinase gefitinib showed antitumor activity in and of patients with previously treated advanced NSCLC. Another of patients had disease as their best response. In both patients with disease and patients with response, improved. gefitinib has been for the treatment of advanced in the United States by the and and in The trials, failed to find a benefit in the for gefitinib added to standard chemotherapy regimens or These major negative findings raised questions regarding the preclinical models that had shown or effects of chemotherapy and response to gefitinib did not with in the trials a improvement in was observed in patients who on to an that, if may be useful in clinical patients with and were more likely to respond to gefitinib. treatment response and the of toxicity was reported in the studies, in contrast to the studies with erlotinib summarized Patients with appear to a distinct who have only limited response to chemotherapy but superior response rates to tyrosine kinase Studies of response in these patients may serve to identify predictive markers of efficacy for the agents in other patient is well and Phase II studies have suggested it may be an to in the second-line Phase III studies comparing gefitinib with in terms of survival and of are studies are to assess the possible role of gefitinib as therapy after chemotherapy for stage IV disease and after for stage III the with gefitinib, erlotinib demonstrated activity in the second-line setting, but trials showed no advantage to erlotinib in with standard chemotherapy. are issues in dosing with both erlotinib and gefitinib because they are given as a dose individual differences in In the second-line trials, response to erlotinib was correlated with the and of an that provides a for the dose in individual patients to a level that detectable large randomized Phase III trial of erlotinib as therapy best supportive care is and the results of this trial will be important in whether the tyrosine kinase can a meaningful in terms of survival improvement of lung cancer is a that in cell and expression of Preclinical studies potential and a number of chemotherapeutic agents, including cisplatin and In this synergy has been demonstrated clinically in cancer and and cancer. Positive results have been reported in combination with chemotherapy. An ongoing study is evaluating as monotherapy in patients with who have failed platinum-based chemotherapy. with the tyrosine kinase inhibitors, molecular mechanisms response to therapy are not well understood. Some that which a different mechanism of may be more effective than the tyrosine kinase when combined with chemotherapy. However, this is a preliminary that has not yet been confirmed by clinical is a against growth In a randomized Phase II trial in patients with advanced metastatic the addition of to standard chemotherapy the time to progression with a increase in response rate as well. Several Phase II trials in combination with chemotherapy or with a targeted agent, such as are A large randomized study comparing standard chemotherapy with standard chemotherapy is by the trials will issues of optimal and with chemotherapy and with other targeted therapies, as well as the of that have been the most issue in the lung cancer is an agent with antitumor activity demonstrated in a number of solid including NSCLC. with and has the and toxicity seen in early studies of pemetrexed, permitting more In single-agent activity has been demonstrated in the and second-line in Phase II trials. A randomized Phase III trial comparing pemetrexed with as second-line therapy in advanced has shown similar activity and has also shown activity in Phase II combination trials with platinum compounds and vinorelbine, and about the optimal of pemetrexed and gemcitabine, and a current Phase III trial is different dosing regimens of these two agents to this trials with other novel agents as are is a antisense designed to to its and protein is an protein implicated in the development of resistance to both chemotherapy and However, conference participants felt that the clinical significance of as a target has not been in lung cancer. expression to with poor prognosis in a number of but the data are and on this in NSCLC. Several participants also that there are issues with effects of antisense of small preliminary studies with are It is not clear whether is best used as a single agent, given the that other can for or in combination with other targeted agents. It is in combination with carboplatin and as therapy in small cell lung cancer and with as second-line therapy in or is a that in preclinical models cell and It has been by the United States and for use in patients with multiple A randomized Phase II study is evaluating with or without in second-line therapy of advanced NSCLC. is also a Phase II trial of single-agent as second-line therapy of advanced small cell lung cancer. Several ongoing combination Phase I trials are evaluating in combination with platinum-based chemotherapy. It is that may more to chemotherapy by However, there are issues with the use of and chemotherapy that study because preclinical data the of or with chemotherapy, on the two are or with pemetrexed, different cell lines have is a kinase that cell In preclinical studies, provides effects when after a whereas it is if given or to the Phase I trials have the effects of when with or with A randomized Phase II study of and is is regarding the optimal dosing for to the lack of pharmacodynamic end to confirm target The conference participants felt that although may not have the that it an optimal agent, this target remains a valid one for in lung cancer. more kinase are clinical trials, and the results are and are investigational designed to and cell and In preclinical studies, growth in small cell lung cancer and cell lines and in xenograft models. Phase II trials have evaluated and in advanced untreated and in small cell lung cancer. responses were reported in any of these trials, and the drugs had no single-agent activity, although the trial of in showed some of disease in a small minority of patients. The latter trial at least of activity in of the patients, that the drug was the effect in surrogate have shown activity in other and biomarkers identified in these tumors may allow identification of a lung cancer for combining the molecularly targeted agents with standard chemotherapy needs to be therapy may have rather than effects if the regimens selected are not on of the and mechanisms of action of both of