Candidate predictive biomarkers for epidermal growth factor receptor inhibitors (EGFRi), skin rash and serum proteomic assays, require further qualification to improve EGFRi therapy in non-small cell lung cancer (NSCLC). In a phase II trial that was closed to accrual because of changes in clinical practice we examined the relationships among candidate biomarkers, quantitative changes in tumor size, progression-free and overall survival.
55 patients with progressive NSCLC after platinum therapy were randomized to receive (Arm A) cetuximab, followed by pemetrexed at progression, or (Arm B) concurrent cetuximab and pemetrexed. All received cetuximab monotherapy for the first 14 days. Pre-treatment serum and weekly rash assessments by standard and EGFRi-induced rash (EIR) scales were collected.
43 patients (20-Arm A, 23-Arm B) completed the 14-day run-in. Median survival was 9.1 months. Arm B had better median overall (Arm B = 10.3 [95% CI 7.5, 16.8]; Arm A = 3.5 [2.8, 11.7] months P = 0.046) and progression-free survival (Arm B = 2.3 [1.6, 3.1]; Arm A = 1.6 [0.9, 1.9] months P = 0.11). The EIR scale distributed ratings among 6 rather than 3 categories but ordinal scale rash severity did not predict outcomes. The serum proteomic classifier and absence of rash after 21 days of cetuximab did.
Absence of rash after 21 days of cetuximab therapy and the serum proteomic classifier, but not ordinal rash severity, were associated with NSCLC outcomes. Although in a small study, these observations were consistent with results from larger retrospective analyses.
Clinicaltrials.gov Identifier NCT00203931
Pemetrexed; Lung Cancer; Cetuximab; Rash; EGFR; Proteomics
Ganetespib is a novel inhibitor of the heat shock protein 90 (Hsp90), a chaperone protein critical to tumor growth and proliferation. In this phase II study, we evaluated the activity and tolerability of ganetespib in previously treated patients with non–small cell lung cancer (NSCLC).
Patients were enrolled into cohort A (mutant EGFR), B (mutant KRAS), or C (no EGFR or KRAS mutations). Patients were treated with 200 mg/m2 ganetespib by intravenous infusion once weekly for 3 weeks followed by 1 week of rest, until disease progression. The primary endpoint was progression-free survival (PFS) at 16 weeks. Secondary endpoints included objective response (ORR), duration of treatment, tolerability, median PFS, overall survival (OS), and correlative studies.
Ninety-nine patients with a median of 2 prior systemic therapies were enrolled; 98 were assigned to cohort A (n = 15), B (n = 17), or C (n = 66), with PFS rates at 16 weeks of 13.3%, 5.9%, and 19.7%, respectively. Four patients (4%) achieved partial response (PR); all had disease that harbored anaplastic lymphoma kinase (ALK) gene rearrangement, retrospectively detected by FISH (n = 1) or PCR-based assays (n = 3), in crizotinib-naïve patients enrolled to cohort C. Eight patients (8.1%) experienced treatment-related serious adverse events (AE); 2 of these (cardiac arrest and renal failure) resulted in death. The most common AEs were diarrhea, fatigue, nausea, and anorexia.
Ganetespib monotherapy showed a manageable side effect profile as well as clinical activity in heavily pretreated patients with advanced NSCLCs, particularly in patients with tumors harboring ALK gene rearrangement.
World-wide, esophageal cancer is a growing epidemic and patients frequently present with advanced disease that is surgically inoperable. Hence, chemotherapy is the predominate treatment. Cytotoxic platinum compounds are mostly used, but their efficacy is only moderate. Newer alkylating agents have shown promise in other tumor types, but little is known about their utility in esophageal cancer.
We utilized archived human esophageal cancer samples and esophageal cancer cell lines to evaluate O-6-methylguanine-deoxyribonucleic acid methyltransferase (MGMT) hypermethylation status and determined sensitivity to the alkylating drug temozolomide (TMZ). Immunoblot analysis was performed to determine MGMT protein expression in cell lines. To assess and confirm the effect of TMZ treatment in a methylated esophageal cancer cell line in vivo, a mouse flank xenograft tumor model was utilized.
Nearly 71% (12/17) of adenocarcinoma and 38% (3/8) of squamous cell carcinoma (SCC) patient samples were MGMT hypermethylated. Out of four adenocarcinoma and nine SCC cell lines tested, one of each histology was hypermethylated. Immunoblot analyses confirmed that hypermethylated cell lines did not express the MGMT protein. In vitro cell viability assays showed the methylated Kyse-140 and FLO cells to be sensitive to TMZ at an IC50 of 52-420 μM, whereas unmethylated cells Kyse-410 and SKGT-4 did not respond. In an in vivo xenograft tumor model with Kyse-140 cells, which are MGMT hypermethylated, TMZ treatment abrogated tumor growth by more than 60%.
