Decreased MAPK Signaling Research Articles

BMP4 and Temozolomide Synergize in the Majority of Patient-Derived Glioblastoma Cultures.

One of the main causes of poor prognoses in patient with glioblastoma (GBM) is drug resistance to current standard treatment, which includes chemoradiation and adjuvant temozolomide (TMZ). In addition, the concept of cancer stem cells provides new insights into therapy resistance and management also in GBM and glioblastoma stem cell-like cells (GSCs), which might contribute to therapy resistance. Bone morphogenetic protein-4 (BMP4) stimulates astroglial differentiation of GSCs and thereby reduces their self-renewal capacity. Exposure of GSCs to BMP4 may also sensitize these cells to TMZ. A recent phase I trial has shown that local delivery of BMP4 is safe, but a large variation in survival is seen in these treated patients and in features of their cultured tumors. We wanted to combine TMZ and BMP4 (TMZ + BMP4) therapy and assess the inter-tumoral variability in response to TMZ + BMP4 in patient-derived GBM cultures. A phase II trial could then benefit a larger group of patients than those treated with BMP4 only. We first show that simultaneous treatment with TMZ + BMP4 is more effective than sequential treatment. Second, when applying our optimized treatment protocol, 70% of a total of 20 GBM cultures displayed TMZ + BMP4 synergy. This combination induces cellular apoptosis and does not inhibit cell proliferation. Comparative bulk RNA-sequencing indicates that treatment with TMZ + BMP4 eventually results in decreased MAPK signaling, in line with previous evidence that increased MAPK signaling is associated with resistance to TMZ. Based on these results, we advocate further clinical trial research to test patient benefit and validate pathophysiological hypothesis.

Spatial genomic, biochemical and cellular mechanisms underlying meningioma heterogeneity and evolution.

Intratumor heterogeneity underlies cancer evolution and treatment resistance, but targetable mechanisms driving intratumor heterogeneity are poorly understood. Meningiomas are the most common primary intracranial tumors and are resistant to all medical therapies, and high-grade meningiomas have significant intratumor heterogeneity. Here we use spatial approaches to identify genomic, biochemical and cellular mechanisms linking intratumor heterogeneity to the molecular, temporal and spatial evolution of high-grade meningiomas. We show that divergent intratumor gene and protein expression programs distinguish high-grade meningiomas that are otherwise grouped together by current classification systems. Analyses of matched pairs of primary and recurrent meningiomas reveal spatial expansion of subclonal copy number variants associated with treatment resistance. Multiplexed sequential immunofluorescence and deconvolution of meningioma spatial transcriptomes using cell types from single-cell RNA sequencing show decreased immune infiltration, decreased MAPK signaling, increased PI3K-AKT signaling and increased cell proliferation, which are associated with meningioma recurrence. To translate these findings to preclinical models, we use CRISPR interference and lineage tracing approaches to identify combination therapies that target intratumor heterogeneity in meningioma cell co-cultures.

Abstract 3779: Patient-derived iPSCs reveal pharmacologic targeting mitochondrial respiratory complex I for treating Rothmund-Thomson syndrome associated osteosarcoma

