Therapeutic Target In Combination Research Articles

Inhibition of endoplasmic reticulum chaperone protein glucose-regulated protein 78 potentiates anti-angiogenic therapy in renal cell carcinoma through inactivation of the PERK/eIF2α pathway.

Tumor microenvironments are characterized by decreased oxygen and nutrition due to the rapid and progressive nature of tumors and also stresses induced by several anti-tumor therapies. These intense cell stressors trigger a protective cell survival mechanism heralded by the unfolded protein response (UPR). The UPR is induced by an accumulation of unfolded proteins in the endoplasmic reticulum (ER) following cell starvation. Although the ER stress response is implicated in cytoprotection, its precise role during anti-angiogenic therapy remains unclear. One of the major proteins involved in ER stress is glucose-regulated protein 78 (GRP78), which binds to unfolded proteins and dissociates from membrane-bound ER stress sensors. To determine the role of ER stress responses during anti-angiogenic therapy and the potential role of GRP78 in combined therapy in renal cell carcinoma (RCC), we used GRP78 overexpressing or knockdown RCC cells under hypoxic or hypoglycemic conditions in vitro and in animal models treated with sunitinib. Here, we report that GRP78 plays a crucial role in protecting RCC cells from hypoxic and hypoglycemic stress induced by anti-angiogenic therapy. Knockdown of GRP78 using siRNA inhibited cancer cell survival and induced apoptosis in RCC cells in vitro and also resulted in ER stress-induced apoptosis and hypoxic/hypoglycemic stress-induced apoptosis by inactivating the PERK/eIF-2α pathway. Finally, GRP78 knockdown showed potent suppression of tumor growth and enhanced the antitumor effect of sunitinib in RCC xenografts. Our findings suggest that GRP78 may serve as a novel therapeutic target in combination with anti-angiogenic therapy for the management of RCC.

Genome-wide analysis of differentially expressed miRNA in PLX4720-resistant and parental human thyroid cancer cell lines

Investigating BRAF((V600E)) inhibitors (BRAFi) as a strategy to treat patients with aggressive thyroid tumors harboring the BRAF((V600E)) mutant currently is in progress, and drug resistance is expected to pose a challenge. MicroRNAs (miRNAs) are involved in development of resistance to a variety of drugs in different malignancies. miRNA expression profiles in the human anaplastic thyroid cancer cell line (8505c) were compared with its PLX4720-resistant counterpart (8505c-R) by the use of Illumina deep sequencing. We conducted a functional annotation and pathway analysis of the putative and experimentally validated target genes of the significantly altered miRNAs. We identified 61 known and 2 novel miRNAs whose expression was altered greatly in 8505c-R. Quantitative reverse-transcription polymerase chain reaction validated altered expression of 7 selected miRNAs in 8505c-R and BCPAP-R (PLX4720-resistant papillary thyroid cancer cell line). We found 14 and 25 miRNAs whose expression levels changed substantially in 8505c and 8505c-R, respectively, after treatment with BRAFi. The mitogen-activated protein kinase and phosphatidylinositol 3-kinase-AKT pathways were among the prominent targets of many of the deregulated miRNAs. We have identified a number of miRNAs that could be used as biomarkers of resistance to BRAFi in patients with thyroid cancer. In addition, these miRNAs can be explored as potential therapeutic targets in combination with BRAFi to overcome resistance.

Relationship between serum response factor and androgen receptor in prostate cancer.

Serum response factor (SRF) is an important transcription factor in castrate-resistant prostate cancer (CRPC). Since CRPC is associated with androgen receptor (AR) hypersensitivity, we investigated the relationship between SRF and AR. Transcriptional activity was assessed by luciferase assay. Cell proliferation was measured by MTT and flow cytometry. Protein expression in patients was assessed by immunohistochemistry. To investigate AR involvement in SRF response to androgen, AR expression was down-regulated using siRNA. This resulted in the abrogation of SRF induction post-DHT. Moreover, DHT stimulation failed to induce SRF transcriptional activity in AR-negative PC346 DCC cells, which was only restored following AR over-expression. Next, SRF expression was down-regulated by siRNA, resulting in AR increased transcriptional activity in castrate-resistant LNCaP Abl cells but not in the parental LNCaP. This negative feedback loop in the resistant cells was confirmed by immunohistochemistry which showed a negative correlation between AR and SRF expression in CRPC bone metastases and a positive correlation in androgen-naïve prostatectomies. Cell proliferation was next assessed following SRF inhibition, demonstrating that SRF inhibition is more effective than AR inhibition in castrate-resistant cells. Our data support SRF as a promising therapeutic target in combination with current treatments.