MGMT methylation may be an important biomarker in subsets of esophageal cancers and targeting by TMZ may be utilized to successfully treat these patients.
Alkylating agents; deoxyribonucleic acid repair genes; in vivo pre-clinical; esophageal cancer; O-6-methylguanine-deoxyribonucleic acid methyltransferase hypermethylation; response to treatment; temozolomide
To assess the efficacy and toxicity of carboplatin, etoposide, and the bcl-2 antisense oligonucleotide oblimersen as initial therapy for extensive-stage small-cell lung cancer (ES-SCLC). bcl-2 has been implicated as a key factor in SCLC oncogenesis and chemotherapeutic resistance.
Patients and Methods
A 3:1 randomized phase II study was performed to evaluate carboplatin and etoposide with (arm A) or without oblimersen (arm B) in 56 assessable patients with chemotherapy-naïve ES-SCLC. Outcome measures including toxicity, objective response rate, complete response rate, failure-free survival, overall survival, and 1-year survival rate.
Oblimersen was associated with slightly more grade 3 to 4 hematologic toxicity (88% v 60%; P = .05). Response rates were 61% (95% CI, 45% to 76%) for arm A and 60% (95% CI, 32% to 84%) for arm B. The percentage of patients alive at 1 year was 24% (95% CI, 12% to 40%) with oblimersen, and 47% (95% CI, 21% to 73%) without oblimersen. Hazard ratios for failure-free survival (1.79; P = .07) and overall survival (2.13; P = .02) suggested worse outcome for patients receiving oblimersen. These results hold when adjusted for other prognostic factors, such as weight loss, in multivariate regression analysis.
Despite extensive data supporting a critical role for Bcl-2 in chemoresistance in SCLC, addition of oblimersen to a standard regimen for this disease did not improve any clinical outcome measure. Emerging data from several groups suggest that this lack of efficacy may be due to insufficient suppression of Bcl-2 in vivo. Additional evaluation of this agent in SCLC is not warranted.
Despite progress in locoregional and systemic therapies, patient survival from lung cancer remains a challenge. Receptor tyrosine kinases are frequently implicated in lung cancer pathogenesis, and some tyrosine kinase inhibition strategies have been effective clinically. The EphB4 receptor tyrosine kinase has recently emerged as a potential target in several other cancers. We sought to systematically study the role of EphB4 in lung cancer. Here, we demonstrate that EphB4 is overexpressed 3-fold in lung tumors compared to paired normal tissues and frequently exhibits gene copy number increases in lung cancer. We also show that overexpression of EphB4 promotes cellular proliferation, colony formation, and motility, while EphB4 inhibition reduces cellular viability in vitro, halts the growth of established tumors in mouse xenograft models when used as a single-target strategy, and causes near-complete regression of established tumors when used in combination with paclitaxel. Taken together, these data suggest an important role for EphB4 as a potential novel therapeutic target in lung cancer. Clinical trials investigating the efficacy of anti-EphB4 therapies as well as combination therapy involving EphB4 inhibition may be warranted.
Lung cell migration is a crucial step for re-epithelialization that in turn is essential for remodeling and repair after lung injury. We hypothesize that secreted autotaxin (ATX), which exhibits lysophospholipase D (lysoPLD) activity, stimulates lung epithelial cell migration through lysophosphatidic acid (LPA) generation-dependent and -independent pathways. Release of endogenous ATX protein and activity was detected in lung epithelial cell culture medium. ATX with V5 tag (ATX-V5) overexpressed conditional medium had higher LPA levels compared to control medium and stimulated cell migration through Gαi-coupled LPA receptors, cytoskeleton rearrangement, phosphorylation of PKCδ and cortactin at the leading edge of migrating cells. Inhibition of PKCδ attenuated ATX-V5 overexpressed conditional medium-mediated phosphorylation of cortactin. In addition, a recombinant ATX mutant, lacking lysoPLD activity, or heat-inactived ATX also induced lung epithelial cell migration. Extracelluar ATX bound to LPA receptor and integrin β4 complex on A549 cell surface. Finally, intratracheal administration of lipopolysaccharide into mouse airway induced ATX release and LPA production in bronchoalveolar lavage fluid. These results suggested a significant role for ATX in lung epithelial cell migration and remodeling through its ability to induce LPA production-mediated phosphorylation of PKCδ and cortactin. In addition we also demonstrated assocation of ATX with epithelial cell surface LPA receptor and integrin β4.