Abstract Background: Rothmund-Thomson syndrome (RTS) is an autosomal recessive genetic disorder characterized by a rash that progresses to poikiloderma; small stature; skeletal anomalies; sparse hair, eyelashes, and/or eyebrows, juvenile cataracts, and an increased risk to cancer. Type 2 RTS patients with biallelic RECQL4 pathogenic variants have a significantly increased incidence of osteosarcoma. However, there is currently no available RTS model that recapitulates the bone malignancy phenotype in this disease, severely limiting the ability of researchers to explore new treatment avenues. Here, we generated RTS patient-derived induced pluripotent stem cells (iPSCs) to dissect the pathological signaling leading to RTS patient-associated osteosarcoma. Methods: iPSCs were reprogrammed from fibroblasts of four individuals from two RTS families, each with one affected RTS proband with RECQL4 mutations and one unaffected parent. These patient iPSCs were differentiated to mesenchymal stem cells (MSCs) and then to osteoblasts, the cell-of-origin of osteosarcoma. All of these MSCs and osteoblasts were subjected to transcriptome analysis by RNA-seq. Based on enriched mitochondrial respiratory gene signatures, we also systematically analyzed the enzymatic activities of the major mitochondrial respiratory protein complexes including complex I/II/III and citrate synthase enzyme activity in RTS osteoblasts by Seahorse assay. Finally, we tested the potential therapeutic effect of clinical oxidative phosphorylation complex I inhibitor IACS-010759 to treat RTS cells and dissect the pharmacological mechanisms involved. Results: RTS iPSC-derived osteoblasts showed defective osteogenic differentiation and a gain of in vitro tumorigenic ability. Transcriptome analysis of RTS osteoblasts revealed decreased bone morphogenesis gene expression but aberrantly upregulated mitochondrial complex I gene expression. Metabolic assays of RTS osteoblasts demonstrated elevated mitochondrial respiratory complex I function, increased oxidative phosphorylation, but decreased ATP production. Inhibition of mitochondrial respiratory complex I activity by IACS-010759 selectively suppressed cellular respiration and cell viability of RTS osteoblasts. Furthermore, systems analysis of IACS-010759-induced changes to RTS osteoblasts suggested that inhibition of mitochondrial complex I in RTS osteoblasts leads to enhanced cell senescence, decreased MAPK signaling and cell cycle associated genes. Conclusion: In summary, we established an RTS iPSC disease platform to dissect the pathological mechanisms involved in RTS-associated osteosarcoma. Our data suggested that mitochondrial complex I is a potential therapeutic target for RTS-associated osteosarcoma and provides future insights for clinical treatment strategies. Citation Format: An Xu, Brittany E. Jewell, Dandan Zhu, Mo-Fan Huang, Yi-Hung Chen, Linchao Lu, Ruiying Zhao, Lisa L. Wang, Dung-Fang Lee. Patient-derived iPSCs reveal pharmacologic targeting mitochondrial respiratory complex I for treating Rothmund-Thomson syndrome associated osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3779.

Exploring the total flavones of Abelmoschus manihot against IAV-induced lung inflammation by network pharmacology

BackgroundAbelmoschus manihot (L.) Medicus (AM) is a medicinal plant with various biological activities, including anti-inflammatory, antioxidant, antiviral and immunomodulatory. Previous studies have identified total flavones as the primary bioactive ingredient of AM (termed TFA). However, its role and mechanism in counteracting Influenza A virus (IAV) infection are yet to be explored. Therefore, the study aims to study the antiviral and anti-inflammatory effects of TFA on IAV in vitro and in vivo.MethodsA network pharmacology-based approach was applied to identify the antiviral mechanism of TFA against IAV. For the mechanism validation, the cytopathic effect reduction assay evaluated the antiviral activity of TFA in vitro. Meanwhile, the mice were intranasally infected with IAV to induce lung infection. The antiviral effect of TFA was observed in vivo. Further investigation whether the reprogramming microbiome in the TFA treatment group affected antiviral, we conducted a microbial-transfer study with co-housing experiments.ResultsBy applying the network pharmacology-based methods (PPI, GO, and KEGG), we identified 167 potential targets of TFA action, among which 62 targets were related to IAV pathogenesis. A core network containing the pro-inflammatory TNFα, IL-6, IL-1β, MAPKs, and RIG-I receptor signaling pathway was further confirmed as the crucial targets for anti-influenza efficacy of TFA. We demonstrate that TFA provided profound protection against pulmonary IAV infection, which alleviated inflammatory responses, decreased MAPK signaling pathway and expedited viral eradiation.ConclusionsOur study unveils a pivotal role for TFA in controlling viral infection and dampening pathology, making it a promising strategy for treating IAV-induced pneumonia.