Monocarboxylate transport inhibition potentiates the cytotoxic effect of 5-fluorouracil in colorectal cancer cells

Cancer cells rely mostly on glycolysis to meet their energetic demands, producing large amounts of lactate that are extruded to the tumour microenvironment by monocarboxylate transporters (MCTs). The role of MCTs in the survival of colorectal cancer (CRC) cells is scarce and poorly understood. In this study, we aimed to better understand this issue and exploit these transporters as novel therapeutic targets alone or in combination with the CRC classical chemotherapeutic drug 5-Fluorouracil.For that purpose, we characterized the effects of MCT activity inhibition in normal and CRC derived cell lines and assessed the effect of MCT inhibition in combination with 5-FU.Here, we demonstrated that MCT inhibition using CHC (α-cyano-4-hydroxycinnamic acid), DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid) and quercetin decreased cell viability, disrupted the glycolytic phenotype, inhibited proliferation and enhanced cell death in CRC cells. These results were confirmed by specific inhibition of MCT1/4 by RNA interference. Notably, we showed that 5-FU cytotoxicity was potentiated by lactate transport inhibition in CRC cells, either by activity inhibition or expression silencing.These findings provide novel evidence for the pivotal role of MCTs in CRC maintenance and survival, as well as for the use of these transporters as potential new therapeutic targets in combination with CRC conventional therapy.

PGRMC1 contributes to doxorubicin-induced chemoresistance in MES-SA uterine sarcoma.

Chemotherapy is one of the major categories of medical oncology and a primary tumor treatment; however, the effectiveness of chemotherapy is restricted by drug resistance. Overcoming resistance to chemotherapy and investigating molecular targeted therapies are challenges currently faced during resistance management. Progesterone receptor membrane component 1 (PGRMC1) is an adapter protein mediating cholesterol synthesis, steroid signaling, and cytochrome p450 activation. Attention has recently focused on the role of PGRMC1 in cell survival, anti-apoptosis, and damage response. In the present study, we used knockdown and overexpression approaches in the following set of uterine sarcoma models to further evaluate the role of PGRMC1 in drug resistance: the doxorubicin-sensitive MES-SA cells and the doxorubicin-resistant MES-SA/DxR-2 µM and MES-SA/DxR-8 µM cells (with different levels of doxorubicin resistance). PGRMC1 repressed doxorubicin-induced cytotoxicity and exhibited an anti-apoptotic effect; it also promoted cell proliferation and cell cycle progression to the S phase. Of note, PGRMC1 overexpression led to the epithelial-mesenchymal transition (EMT) of the sensitive MES-SA cells, thus facilitating their migration and invasion. The combination of PGRMC1 knockdown and the P-glycoprotein inhibitor verapamil significantly decreased the viability of P-glycoprotein-overexpressing MES-SA/DxR-8 μM cells after doxorubicin treatment. Taken together, our results show that PGRMC1 contributed to chemoresistance through cell proliferation, anti-apoptosis, and EMT induction, leading to the suggestion that PGRMC1 may serve as a therapeutic target in combination with an inhibitor in different drug resistance pathways and indicating the usefulness of predictive resistance biomarkers in uterine sarcoma.

HspX knock-out in Mycobacterium tuberculosis leads to shorter antibiotic treatment and lower relapse rate in a mouse model – A potential novel therapeutic target

Effective global tuberculosis control is hindered by the need for prolonged chemotherapy which leads to poor patient compliance. Therefore novel drug targets that shorten the duration of chemotherapy and reduce disease relapse rates are highly desirable. We have previously shown that HspX, an alpha-crystallin-like protein, is associated with growth suppression of Mycobacterium tuberculosis in mouse models. We determined to evaluate hspX as a novel target for controlling M. tuberculosis growth in combination with traditional antibiotic therapy in the Cornell mouse model. The hspX deletion mutant (ΔhspX) was used as a model of potential hspX inhibition. Normal BALB/c mice were infected with ΔhspX or the wild type (WT) strain. Three weeks after infection, the mice were treated with rifampicin, isoniazid and pyrazinamide for 14 weeks followed by 8 weeks of hydrocortisone. The effect of chemotherapy was measured by organ bacterial counts and the relapse rate. Antibiotic treatment of mice infected with ΔhspX resulted in faster visceral clearance; organs were disease free 8 weeks post-treatment for ΔhspX infection compared to 14 weeks for the WT strain. Disease relapse rate was significantly lower in ΔhspX infection (60.7%) compared to WT infection (92.6%). HspX may be a promising therapeutic target in combination with traditional antibiotic therapy to shorten the length of treatment and reduce disease relapse.