ATX (autotoxin); lysoPLD; LPA; cell migration; signal transduction
Cytoskeletal and focal adhesion abnormalities are observed in several types of cancer, including lung cancer. We have previously reported that paxillin (PXN) was mutated, amplified, and overexpressed in a significant number of lung cancer patient samples, that PXN protein was upregulated in more advanced stages of lung cancer compared with lower stages, and that the PXN gene was also amplified in some pre-neoplastic lung lesions. Among the mutations investigated, we previously found that PXN variant A127T in lung cancer cells enhanced cell proliferation and focal adhesion formation and colocalized with the anti-apoptotic protein B Cell Lymphoma 2 (BCL-2), which is known to localize to the mitochondria, among other sites. To further explore the effects of activating mutations of PXN on mitochondrial function, we cloned and expressed wild-type PXN and variants containing the most commonly occurring PXN mutations (P46S, P52L, G105D, A127T, P233L, T255I, D399N, E423K, P487L, and K506R) in a GFP-tagged vector using HEK-293 human embryonic kidney cells. Utilizing live-cell imaging to systematically study the effects of wild-type PXN vs. mutants, we created a model that recapitulates the salient features of the measured dynamics and conclude that compared with wild-type, some mutant clones confer enhanced focal adhesion and lamellipodia formation (A127T, P233L, and P487L) and some confer increased association with BCL-2, Dynamin-related Protein-1 (DRP-1), and Mitofusion-2 (MFN-2) proteins (P233L and D399N). Further, PXN mutants, through their interactions with BCL-2 and DRP-1, could regulate cisplatin drug resistance in human lung cancer cells. The data reported herein suggest that mutant PXN variants play a prominent role in mitochondrial dynamics with direct implications on lung cancer progression and hence, deserve further exploration as therapeutic targets.
mitochondria; paxillin; gene mutation; cell motility; fission; fusion; mitochondrial dynamics
Malignant pleural mesothelioma (MPM) often develops decades following exposure to asbestos. Current best therapy produces a response in only half of patients, and the median survival with this therapy remains under a year. A search for novel targets and therapeutics is underway, and recently identified targets include VEGF, Notch, and EphB4-Ephrin-B2. Each of these targets has dual activity, promoting tumor cell growth as well as tumor angiogenesis.
We investigated EphB4 expression in 39 human mesothelioma tissues by immunohistochemistry. Xenograft tumors established with human mesothelioma cells were treated with an EphB4 inhibitor (monomeric soluble EphB4 fused to human serum albumin, or sEphB4-HSA). The combinatorial effect of sEphB4-HSA and biologic agent was also studied.
EphB4 was overexpressed in 72% of mesothelioma tissues evaluated, with 85% of epithelioid and 38% of sarcomatoid subtypes demonstrating overexpression. The EphB4 inhibitor sEphB4-HSA was highly active as a single agent to inhibit tumor growth, accompanied by tumor cell apoptosis and inhibition of PI3K and Src signaling. Combination of sEphB4-HSA and the anti-VEGF antibody (Bevacizumab) was superior to each agent alone and led to complete tumor regression.
EphB4 is a potential therapeutic target in mesothelioma. Clinical investigation of sEphB4-HSA as a single agent and in combination with VEGF inhibitors is warranted.
EphB4; Mesothelioma; sEphB4; Cancer therapy
Lung cancer is a heterogeneous group of diseases. There has been much research in lung cancer over the past decade which has advanced our ability to treat these patients with a more personalized approach. The scope of this paper is to review the literature and give a broad understanding of the current molecular targets for which we currently have therapies as well as other targets for which we may soon have therapies. Additionally, we will cover some of the issues of resistance with these targeted therapies. The molecular targets we intend to discuss are epidermal growth factor receptor (EGFR), Vascular endothelial growth factor (VEGF), anaplastic large-cell lymphoma kinase (ALK), KRAS, C-MET/RON, PIK3CA. ROS-1, RET Fibroblast growth factor receptor (FGFR). Ephrins and their receptors, BRAF, and immunotherapies/vaccines. This manuscript only summarizes the work which has been done to date and in no way is meant to be comprehensive.
Cellular mechanism; HGF; MET; oncogene; receptor tyrosine kinase; targeted cancer therapy
Patients with lung cancer who develop brain metastases have a poor prognosis. Those patients with progressive brain metastases tend to have a dismal prognosis. Currently, there is no standard of care for the treatment of these patients.