Patient-derived iPSCs link elevated mitochondrial respiratory complex I function to osteosarcoma in Rothmund-Thomson syndrome.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive genetic disorder characterized by poikiloderma, small stature, skeletal anomalies, sparse brows/lashes, cataracts, and predisposition to cancer. Type 2 RTS patients with biallelic RECQL4 pathogenic variants have multiple skeletal anomalies and a significantly increased incidence of osteosarcoma. Here, we generated RTS patient-derived induced pluripotent stem cells (iPSCs) to dissect the pathological signaling leading to RTS patient-associated osteosarcoma. RTS iPSC-derived osteoblasts showed defective osteogenic differentiation and gain of in vitro tumorigenic ability. Transcriptome analysis of RTS osteoblasts validated decreased bone morphogenesis while revealing aberrantly upregulated mitochondrial respiratory complex I gene expression. RTS osteoblast metabolic assays demonstrated elevated mitochondrial respiratory complex I function, increased oxidative phosphorylation (OXPHOS), and increased ATP production. Inhibition of mitochondrial respiratory complex I activity by IACS-010759 selectively suppressed cellular respiration and cell proliferation of RTS osteoblasts. Furthermore, systems analysis of IACS-010759-induced changes in RTS osteoblasts revealed that chemical inhibition of mitochondrial respiratory complex I impaired cell proliferation, induced senescence, and decreased MAPK signaling and cell cycle associated genes, but increased H19 and ribosomal protein genes. In summary, our study suggests that mitochondrial respiratory complex I is a potential therapeutic target for RTS-associated osteosarcoma and provides future insights for clinical treatment strategies.

Effects of somatostatin, curcumin, and quercetin on the fatty acid profile of breast cancer cell membranes.

Breast cancer is a worldwide commonly found malignancy in women and effective treatment is regarded as a huge clinical challenge even in the presence of several treatment options. Extensive literature is available demonstrating polyphenols as phytopharmaceutical anticancer agents. Among the polyphenols, quercetin and curcumin have been reported to have a strong potential against breast cancer. However, so far, no comprehensive study has been performed to demonstrate the anticarcinogenic effects of curcumin, quercetin, and their combinations with somatostatin on the fatty acid profile of breast cancer cell membranes. We used MCF-7 and MDA-MB231 breast cancer cells incubated with curcumin and quercetin for 24 h, in the absence and presence of somatostatin, at their EC50 concentrations to evaluate membrane fatty acid based functional lipidomics together with the followup of EGFR and MAPK signaling pathways. The two cell lines gave different membrane free fatty acid reorganization. In MCF-7 cells, the following changes were observed: an increase of ω6 linoleic acid in the cells incubated with somatostatin + quercetin and quercetin and a decrease of ω3 acids in the cells incubated with somatostatin + curcumin compared to somatostatin and significant increases of monounsaturated fatty acid (MUFA), mono-trans arachidonic acid levels and docosapentaenoic acid for the cells incubated with somatostatin + quercetin compared to the control cells. In MDA-MB231 cells, incubations with curcumin, quercetin, and somatostatin + quercetin induced the most significant membrane remodeling with the increase of stearic acid, diminution of ω6 linoleic, arachidonic acids, and ω3 (docosapentaenoic and docosahexaenoic acids). Distinct signaling pathway changes were found for these cell lines. In MCF-7 cells, separate or combined incubations with somatostatin and quercetin, significantly decreased EGFR and incubation with curcumin decreased MAPK signaling. In MDA-MB231 cells, incubation with curcumin decreased AKT1 and p-AKT1 (Thr308) levels. Incubation with curcumin and quercetin decreased the EGFR levels. Our results showed that cytostatic and antioxidant treatments can be combined to induce membrane fatty acid changes, including lipid isomerization as specific free radical driven process, and to influence signaling pathways. This study aimed to contribute to the literature on these antioxidants in the treatment of breast cancer to clarify the effects and mechanisms in combination with somatostatin.

IQGAP1 Scaffold-MAP Kinase Interactions Enhance Multiple Myeloma Clonogenic Growth and Self-Renewal.