Galectin-3 as a marker and potential therapeutic target in breast cancer.

Galectin-3 has a relatively high level of expression in triple-negative breast cancers and is a potential marker for this disease. However, the clinical and prognostic implications of galectin-3 expression in breast cancer remain unclear. We examined mastectomy specimens from 1086 breast cancer cases and matching, adjacent non-cancerous tissues using immunohistochemistry. Overall, triple-negative breast cancers expressed galectin-3 more strongly than did other breast cancers types (63.59% vs 21.36%, P = 0.001). Galectin-3 expression was not found to be an independent prognostic factor for breast cancer by Cox regression analysis, but was associated with chemotherapeutic resistance. Apoptosis was only weakly induced by arsenic trioxide (ATO) treatment in galectin-3-positive breast cancer cells (MDA-MB-231 and MCF-7), although ATO treatment up-regulated galectin-3 expression. Knockdown of galectin-3 in MDA-MB-231 cells sensitized them to killing by ATO. These findings support a possible role for galectin-3 as a marker for triple-negative breast cancer progression and as a therapeutic target in combination with ATO treatment, although the mechanisms that underlie this synergy require further investigation.

Hedgehog signalling in primary cervix xenograft (OCICx) models: A potential new therapeutic target in combination with chemoradiotherapy.

11120 Background: Overcoming treatment resistance and repopulation in patients undergoing chemo-radiotherapy for primary treatment of cervical cancer is critical in order to improve long-term survival. Upregulation of Hh gene expression in women with cervical cancer prior to chemoradiotherapy is associated with a worse outcome. Aim: to investigate inhibition of the Hh pathway in combination with chemoradiotherapy in orthotopic primary cervical cancer xenograft models (OCICX). Methods: mRNA expression levels of Hh pathway members were characterized using qRT-PCR analysis in OCICx models. To simulate clinical treatment 5E1 (antibody blocking SHH and IHH ligand interaction with PTCH1) was administered to mice bearing cervix xenografts (20 mg/kg/weekly) with localized fractionated irradiation (15 x 2Gy fractions given 5 days/wk over 3 wks). Tumor growth delay, lymph node metastases, survival and toxicity were assessed. Results: The OCICX models exhibited matched levels of Hh gene expression compared to the pa...

Clinical Relevance Of Cachexia Assessed By An Anthropometric Tool In Elderly Patients With Diffuse Large B-Cell Lymphoma Treated By Immunochemotherapy