In this manuscript, we present a retrospective evaluation of 10 patients treated at our institution with a combination of temozolomide and/or erlotinib after disease progression in the central nervous system following radiation therapy.
Median overall survival was 28 weeks. Median time to progression in the central nervous system was 14 weeks. Median time to progression systemically was 7.5 weeks. Some patients demonstrated prolonged stability of disease.
A palliative regimen of temozolomide and/or erlotinib could be considered in progressive central nervous system metastases from lung cancer.
Lung cancer; Brain metastases; Temozolomide; Erlotinib; Epidermal growth factor receptor
Background: Previously, we identified a sequence variant (N375S) of c-Met gene, however, its association with lung cancer risk and prognosis remain undefined.
Patients and Methods: We investigated the genotype distribution of the c-Met-N375S sequence variant in 206 lung cancer patients and 207 non-cancer controls in the Taiwanese population by DNA sequencing.
Results: Lung cancer patients with variant A/G and G/G genotypes showed 1.08-fold increased cancer risk when compared to patients with the wild-type A/A genotype (95% CI, 0.60-1.91). There were no significant differences in postoperative survival between c-Met-N375S and wild-type patients. In the cell model, the c-Met-N375S cells showed a decrease in cell death upon treatment with MET inhibitor SU11274 compared to wild-type cells.
Conclusion: Our data suggest that the c-Met-N375S sequence variant may not play a significant role in cancer susceptibility and the prognosis of lung cancer patients. The correlation with chemoresponse of c-Met-N375S is worth further investigation in patients receiving MET therapy.
c-Met; N375S; cancer risk; prognosis; lung cancer.
Our objective was to investigate the application of three-dimensional (3D) stereoscopic volume rendering with perceptual colorization on preoperative imaging for malignant pleural mesothelioma. At present, we have prospectively enrolled 6 patients being considered for resection of malignant pleural mesothelioma that have undergone a multidetector-row computed tomography (CT) scan of the chest. The CT data sets were volume rendered without preprocessing. The resultant 3D rendering was displayed stereoscopically and used to provide information regarding tumor extent, morphology, and anatomic involvement. To demonstrate this technique, this information was compared with the corresponding two-dimensional CT grayscale axial images from two of these patients. Three-dimensional stereoscopic reconstructions of the CT data sets provided detailed information regarding the local extent of tumor that could be used for preoperative surgical planning. Three-dimensional stereoscopic volume rendering for malignant pleural mesothelioma is a novel approach. Combined with our innovative perceptual colorization algorithm, stereoscopic volumetric analysis potentially allows for the accurate determination of the extent of pleural mesothelioma with results difficult to duplicate using grayscale, multiplanar CT images.
Mesothelioma; Imaging; Lung cancer; Diagnosis; Computed tomography; CAT scan; Imaging
NOL7 is a putative tumor suppressor gene (TSG) localized to 6p23, a region with frequent loss of heterozygosity (LOH) in a number of cancers, including cervical cancer (CC). We have previously demonstrated that reintroduction of NOL7 into CC cells alters the angiogenic phenotype and suppressed tumor growth in vivo by 95%. Therefore, to understand its mechanism of inactivation in CC, we investigated the genetic and epigenetic regulation of NOL7. NOL7 mRNA and protein levels were assessed in thirteen CC cell lines and twenty-three consecutive CC specimens by RTQ-PCR, western blotting, and IHC. Methylation of the NOL7 promoter was analyzed by bisulfite sequencing and mutations were identified through direct sequencing. A CpG island with multiple CpG dinucleotides spanned the 5′UTR and first exon of NOL7. However, bisulfite sequencing failed to identify persistent sites of methylation. Mutational sequencing revealed that 40% of the CC specimens and 31% of the CC cell lines harbored somatic mutations that may affect the in vivo function of NOL7. Endogenous NOL7 mRNA and protein expression in CC cell lines was significantly decreased in 46% of the CC cell lines. Finally, immunohistochemistry demonstrated strong NOL7 nucleolar staining in normal tissues that decreased with histologic progression towards CC. NOL7 is inactivated in CC in accordance with Knudson's two-hit hypothesis through LOH and mutation. Together with evidence of its in vivo tumor suppression, these data support the hypothesis that NOL7 is the legitimate TSG located on 6p23.