agents. preclinical studies are needed to explore of molecularly targeted agents with the agents may in fact with the effects of chemotherapy if their effect is to cell trials with such as or A have had disappointing In primary with or to increase the of lung cancer in interest in for lung cancer has focused on the selective and is highly expressed in lung In expression of has been with decreased Some studies have reported a in lung cancer with use of or whereas other studies have failed to find a effect for It is what the optimal dosing is for the either as primary or in with chemotherapy. A trial of a is also expression is also in the of using the agents for One trial has been to test the tyrosine kinase gefitinib in the of of the and another trial the study design would the These two trials, the of Trials Lung Cancer, have been on of issues raised by the United States and Although these are both studies, the and for studies in these trials is also discussion of as a possible target for of current treatment trials with antisense with positive results and data. All such trials need to issues for a disappointing trial results seen with a number of the molecularly targeted have the of our current understanding of lung cancer biology. A more thorough of the multiple growth and of the of and of mechanisms is to targeted effective for more than small, of patients. More research needs to be at understanding mechanisms of tumor growth and resistance. developed mouse models will be important for this in addition to a more determined effort to obtain biopsies and to study available archival tissue using agents have if activity. are well and they are in the The primary research issue that needs to be addressed is determining the clinical or markers that predict which patients are likely to respond to these agents. is a general conference participants that tumor tissue studies at of response to should be given a priority to specific patient with a greater of response. Although to the group felt strongly that studies which correlated outcome to end had the ability to improve our understanding of lung cancer and, the treatment of patients with this at the Conference on Novel Agents in the Treatment of Lung Cancer, October 17–18, 2003, Cambridge, for of and of these were made possible by from and and were by

Background: Inhibiting polo-like kinase 1 PLK1 may be an effective treatment for non-small cell lung cancer (NSCLC). PLK1 is a key regulator of mitosis and DNA damage checkpoints. PLK1 inhibitors are well tolerated, but only a few unselected patients with NSCLC respond to single-agent therapy. However, predictive biomarkers have not been used to select patients who are likely to experience a response to PLK1 inhibitors, and the mechanisms of resistance to PLK1 inhibitors have not been elucidated, making these unknowns a major gap in knowledge. To address this gap, we compared basal gene and protein expression in 63 NSCLC cell lines and discovered that mesenchymal NSCLC cell lines were more sensitive to PLK1 inhibitors than epithelial cell lines in vitro and in vivo. The induction of apoptosis in some NSCLC cell lines at very low drug concentrations and the need to find better therapy for mesenchymal NSCLC motivated us to further study PLK1 inhibition. Methods: To identify the pathways involved in PLK1 inhibitor-induced apoptosis, we used 3 pairs of isogenic NSCLC cell lines in which we had induced a mesenchymal phenotype using TGF-β. These isogenic lines were treated with the PLK1 inhibitor (volasertib) for 24 hours and levels of 301 proteins and phosphoproteins were simultaneously measured before and after treatment with volasertib using reverse phase protein array (RPPA). Results: The induction of a mesenchymal phenotype using TGF-β increased PLK1 inhibition-induced DNA damage and apoptosis in 3 NSCLC cell lines. To further elucidate mechanisms of resistance to PLK1 inhibition, we compared gene and protein expression in these isogenic cell lines, before and after PLK1 inhibition. There were 35, 12 and 43 proteins differentially regulated following PLK1 inhibition in epithelial vs. mesenchymal lines in HCC366, H1975, and HCC4006 cell lines, respectively at False Discovery rate 0.1. Phosphorylated FAK (Y397) and c-Met (Y1234/1235) were consistently inhibited following PLK1 inhibition in the mesenchymal lines but activated in the epithelial lines. These changes were confirmed by Western blotting. Total FAK and c-Met protein and mRNA levels were not affected, demonstrating post-translational changes. The inhibition of c-Met using EMD 1214063 led to FAK inhibition but FAK inhibition did not affect c-Met activation. The combination of c-Met inhibitor and volasertib increases sensitivity in NSCLC cell lines tested. The combinations led to more apoptosis than the single-agent inhibitors. Conclusions: NSCLC cell lines have diverse sensitivities to PLK1 inhibition, which is consistent with the results of clinical trials of PLK1 inhibitors in solid tumors, but no studies to date explain these diverse responses to PLK1 inhibition. We have identified c-Met activation as a previously unknown pathway of resistance to PLK1 inhibition in epithelial NSCLC. Citation Format: Ratnakar Singh, Li Shen, Pan Tong, Jing Wang, Faye M. Johnson. c-Met activation mediates resistance to polo-like kinase 1 inhibitor-induced apoptosis in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4095. doi:10.1158/1538-7445.AM2017-4095

Objective To investigate the immunoreactivity of platelet-derived growth factor-A (PDGF-A) and its receptor at human and mouse neuromuscular junctions and to explore their regulatory effects on neuromuscular junctions. Methods Specific polyclonal antibodies were used to detect PDGF-A and PDGF a-receptor expression. Double immunohistochemistry for platelet-derived growth factor and the acetylcholine receptor was performed on normal human muscle biopsy specimens. Double fluorescence labeling was applied to detect immunoreactivity for PDGF-A, its receptor and acetylcholine receptors. Results PDGF-A and its receptor was closely co-localized with acetylcholine receptors at human and mouse neuromuscular junctions. Conclusion PDGF-A and its receptor concentrated at human and mouse neuromuscular junctions. PDGF might be involved in the interaction between the presynaptic and postsynaptic components, PDGF-A and its receptor might play regulatory role in signaling at neuromuscular junctions in normal muscle. Key words: Platelet-derived growth factor; Receptor; Neuromuscular junction