BackgroundCancer Cachexia is a metabolic syndrome that can be present even in absence of weight loss and associated with significantly impaired survival. Muscle wasting represents a key-symptom of this syndrome and we recently demonstrated the strong prognosis impact of sarcopenia assessed by computed tomography (CT)-scan in diffuse large B-cell lymphoma (DLBCL) (Lanic et al. Leukemia & Lymphoma 2013). Conversely, the clinical relevance of loss of fat mass (adipopenia) remains unclear. The aim of this study was (i) to investigate the prognostic impact of a multidimensional tool combining a nutritional parameter (albuminemia) and body composition measurements (skeletal muscle and body fat composition) in elderly patients with DLBCL treated by chemotherapy and rituximab (R) (ii) to document the evolution of sarcopenia after immunochemotherapy. MethodsThis retrospective analysis included 80 DLBCL patients older than 70 years (y) and treated by R-CHOP or R-miniCHOP. Skeletal muscle (SM), visceral (V) and subcutaneal (S) adipose (A) tissues were measured by analysis of stored CT images at the Lumbar vertebrae 3 (L3) level. The surface of the muscular and adipose tissues was selected according to CT Hounsfield unit. Values were normalized for stature to calculate the L3 SM index (LSMI, in cm2/m2), the LVAI and the LSAI and used to define sarcopenia and visceral/subcutaneal adipopenia. ResultsThe characteristics of the patients were as follows: median age = 78 y [70-95]; 36 males; IPI 0-2 = 22, 3-5 = 58; treatment by R-CHOP (n = 45) or R-miniCHOP (n = 35); median body mass index (BMI; in kg/m2) = 23.9. According to the sex-specific defined cut-offs for LSMI (< 55.8 cm²/m² for men and 38.9 cm²/m² for women), 44 DLBCL patients (55 %, 23 males) were considered as sarcopenic. With a median follow-up of 39 months, the 2y overall survival (OS) in the sarcopenic population was 46% as compared to 84% in the non-sarcopenic group (HR = 3.12; CI95%, 1.66-5.88; p=0.0004). The median LSAI was 76.3 cm2/m2 [10-167] in females and 47.4 cm2/m2[22-100] in males. The median LVSAI was 43.5 cm2/m2[3-141] in females and 50.4 cm2/m2[14-159] in males. Adipopenia, defined by a low LVAI and/or a low LSAI was also highly predictive of the outcome. The 2y OS of the low LVAI population was 48% as compared to 82% for the non-adipopenic group (HR = 2.20; CI95%, 1.19-4.05; p=0.01). The 2y OS in the low LSAI population was 48% as compared to 80% in the non-adipopenic group (HR = 2.28; CI95%, 1.23-4.21; p=0.008). A Three-point cachexia score (CS) including adipopenia, sarcopenia and hypo-albuminemia (defined by an albuminemia < 35 g/L) was build and delineates three distinct risk-groups (Figure 1). More importantly the CS remains predictive of the prognosis in a multivariate analysis including BMI (< or >= 25 kg/m2), age (< or >= 80y), IPI and gender (HR=2.5; CI95%= 1.14-5.39; p =0.02). LMSI was subsequently reassessed in thirty seven patients during the routine CT scan follow-up [mean = 10 months after pre-treatment CT scan (range 2.8-19.2)]. 15 (40%) patients displayed a 5% decrease of their LSMI, whereas 13 (35%) and 9 (25%) displayed no significant change or increase (>5%) of the LMSI respectively. [Display omitted] ConclusionOur study demonstrates that sarcopenia and adipopenia estimated by CT-scan define cachexia more accurately than BMI or weight loss in elderly DLBCL patients. These factors can be integrated in a cachexia scoring tool which predicts the outcome independently of the BMI and of the IPI. CT scan follow-up indicates that cachexia is a reversible process that should be integrated as part of the therapeutic target in combination with lymphoma treatment. A prospective multicentric trial (registered as NCT01715961/Clinical.gov) is ongoing to validate these anthropometric and nutritional parameters and compare to geriatric assessment scales. Disclosures:No relevant conflicts of interest to declare.

Abstract 2059: Kinase library siRNA screen identifies KSR1 as a synergistic therapeutic target in combination with TIC10.

Abstract Apo2L/TRAIL is an endogenous antitumor protein that possesses efficacy-limiting properties such as short serum half-life, stability, cost, and limitations in delivery when used as a recombinant protein therapeutic. TIC10 is a first-in-class small molecule inducer of the human TRAIL gene that improves these unfavorable drug properties to ultimately increase the efficacy and utility of TRAIL. TIC10 upregulates TRAIL gene transcription by activating the pro-apoptotic transcription factor Foxo3a, which is achieved through dual inhibition of Akt and MEK/ERK. We conducted a siRNA screen using a kinase library to identify kinases that affect TIC10 response in cancer cells assayed by cell viability assays to gain mechanistic insight into the molecular mechanisms of TIC10 and potentially identify a combinatorial target to improve the activity of TIC10. Execution and validation of the siRNA screen revealed 4 kinases (DGKD, SGKL, STK23, KSR) that improved the therapeutic response to TIC10 upon siRNA-mediated knockdown at 36 hours, but not 12 hours, post-treatment in HCT116 human colon cancer cells. We next assayed the effect of knocking down these 4 kinases on cancer cell cytotoxic response to recombinant human TRAIL. These studies revealed no effect with kinase knockdowns, suggesting that the mechanisms of sensitization lie upstream of TIC10-mediated TRAIL production. Network analysis of the “hit” kinases and the previously described mechanism of TIC10 revealed rational overlap of signaling pathways and kinetics. Among the 4 identified kinases, KSR1 possessed the most direct connection to the putative mechanism of TIC10 that involves the dual inhibition of Akt and the MAPK pathway. KSR1 is a MAPK scaffold protein that positively regulates the MAPK signaling pathway. Based on these observations, we are testing the hypothesis that inhibiting KSR1 in combination with TIC10 cooperatively inhibits MAPK signaling to increase TRAIL production. Together, these results suggest that KSR1 is a key player in the cytotoxic response to TIC10 and that combining TIC10 and an inhibitor of KSR1 may result in improved therapeutic response. Citation Format: Joshua E. Allen, Wafik S. El-Deiry. Kinase library siRNA screen identifies KSR1 as a synergistic therapeutic target in combination with TIC10. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2059. doi:10.1158/1538-7445.AM2013-2059