NOL7; Hypermethylation; Mutation
Amplification of the MET proto-oncogene in gastroesophageal cancer (GEC) may constitute a molecular marker for targeted therapy. We examined a GEC cohort with follow-up and reported the clinical response of four additional patients with MET-amplified tumors to the small molecule inhibitor crizotinib as part of an expanded phase I cohort study.
Patients and Methods
From 2007 to 2009, patients with GEC were genetically screened as a consecutive series of 489 tumors (stages 0, I, and II, 39%; III, 25%; IV, 36%; n = 222 esophageal, including n = 21 squamous carcinomas). MET, EGFR, and HER2 amplification status was assessed by using fluorescence in situ hybridization.
Ten (2%) of 489 patients screened harbored MET amplification; 23 (4.7%) harbored EGFR amplification; 45 (8.9%) harbored HER2 amplification; and 411 (84%) were wild type for all three genes (ie, negative). MET-amplified tumors were typically high-grade adenocarcinomas that presented at advanced stages (5%; n = 4 of 80). EGFR-amplified tumors showed the highest fraction of squamous cell carcinoma (17%; n = 4 of 23). HER2, MET, and EGFR amplification were, with one exception (MET and EGFR positive), mutually exclusive events. Survival analysis in patients with stages III and IV disease showed substantially shorter median survival in MET/EGFR-amplified groups, with a rank order for all groups by median survival (from most to least aggressive): MET (7.1 months; P < .001) less than EGFR (11.2 months; P = .16) less than HER2 (16.9 months; P = .89) when compared with the negative group (16.2 months). Two of four patients with MET-amplified tumors treated with crizotinib experienced tumor shrinkage (−30% and −16%) and experienced progression after 3.7 and 3.5 months.
MET amplification defines a small and aggressive subset of GEC with indications of transient sensitivity to the targeted MET inhibitor crizotinib (PF-02341066).
A 48 year-old female with chemo-refractory metastatic gastric cancer to the liver was treated on a Phase I clinical trial with MetMAb, a monoclonal antibody targeting the Met tyrosine kinase receptor. The primary tumor had high MET gene polysomy and evidence for an autocrine production of HGF, the growth factor ligand of Met. A complete response was obtained lasting two years; the cancer recurred as a peritoneal deposit invading into the transverse colon and a gastrohepatic ligament node. Compassionate use of MetMAb therapy at recurrence achieved a mixed response - a partial response of the two initial lesions, but with development of multiple new foci of carcinomatosis. Tissue and serum studies evaluating the Met signaling pathway did correlate with MetMAb treatment response initially and at the time of recurrence.
Gastric adenocarcinoma; Met; HGF; MetMab
MetMAb (OA-5D5) is a one-armed monoclonal antibody developed to bind to and inhibit c-MET receptor tyrosine kinase. Though early in clinical testing, this agent holds great promise in diseases thought to be driven by c-MET activation, as evidenced by the phase II results in non-small cell lung cancer, (NSCLC) where a benefit in overall survival was observed in patients with MET diagnostic positive disease. Thus far, both alone and in combination with other targeted agents, this drug has been well tolerated and no new significant safety signals have been identified.
The review summarizes the structure and function of the c-MET receptor and its ligand HGF, provides an overview of select targeted monotherapies developed to interfere in the MET-HGF signaling pathway, discusses pre-clinical and clinical data surrounding MetMAb, and concludes with an expert opinion regarding this novel agent.
MetMAb has been well tolerated and based on phase II data testing it, in combination with erlotinib in advanced NSCLC, may have a role in improving survival in patients with disease driven by c-MET activation. However, phase III validation is underway and the results of these studies will help elucidate which patients will benefit most from this novel agent.
MetMAb; c-MET; HGF; targeted therapy; monoclonal antibody; personalized medicine; non-small cell lung cancer
An area of need in cancer informatics is the ability to store images in a comprehensive database as part of translational cancer research. To meet this need, we have implemented a novel tandem database infrastructure that facilitates image storage and utilisation.
We had previously implemented the Thoracic Oncology Program Database Project (TOPDP) database for our translational cancer research needs. While useful for many research endeavours, it is unable to store images, hence our need to implement an imaging database which could communicate easily with the TOPDP database.
The Thoracic Oncology Research Program (TORP) imaging database was designed using the Research Electronic Data Capture (REDCap) platform, which was developed by Vanderbilt University. To demonstrate proof of principle and evaluate utility, we performed a retrospective investigation into tumour response for malignant pleural mesothelioma (MPM) patients treated at the University of Chicago Medical Center with either of two analogous chemotherapy regimens and consented to at least one of two UCMC IRB protocols, 9571 and 13473A.