Despite improved outcomes in newly diagnosed multiple myeloma, virtually all patients relapse and ultimately develop drug-resistant disease. Aberrant RAS/MAPK signaling is activated in the majority of relapsed/refractory multiple myeloma patients, but its biological consequences are not fully understood. Self-renewal, as defined by the long-term maintenance of clonogenic growth, is essential for disease relapse, and we examined the role of RAS/MAPK activation on multiple myeloma self-renewal by targeting IQ motif-containing GTPase-activating protein 1 (IQGAP1), an intracellular scaffold protein required for mutant RAS signaling. We found that loss of IQGAP1 expression decreased MAPK signaling, cell-cycle progression, and tumor colony formation. Similarly, a peptide mimicking the WW domain of IQGAP1 that interacts with ERK inhibited the clonogenic growth and self-renewal of multiple myeloma cell lines and primary clinical specimens in vitro as well as tumor-initiating cell frequency in immunodeficient mice. During multiple myeloma progression, self-renewal may be enhanced by aberrant RAS/MAPK signaling and inhibited by targeting IQGAP1. Mol Cancer Ther; 15(11); 2733-9. ©2016 AACR.

Molecular subclasses of hepatocellular carcinoma predict sensitivity to fibroblast growth factor receptor inhibition.

A recent gene expression classification of hepatocellular carcinoma (HCC) includes a poor survival subclass termed S2 representing about one-third of all HCC in clinical series. S2 cells express E-cadherin and c-myc and secrete AFP. As the expression of fibroblast growth factor receptors (FGFRs) differs between S2 and non-S2 HCC, this study investigated whether molecular subclasses of HCC predict sensitivity to FGFR inhibition. S2 cell lines were significantly more sensitive (p < 0.001) to the FGFR inhibitors BGJ398 and AZD4547. BGJ398 decreased MAPK signaling in S2 but not in non-S2 cell lines. All cell lines expressed FGFR1 and FGFR2, but only S2 cell lines expressed FGFR3 and FGFR4. FGFR4 siRNA decreased proliferation by 44% or more in all five S2 cell lines (p < 0.05 for each cell line), a significantly greater decrease than seen with knockdown of FGFR1-3 with siRNA transfection. FGFR4 knockdown decreased MAPK signaling in S2 cell lines, but little effect was seen with knockdown of FGFR1-3. In conclusion, the S2 molecular subclass of HCC is sensitive to FGFR inhibition. FGFR4-MAPK signaling plays an important role in driving proliferation of a molecular subclass of HCC. This classification system may help to identify those patients who are most likely to benefit from inhibition of this pathway.

Molecular mechanism of anti-cancer activity of phycocyanin in triple-negative breast cancer cells.

BackgroundTriple-negative breast cancers represent an important clinical challenge, as these cancers do not respond to conventional endocrine therapies or other available targeted agents. Phycocyanin (PC), a natural, water soluble and non-toxic molecule is shown to have potent anti-cancer property.MethodsIn this study, we determined the efficacy of PC as an anti-neoplastic agent in vitro on a series of breast cancer cell lines. We studied effects of PC in inducing DNA damage and apoptosis through western blot and qPCR. Also, anti-metastatic and anti-angiogenic properties were studied by classic wound healing and vasculogenic mimicry assays.ResultsWe found that triple negative MDA-MB-231 cells were most sensitive to PC (IC50 : 5.98 ± 0.95 μM) as compared to other cells. They also showed decreased cell proliferation and reduced colony formation ability upon treatment with PC. Profile of Cell cycle analysis showed that PC caused G1 arrest which could be attributed to decreased mRNA levels of Cyclin E and CDK-2 and increased p21 levels. Mechanistic studies revealed that PC induced apoptosis as evident by increase in percentage of annexin positive cells, increase in γ-H2AX levels, and by changing the Bcl-2/Bax ratio followed by release of cytochrome C and increased Caspase 9 levels. MDA MB 231 cells treated with PC resulted in decreased cell migration and increased cell adhesive property and also showed anti-angiogenic effects. We also observed that PC suppressed cyclooxygenase-2 (COX-2) expression and prostaglandin E(2) production. All these biological effects of phycocyanin on MDA MB 231 cells could be attributed to decreased MAPK signaling pathway. We also observed that PC is non-toxic to non-malignant cells, platelets and RBC’s.ConclusionTaken together, these findings demonstrate, for the first time, that PC may be a promising anti-neoplastic agent for treatment of triple negative breast cancers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1784-x) contains supplementary material, which is available to authorized users.