Valeur pronostique de la détection de cellules tumorales circulantes au cours du mélanome métastatique

Investigating BRAF(V600E) inhibitors (BRAFi) as a strategy to treat patients with aggressive thyroid tumors harboring the BRAF(V600E) mutant currently is in progress, and drug resistance is expected to pose a challenge. MicroRNAs (miRNAs) are involved in development of resistance to a variety of drugs in different malignancies.miRNA expression profiles in the human anaplastic thyroid cancer cell line (8505c) were compared with its PLX4720-resistant counterpart (8505c-R) by the use of Illumina deep sequencing. We conducted a functional annotation and pathway analysis of the putative and experimentally validated target genes of the significantly altered miRNAs.We identified 61 known and 2 novel miRNAs whose expression was altered greatly in 8505c-R. Quantitative reverse-transcription polymerase chain reaction validated altered expression of 7 selected miRNAs in 8505c-R and BCPAP-R (PLX4720-resistant papillary thyroid cancer cell line). We found 14 and 25 miRNAs whose expression levels changed substantially in 8505c and 8505c-R, respectively, after treatment with BRAFi. The mitogen-activated protein kinase and phosphatidylinositol 3-kinase-AKT pathways were among the prominent targets of many of the deregulated miRNAs.We have identified a number of miRNAs that could be used as biomarkers of resistance to BRAFi in patients with thyroid cancer. In addition, these miRNAs can be explored as potential therapeutic targets in combination with BRAFi to overcome resistance.

Enhancement of Carboplatin- and Quercetin-Induced Cell Death by Roscovitine Is Akt Dependent and p53 Independent in Hepatoma Cells

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and has an annual occurrence of one million new cases. Novel therapeutic strategies of increased efficacy in the treatment of HCC-bearing patients would certainly be helpful. Hence, the authors explored the effect of combination treatment of roscovitine with chemotherapeutic drugs or quercetin (Qctn) in hepatoma cells, HepG2 and Hep3B. Cell viability was assessed by MTT assay, cell growth assay, and nuclear morphological changes by DAPI staining. The altered expression of signaling proteins and apoptotic molecules was established by Western blotting. Roscovitine pretreatment considerably enhanced the drugs and Qctn-induced cell death in HepG2 and Hep3B cells. The exploratory studies revealed that augmented cell killing in HepG2 and Hep3B was mediated via Akt pathway and was independent of p53. pAkt was found to be significantly downregulated in combination treatment of roscovitine with carboplatin or Qctn. Corresponding to reduced expression of pAkt, the downstream molecules Bcl-2 and proactive forms of caspase 9 and caspase 3 were also downregulated indicating apoptosis. The present study reports for the first time, in hepatoma cells, the potentiation of carboplatin- and Qctn-induced cell death by the cell cycle inhibitor roscovitine. Roscovitine can thus be considered as a potential therapeutic target in combination with chemotherapeutic drugs or Qctn for treatment of HCC.

Abstract 3147: HER2 gene amplification and CDC25A overexpression in human breast cancer

Abstract HER2 gene amplification/overexpression has been identified in 10-34% of invasive breast cancers and correlates with reduced disease-free and overall survival. The HER2-targeted therapy with trastuzumab decreases the risk of relapse and prolongs survival. Resistance to trastuzumab is a significant barrier to the effective HER2-positive breast cancer treatment: response rate ranges from 20-35%, underlining that HER2-positive breast cancer is hardly a homogeneous disease and, more importantly, that other HER2-related pathways need to be unveiled. Recent studies provided evidence that HER2 overexpression increases skin tumorigenesis through CDC25A maintenance: HER2 suppresses DNA damage-induced checkpoint activation through lack of CDC25A degradation. CDC25A phosphatase, an essential component of the cell cycle machinery, is also a key player in integrating the specific signals of checkpoint control in response to DNA damage. CDC25A misregulation contributes to genomic instability and cancer development. Oncogene activation during early stages of tumor development causes DNA replication stress resulting in the DNA damage response (DDR) induction and the selection of cells defecting in their DDR could lead to malignant progression. Accordingly, CDC25A overexpression in primary human breast cells promotes the DDR alteration, increasing the chances of tumorigenic conversion by enhancing genomic instability at common fragile sites. The aim of our work was to verify a relation between HER2 and CDC25A in human breast cancer. To this end HER2 amplification and CDC25A overexpression were determined in 220 breast cancer patients. We found a statistically significant correlation between HER2 gene status and CDC25A expression (p=0,000): 64,4% of HER2 amplified tumours show CDC25A overexpression and 75,5% of tumours with no HER2 amplification do not overexpress CDC25A. Furthermore HER2 status and CDC25A expression significantly correlate with prognosis (p=0,003 and p=0,047 respectively). A mortality risk stratification was identified when four different combinations of HER2 status and CDC25A expression were evaluated for their relative effect on survival: the group with the highest mortality was the one in which breast carcinomas showed both HER2 amplification and CDC25A overexpression (p=0,019). Our in vitro studies demonstrate that HER2 can stabilize CDC25A protein levels in breast cancer cell lines, through PI3K-AKT pathway: decreased HER2-mediated signaling by HER2 inhibitors resulted in CDC25A decreased expression; remarkably CDC25A inactivation by specific inhibitor can restore response to IR-induced DNA damage in HER2-positive, both trastuzumab sensitive and resistant, breast cancer cell lines. These findings suggest a CDC25A role for a more specific characterization of HER2-positive breast cancers, warranting further study of its potential as therapeutic target in combination with HER2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3147. doi:10.1158/1538-7445.AM2011-3147