A cohort of 22 MPM patients was identified using clinical data in the TOPDP database. After measurements were acquired, two representative CT images and 0–35 histological images per patient were successfully stored in the TORP database, along with clinical and demographic data.
We implemented the TORP imaging database to be used in conjunction with our comprehensive TOPDP database. While it requires an additional effort to use two databases, our database infrastructure facilitates more comprehensive translational research.
The investigation described herein demonstrates the successful implementation of this novel tandem imaging database infrastructure, as well as the potential utility of investigations enabled by it. The data model presented here can be utilised as the basis for further development of other larger, more streamlined databases in the future.
Bronchioloalveolar carcinoma (BAC), a subtype of non-small cell lung cancer (NSCLC), is a difficult disease to treat with low response rates with cytotoxic chemotherapy. Bortezomib, a proteasome inhibitor, has demonstrated objective responses in BAC patients in early phase clinical trials. We conducted a phase II study of bortezomib inpatients with advanced stage BAC.
Patients with advanced BAC, adenocarcinoma with BAC features or BAC with adenocarcinoma features and less than two prior regimens were eligible. Prior epidermal growth factor receptor (EGFR) inhibitor therapy was allowed. Bortezomib was administered intravenously at 1.6 mg/m2 on days 1 and 8 of every 21 days cycle until disease progression or unacceptable toxicity. The primary endpoint was response rate. The Simon two-stage design was utilized.
Forty-two patients were enrolled and the study was halted early for slow accrual. Patient characteristics were: female 55%, median age 68 years, and ECOG performance status of 0 and 1 in 31 and 11 patients respectively. Twenty-six(62%)patients had received prior therapy with an EGFR inhibitor. A median of 4 cycles of therapy were administered. Objective responses were noted in 5% while 57% had disease stabilization. The median progression-free survival and overall survival were 5.5 months and 13.6 months respectively. Grade 3 diarrhea and fatigue were noted in 3 and 5 patients respectively.
Bortezomib is tolerated well and is associated with modest anti-cancer activity in advanced BAC, including inpatients that progressed on EGFR inhibitor therapy.
bortezomib; proteasome inhibition; BAC; bronchioloalveolar carcinoma; NSCLC
Malignant pleural mesothelioma (MPM) is a devastating disease with an overall poor prognosis. Despite the recent advances in targeted molecular therapies, there is a clear and urgent need for the identification of novel mesothelioma targets for the development of highly efficacious therapeutics.
In this study, we report that the expression of Sphingosine Kinase 1 (SphK1) protein was preferentially elevated in MPM tumor tissues (49 epithelioid and 13 sarcomatoid) compared to normal tissue (n = 13). In addition, we also observed significantly elevated levels of SphK1 and SphK2 mRNA and SphK1 protein expression in MPM cell lines such as H2691, H513 and H2461 compared to the non-malignant mesothelial Met5 cells. The underlying mechanism appears to be mediated by SphK1 induced upregulation of select gene transcription programs such as that of CBP/p300 and PCAF, two histone acetyl transferases (HAT), and the down regulation of cell cycle dependent kinase inhibitor genes such as p27Kip1 and p21Cip1. In addition, using immunoprecipitates of anti-acetylated histone antibody from SphK inhibitor, SphK-I2 treated Met5A and H2691 cell lysates, we also showed activation of other cell proliferation related genes, such as Top2A (DNA replication), AKB (chromosome remodeling and mitotic spindle formation), and suppression of p21 CIP1 and p27KIP1. The CDK2, HAT1 and MYST2 were, however, unaffected in the above study. Using SphK inhibitor and specific siRNA targeting either SphK1 or SphK2, we also unequivocally established that SphK1, but not SphK2, promotes H2691 mesothelioma cell proliferation. Using a multi-walled carbon nanotubes induced peritoneal mesothelioma mouse model, we showed that the SphK1−/− null mice exhibited significantly less inflammation and granulamatous nodules compared to their wild type counterparts.
The lipid kinase SphK1 plays a positive and essential role in the growth and development of malignant mesothelioma and is therefore a likely therapeutic target.