Abstract 2138: RasGRP3 mediates MAPK pathway activation in GNAQ mutant uveal melanoma

Abstract Uveal melanoma (UM) is the most common intraocular malignancy in adults and no effective treatment options are available for metastatic disease. Over 80% of UM show mutations in the Gαq family members GNAQ and GNA11. MAP-kinase pathway activation in part mediated by protein kinase C (PKC) has been shown as one critical contributing factor to GNAQ-mediated oncogenesis. However PKC inhibition alone does not completely suppress MAPK signaling. A more refined understanding of the signaling cascade linking MAPK signaling to mutant GNAQ or GNA11 is required to develop more effective strategies for targeted therapy. Among more than 10 different PKC isoforms, we identified both PKC δ and PKC ϵ to be required and sufficient to activate MAPK pathway in GNAQ mutant melanomas by using siRNA mediated knock-down and co-transfection of GNAQQ209L and specific PKC isoform cDNAs. Overexpression of GNAQQ209L in 293FT cells increased Ras-GTP level and knock down of three Ras isoforms in GNAQ mutant uveal melanoma cell lines decreased MAPK signaling. Microarray analysis of 5 different GNAQ/11 mutant and 5 NRAS/BRAF-mutant melanoma cells revealed RasGRP3, a Ras-guanyl nucleotide exchange factor (RasGEF), ranks at the top of 487 differentially expressed genes between (cut off : p&amp;lt;0.05, fold change &amp;gt;2 or &amp;lt;-2). Quantitative RT-PCR and western blot confirmed markedly (&amp;gt;100 fold) elevated RasGRP3 levels in GNAQ/11 mutant melanoma cells, while other RasGEFs were not significantly altered. Knock down of RasGRP3 decreased MAPK signaling and proliferation in GNAQ mutant melanoma cells, while it no effect on BRAF mutant cells. Mutating the PKC phosphorylation site of RasGRP3 (T133) partially attenuated RasGRP3-mediated MAPK signaling. Overexpression of PKC δ and PKC ϵ increased RasGRP3 T133 phosphorylation but not PKC α and PKC ζ. While PKC inhibition completely abrogated RasGRP3 T133 phosphorylation, it did not fully suppress RasGRP3-mediated MAPK signaling, indicating that its activating effect only partly requires PKC. We found that RasGRP3 activation mediated by its diacylglycerol(DAG) binding C1 domain provides an independent conduit of activation, which is augmented by PKC-mediated phosphorylation at T133. DAG is a second messenger that is released by phospholipase C, a direct effector of activated GNAQ/11. Finally, we found that the markedly elevated RasGRP3 expression in GNAQ mutant melanoma cell lines is a direct consequence of the oncogenic signaling and is mediated by PKC δ and ϵ. Hence, our data identify RasGRP3 as a critical signaling node linking oncogenic signaling downstream of GNAQ/11 to the Ras/Raf/MEK/ERK pathway through three mechanisms: phosphorylation by PKC δ and ϵ, binding of the second messenger DAG, and by upregulation of its protein level as a consequence of oncogenic GNAQ-mediated activation of PKC δ and PKC ϵ. The findings nominate RasGRP3 as a possible therapeutic target for cancers driven by oncogenic GNAQ/11. Citation Format: Xu Chen, Qiuxia Wu, Philippe Depeille, Jeroen P. Roose, Boris C. Bastian. RasGRP3 mediates MAPK pathway activation in GNAQ mutant uveal melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2138. doi:10.1158/1538-7445.AM2015-2138