Integrin β1-dependent invasive migration of irradiation-tolerant human lung adenocarcinoma cells in 3D collagen matrix

Radiotherapy is one of the effective therapies used for treating various malignant tumors. However, the emergence of tolerant cells after irradiation remains problematic due to their high metastatic ability, sometimes indicative of poor prognosis. In this study, we showed that subcloned human lung adenocarcinoma cells (A549P-3) that are irradiation-tolerant indicate high invasive activity in vitro, and exhibit an integrin β1 activity-dependent migratory pattern. In collagen gel overlay assay, majority of the A549P-3 cells displayed round morphology and low migration activity, whereas a considerable number of A549P-3IR cells surviving irradiation displayed a spindle morphology and high migration rate. Blocking integrin β1 activity reduced the migration rate of A549P-3IR cells and altered the cell morphology allowing them to assume a round shape. These results suggest that the A549P-3 cells surviving irradiation acquire a highly invasive integrin β1-dependent phenotype, and integrin β1 might be a potentially effective therapeutic target in combination with radiotherapy.

Abstract LB-345: Functional genomics reveals acute dependencies on polyadenylation for tumor cell mitotic progression

Abstract We are using genome-wide synthetic lethal paclitaxel chemosensitizer screens to understand the protein networks that support mitosis in lung tumor cells. Using this approach, we have identified several components of the polyadenylation machinery that are required for a high-fidelity mitosis in the presence of a synthetic lethal dose of paclitaxel. Here, we show depletion of multiple polyadenylation proteins in combination with low-dose paclitaxel causes a G2/M cell cycle block. We show depletion of one of these polyadenylation proteins, symplekin, has profound effects on mitotic spindle assembly resulting in over-duplicated centrosomes and impaired microtubule growth. Furthermore, we use a panel of lung lines to show symplekin is uniquely required for tumor cell mitosis as compared to normal cell mitosis. Our mechanistic studies suggest symplekin may mediate these effects on tumor cell mitosis by interacting with the centrosomally-associated protein transforming acidic coiled coil 3 protein (TACC3). Interestingly, TACC3 is typically expressed during development but becomes aberrantly upregulated in tumor cells. We propose TACC3 may become upregulated during tumorigenesis and engage symplekin to support mitosis in tumor cells. These results suggest polyadenylation components may represent an attractive therapeutic target in combination with already available anti-mitotic agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-345.

Abstract 2564: High-throughput siRNA library screen for discovery and identification of new melanoma therapeutic targets in combination with antioxidant pretreatment