Currently, non–small-cell lung cancer (NSCLC) is the leading cause of cancer-related death in the United States. Angiogenesis, the formation of new vasculature, is a complex and tightly regulated process that promotes metastasis and disease progression in lung cancer and other malignancies. Developmental antiangiogenic agents have shown activity in NSCLC, and bevacizumab, an antiangiogenic monoclonal antibody, is approved for the treatment of patients with advanced disease. However, predictive biomarkers are needed to guide the administration of antiangiogenic agents. It is possible that angiogenic molecules could accurately predict patient response to targeted antiangiogenic therapies, which would allow for individualized and perhaps more effective treatment. Angiogenic signaling molecules may also have value as prognostic indicators, which may be useful for the management of NSCLC. Here we provide an overview of angiogenic molecules currently being investigated as prognostic biomarkers in NSCLC and discuss their potential to guide treatment choices.
angiogenesis; NSCLC; biomarker; antiangiogenic therapy; vascular endothelial growth factor; platelet-derived growth factor; fibroblast growth factor
VEGFR-2 plays a crucial role in mediating angiogenic endothelial cell responses via the VEGF pathway and angiogenesis inhibitors targeting VEGFR-2 are in clinical use. As angiogenesis is a host-driven process, functional heritable variation in KDR, the gene encoding VEGFR-2, may affect VEGFR-2 function, and ultimately, the extent of tumor angiogenesis.
We resequenced KDR using 24 DNAs each from healthy Caucasian, African American and Asian groups. Non-synonymous genetic variants were assessed for function using phosphorylation assays. Luciferase reporter gene assays were used to examine effects of variants on gene expression. KDR mRNA and protein expression, and microvessel density (MVD) were measured in non-small cell lung cancer (NSCLC) tumor samples and matching patient DNA samples were genotyped to test for associations with variants of interest.
KDR resequencing led to the discovery of 120 genetic variants, of which 25 had not been previously reported. Q472H had increased VEGFR-2 protein phosphorylation and associated with increased MVD in NSCLC tumor samples. −2854C and −2455A increased luciferase expression and associated with higher KDR mRNA levels in NSCLC samples. −271A reduced luciferase expression and associated with lower VEGFR-2 levels in NSCLC samples. −906C and 23408G, associated with higher KDR mRNA levels in NSCLC samples.
This study has defined KDR genetic variation in three populations and identified common variants that impact on tumoral KDR expression and vascularization. These findings may have important implications for understanding the molecular basis of genetic associations between KDR variation and clinical phenotypes related to VEGFR-2 function.
angiogenesis; KDR; resequencing; gene expression; NSCLC; functional validation
XL184 (cabozantinib) is a potent inhibitor of MET, vascular endothelial growth factor receptor 2 (VEGFR2), and RET, with robust antiangiogenic, antitumor, and anti-invasive effects in preclinical models. Early observations of clinical benefit in a phase I study of cabozantinib, which included patients with medullary thyroid cancer (MTC), led to expansion of an MTC-enriched cohort, which is the focus of this article.
Patients and Methods
A phase I dose-escalation study of oral cabozantinib was conducted in patients with advanced solid tumors. Primary end points included evaluation of safety, pharmacokinetics, and maximum-tolerated dose (MTD) determination. Additional end points included RECIST (Response Evaluation Criteria in Solid Tumors) response, pharmacodynamics, RET mutational status, and biomarker analyses.
Eighty-five patients were enrolled, including 37 with MTC. The MTD was 175 mg daily. Dose-limiting toxicities were grade 3 palmar plantar erythrodysesthesia (PPE), mucositis, and AST, ALT, and lipase elevations and grade 2 mucositis that resulted in dose interruption and reduction. Ten (29%) of 35 patients with MTC with measurable disease had a confirmed partial response. Overall, 18 patients experienced tumor shrinkage of 30% or more, including 17 (49%) of 35 patients with MTC with measurable disease. Additionally, 15 (41%) of 37 patients with MTC had stable disease (SD) for at least 6 months, resulting in SD for 6 months or longer or confirmed partial response in 68% of patients with MTC.
Cabozantinib has an acceptable safety profile and is active in MTC. Cabozantinib may provide clinical benefit by simultaneously targeting multiple pathways of importance in MTC, including MET, VEGFR2, and RET. A global phase III pivotal study in MTC is ongoing (ClinicalTrials.gov number NCT00215605).
c-Cbl is an E3 ubiquitin ligase of many tyrosine kinase receptors. We previously detected c-Cbl mutation and low protein expression in non-small cell lung cancer (NSCLC). Therefore, we hypothesized that the overexpression of c-Cbl wild type (WT) may exhibit tumor inhibition.
Wound healing and transwell assays were conducted to examine cell motility after c-Cbl WT transfection in NSCLC cell lines. Cell cycle was investigated by flow cytometry. A549 and H1299-luc c-Cbl WT xenograft and experimental metastasis model were performed to investigate tumor growth and metastasis inhibition in vivo.
Wound healing and transwell assays showed inhibition of migration in A549 and H226br cells 4-24 hr post-transfection. Ectopic c-Cbl WT expression reduced cell proliferation at 48 hr in A549 cells. Importantly, A549 and H1299-luc cells with ectopic c-Cbl WT expression showed inhibition of tumor growth in vivo. A549 cells overexpressing c-Cbl WT inhibited tumor metastasis in animal models.
Our study demonstrates for the first time that c-Cbl WT protein overexpression inhibits tumor metastasis and tumor growth in lung cancer xenograft models. Our results provide evidence that ectopic expression of c-Cbl WT protein can be potentially applied as targeted therapy for lung cancer treatment.
c-Cbl; proliferation; migration; metastasis; gene therapy
Cancer-related genes show racial differences. Therefore, identification and characterization of DNA copy number alteration regions in different racial groups helps to dissect the mechanism of tumorigenesis.
Array-comparative genomic hybridization (array-CGH) was analyzed for DNA copy number profile in 40 Asian and 20 Caucasian lung cancer patients. Three methods including MetaCore analysis for disease and pathway correlations, concordance analysis between array-CGH database and the expression array database, and literature search for copy number variation genes were performed to select novel lung cancer candidate genes. Four candidate oncogenes were validated for DNA copy number and mRNA and protein expression by quantitative polymerase chain reaction (qPCR), chromogenic in situ hybridization (CISH), reverse transcriptase-qPCR (RT-qPCR), and immunohistochemistry (IHC) in more patients.
We identified 20 chromosomal imbalance regions harboring 459 genes for Caucasian and 17 regions containing 476 genes for Asian lung cancer patients. Seven common chromosomal imbalance regions harboring 117 genes, included gain on 3p13-14, 6p22.1, 9q21.13, 13q14.1, and 17p13.3; and loss on 3p22.2-22.3 and 13q13.3 were found both in Asian and Caucasian patients. Gene validation for four genes including ARHGAP19 (10q24.1) functioning in Rho activity control, FRAT2 (10q24.1) involved in Wnt signaling, PAFAH1B1 (17p13.3) functioning in motility control, and ZNF322A (6p22.1) involved in MAPK signaling was performed using qPCR and RT-qPCR. Mean gene dosage and mRNA expression level of the four candidate genes in tumor tissues were significantly higher than the corresponding normal tissues (P<0.001~P=0.06). In addition, CISH analysis of patients indicated that copy number amplification indeed occurred for ARHGAP19 and ZNF322A genes in lung cancer patients. IHC analysis of paraffin blocks from Asian Caucasian patients demonstrated that the frequency of PAFAH1B1 protein overexpression was 68% in Asian and 70% in Caucasian.
Our study provides an invaluable database revealing common and differential imbalance regions at specific chromosomes among Asian and Caucasian lung cancer patients. Four validation methods confirmed our database, which would help in further studies on the mechanism of lung tumorigenesis.
Array-CGH; Lung cancer; Asian; Caucasian; Oncogene
Fyn is a member of the Src family of kinases that we have previously shown to be overexpressed in prostate cancer. This study defines the biological impact of Fyn inhibition in cancer using a PC3 prostate cancer model.
Fyn expression was suppressed in PC3 cells using an shRNA against Fyn (PC3/FYN-). Knockdown cells were characterized using standard growth curves and time-lapse video microscopy of wound assays and Dunn Chamber assays. Tissue microarray analysis was used to verify the physiologic relevance of the HGF/MET axis in human samples. Flank injections of nude mice were performed to assess in vivo growth characteristics.
HGF was found to be sufficient to drive Fyn mediated events. Compared to control transductants (PC3/Ctrl), PC3/FYN- showed a 21% decrease in growth at 4 days (P=0.05). PC3/FYN- cells were 34% longer than control cells (P=0.018) with 50% increase in overall surface area (P<0.001). Furthermore, when placed in a gradient of HGF, PC3/FYN- cells showed impaired directed chemotaxis down an HGF gradient in comparison to PC3/Ctrl (P=0.001) despite a 41% increase in cellular movement speed. In vivo studies showed 66% difference of PC3/FYN- cell growth at 8 weeks using bidimensional measurements (P=0.002).
Fyn plays an important role in prostate cancer biology by facilitating cellular growth and by regulating directed chemotaxis- a key component of metastasis. This finding bears particular translational importance when studying the effect of Fyn inhibition in human subjects.
Fyn; prostate cancer; cell shape; cell motility; HGF