Abstract Melanoma is one of the most prevalent cancers in the United States, having the fifth highest cancer incidence in males and sixth highest incidence in females. Primary cutaneous melanoma can often be cured by surgery, but for those patients in whom metastasis has occurred, mortality is high in part due to numerous chemoresistance mechanisms. Thus, there is an urgent need to discover and identify novel therapeutic targets for the efficient treatment of this lethal disease. Gene silencing by RNA interference (RNAi) is a powerful tool to identify novel genes involved in specific biological processes. In our study, we used high-throughput druggable siRNA library (Dharmacon) targeting 6028 human genes to screen metastatic melanoma cell lines and found that knockdown of gene expression by siRNA pools targeting 885 genes significantly inhibited melanoma growth (p&amp;lt;0.005). Among them, 67 siRNA pools resulted in &amp;gt;50% inhibition of cell viability in the melanoma A375 cells which we employed for the screening analysis. We validated the top-six cancer-related genes (RRM2, BCAR3, PSMA2, UBB, RRM1, TCP1) as potential melanoma therapeutic target candidates, as knockdown of these 6 genes significantly inhibited cell growth and induced apoptosis in multiple melanoma cell lines. We have hypothesized that antioxidant treatment relieves resistance mechanisms, and showed that among the screened &amp;gt;6000 siRNA pools, treatment with 20 pools significantly increased the growth inhibition in the presence of nontoxic levels of bardoxolone methyl (p&amp;lt;0.005), a novel semi-synthetic triterpenoid (previously known as CDDO-Me) with potent antioxidant activity generously provided by Reata Pharmaceuticals. We also selected 5 additional cancer-related genes (GNPAT, SUMO1, SPINT2, FLI1 and SSX1) for further analysis and validated them as additional candidates for therapeutic targeting in the presence of antioxidant. Our results thus demonstrate the feasibility of the high-throughput siRNA library screen system in discovering new melanoma therapeutic targets and shows that these new candidate genes are promising for the development of novel therapeutic targets for melanoma treatment, many of which are revealed by antioxidant pretreatment. Results from our study provide the basis for significant advances in identifying targets for the treatment of melanoma patients. (This study was partially supported by the grant from the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation and the NIH Melanoma SPORE grant P50, CA093459). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2564.

Expression and function of phosphodiesterases in nitrofen‐induced congenital diaphragmatic hernia in rats

Congenital diaphragmatic hernia (CDH) is an anomaly associated with pulmonary hypoplasia and pulmonary hypertension (PH). The limited efficacy of current approaches to treat PH in CDH, including inhaled nitric oxide (NO), drives the search for other therapies. Phosphodiesterases (PDEs) degrade cyclic nucleotide second messenger cAMP and cGMP downstream of NO thereby limiting the vasodilatory response to NO. To identify therapeutic targets by cataloguing the expression and function of PDE isoforms in the pulmonary vasculature in nitrofen-induced CDH in fetal rats. Quantitative RT-PCR revealed PDE1-5 and PDE9 mRNA expression in pulmonary arteries (PAs) of control and nitrofen-induced CDH term fetal rats. In this order of potency, the PDE inhibitors Sildenafil (PDE5) > EHNA (PDE2) > Rolipram (PDE4) > Cilostamide (PDE3) all dilated isolated third generation PA after pre-constriction with the thromboxane analog U46619. Hyperoxic pre-incubation of PAs significantly attenuated vasodilatation induced by the PDE5 inhibitor Sildenafil (65% vs. 33%, P < 0.004). CDH PAs dilated significantly less to PDE2 inhibitor EHNA compared to control (51% vs. 72%, P < 0.05). Subsequently PDE2 protein expression was higher in PAs of CDH animals. Most PDE isoforms exist in the PAs of fetal rats and their inhibition causes pulmonary vasodilatation. PDE5 inhibition was the most potent vasodilator, however, there were no differences between groups. PDE5-induced vasodilatation was attenuated by hyperoxic pre-incubation. PDE inhibitors might be considered therapeutic targets in combination with iNO in neonates with CDH.

Update on Anti-EGFR Targeted Therapy

Targeted therapy has evolved recently as an important treatment modality for cancer, and the most extensively studied pathways for targeted therapy are those related to epidermal growth factor receptor (EGFR). EGFR is widely expressed in different tumor types and strong expression is associated with higher risk of recurrence/metastasis, poorer survival, and resistance to chemotherapy/radiotherapy. Both monoclonal antibody against EGFR and/or tyrosine kinase inhibitors are now in use clinically for treatment of advanced cancer. With the increasing usage of these agents and the availability of more new agents in the future, knowledge on anti-EGFR therapy is highly relevant to the current state of cancer therapy and to new research directions in the field of oncology. The focus of this special issue is on the clinical applications of approved or investigational anti-EGFR therapy in solid tumors with emphasis on efficacy, toxicity, response, assessment, multimodality treatment, prognostic factors and predictive markers. The review by A. Harandi et al. provides a comprehensive and updated overview of the frequently used EGFR inhibitors and summarizes clinical efficacy data of these agents and their associated toxicity and management. This review clearly shows the impact of anti-EGFR therapy on cancer treatment since improved treatment results can be seen with the use of anti-EGFR therapy in many common cancers. One of these cancers is colorectal cancer and the approval of anti-EGFR monoclonal antibodies in the treatment of metastatic colorectal cancer has expanded the armamentarium against this disease. H. Loong et al. in their review summarize the historical progress and recent clinical developments of anti-EGFR therapies in the treatment of metastatic CRC and discuss the novel strategies of targeting the EGFR pathway to improve efficacy as well as ongoing research in identifying specific molecular predictors of response. While the approved use of drugs like the dual kinase inhibitor Lapatinib represents significant advances in the clinical management of breast cancer, confirmatory studies must be considered to foster the use of anti-EGFR therapies including safety, pharmacokinetics, and clinical efficacy in this common cancer. The article by J. Flynn et al. reviews the mechanism of anti-EGFR therapy in breast cancer and summarizes recent advances including the development of improved high-throughput analyses for identifying novel anti-EGFR activity, as well as advances in DNA/RNA-microarray technology for classification purposes, which will contribute to the overall understanding and development of targeted therapy in treatment of breast cancer. Cancers of the esophagus and stomach present a major health burden worldwide. The impact of cytotoxic agents on the disease has been modest. EGFR pathway has also been implicated in pathophysiology of esophageal and stomach cancers. Recently EGFR inhibitors have been explored in patients with esophageal and gastric cancers, and the results are summarized by T. Dragovich and C. Campen in their review. It also appears that tumors of the distal esophagus and gastroesophageal junction are more sensitive to EGFR blockade than distal gastric adenocarcinoma. Similar to cytotoxic agenst, drug resistance remains an important issue and research field in targeted therapy. The article by J. Rolff et al. studied the impact of different resistance markers at protein and mRNA level in patient-derived nonsmall cell lung cancer xenografts in response to erlotinib and other cytotoxic agents. The results suggest that the expression levels of multidrug resistant proteins and mRNA do not play an important role in the drug resistance of nonsmall cell lung cancer. Increased transforming growth factor-β (TGF-β) expression and EGFR amplification accompany the emergence of highly aggressive human carcinomas, and cooperative signaling between these two growth factor/receptor systems promotes cell migration and synthesis of stromal remodeling factors that in turn regulate tumor invasion, neo-angiogenesis and inflammation. R. Samarakoon et al. studied the role of plasminogen activator inhibitor-1 in carcinogensis and explored its potential role as a novel therapeutic target in combination with EGFR inhibition. We hope that this special issue will stimulate interests and new researches in the development of anti-EGFR therapy in cancers, particularly the development of personalized medicine based on predictive biomarkers so that therapy can be tailored and optimized in every patient. Daniel Chua Peter Fasching Brigette Ma Sumitra Thongprasert Lori Wirth

Contribution of Galactofuranose to the Virulence of the Opportunistic Pathogen Aspergillus fumigatus

The filamentous fungus Aspergillus fumigatus is responsible for a lethal disease called invasive aspergillosis that affects immunocompromised patients. This disease, like other human fungal diseases, is generally treated by compounds targeting the primary fungal cell membrane sterol. Recently, glucan synthesis inhibitors were added to the limited antifungal arsenal and encouraged the search for novel targets in cell wall biosynthesis. Although galactomannan is a major component of the A. fumigatus cell wall and extracellular matrix, the biosynthesis and role of galactomannan are currently unknown. By a targeted gene deletion approach, we demonstrate that UDP-galactopyranose mutase, a key enzyme of galactofuranose metabolism, controls the biosynthesis of galactomannan and galactofuranose containing glycoconjugates. The glfA deletion mutant generated in this study is devoid of galactofuranose and displays attenuated virulence in a low-dose mouse model of invasive aspergillosis that likely reflects the impaired growth of the mutant at mammalian body temperature. Furthermore, the absence of galactofuranose results in a thinner cell wall that correlates with an increased susceptibility to several antifungal agents. The UDP-galactopyranose mutase thus appears to be an appealing adjunct therapeutic target in combination with other drugs against A. fumigatus. Its absence from mammalian cells indeed offers a considerable advantage to achieve therapeutic selectivity.

Calcineurin Inhibition or Mutation Enhances Cell Wall Inhibitors against Aspergillus fumigatus

Calcineurin mutation or inhibition enhanced the antifungal morphological effect of cell wall inhibitors caspofungin or nikkomycin Z against Aspergillus fumigatus. Quantification of 1,3-beta-d-glucan revealed decreased amounts in the calcineurin A (DeltacnaA) mutant. Calcineurin can be an excellent adjunct therapeutic target in combination with other cell wall inhibitors against A. fumigatus.