Abstract 5435: Molecular subclasses of human hepatocellular carcinoma predict sensitivity to fibroblast growth factor receptor inhibition

Abstract Background: A recent gene expression classification of hepatocellular carcinoma (HCC) includes two poor survival subclasses, termed S1 and S2. S1 cells are more locally invasive, while S2 cells express E-cadherin and secrete AFP. As the expression of fibroblast growth factor receptors (FGFRs) differs between the S1 and S2 gene signatures, this study investigated whether molecular subclasses of HCC could predict sensitivity to FGFR inhibition. Methods: The effects of AZD4547 and BGJ398, two small molecule inhibitors of FGFR1-4, were assessed in four S1 and five S2 human HCC cell lines. Western blotting was performed for FGFR1-4 and phosphorylated p42/44 as a marker of MAPK signaling. Small interfering RNA (siRNA) transfection was used to assess the effects of individual FGFR knockdown. Results: Cell proliferation was inhibited at least two-fold more in S2 than in S1 cell lines at all doses tested above 1 uM of BGJ398 and AZD4547 (p&amp;lt;0.002). BGJ398 also decreased MAPK signaling in S2 but not in S1 cell lines. In xenografts from the S2 cell line HuH-7, BGJ398 reduced tumor volume by 70% over 14 days and this effect was associated with decreased MAPK signaling and a 36% decline in the number of Ki67 positive cells. BGJ398 had no effect on xenografts from the S1 cell line SK-Hep. All cell lines expressed FGFR1 and FGFR2, but only S2 cell lines expressed FGFR3 and FGFR4. FGFR4 siRNA decreased proliferation by 59 ± 12% in all five S2 cell lines (p&amp;lt;0.00001), a significantly greater decrease than seen with knockdown of FGFR1, FGFR2 or FGFR3. FGFR4 knockdown also decreased MAPK signaling in S2 cell lines. Conclusions: The S2 molecular subclass of HCC is sensitive to FGFR inhibition. FGFR4-MAPK signaling plays an important role in driving proliferation of S2 cells. This classification system may help identify those patients who are most likely to benefit from inhibition of the FGFR pathway. Citation Format: Benjamin Schmidt, Lan Wei, Danielle K. DePeralta, Yujin Hoshida, Michael Lanuti, Kenneth K. Tanabe, Bryan C. Fuchs. Molecular subclasses of human hepatocellular carcinoma predict sensitivity to fibroblast growth factor receptor inhibition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5435. doi:10.1158/1538-7445.AM2014-5435

Altered REDD1, myostatin, and Akt/mTOR/FoxO/MAPK signaling in streptozotocin-induced diabetic muscle atrophy

Type 1 diabetes, if poorly controlled, leads to skeletal muscle atrophy, decreasing the quality of life. We aimed to search highly responsive genes in diabetic muscle atrophy in a common diabetes model and to further characterize associated signaling pathways. Mice were killed 1, 3, or 5 wk after streptozotocin or control. Gene expression of calf muscles was analyzed using microarray and protein signaling with Western blotting. We identified translational repressor protein REDD1 (regulated in development and DNA damage responses) that increased seven- to eightfold and was associated with muscle atrophy in diabetes. The diabetes-induced increase in REDD1 was confirmed at the protein level. This result was accompanied by the increased gene expression of DNA damage/repair pathways and decreased expression in ATP production pathways. Concomitantly, increased phosphorylation of AMPK and dephosphorylation of the Akt/mTOR/S6K1/FoxO pathway of proteins were observed together with increased protein ubiquitination. These changes were especially evident during the first 3 wk, along with the strong decrease in muscle mass. Diabetes also induced an increase in myostatin protein and decreased MAPK signaling. These, together with decreased serum insulin and increased serum glucose, remained altered throughout the 5-wk period. In conclusion, diabetic myopathy induced by streptozotocin led to alteration of multiple signaling pathways. Of those, increased REDD1 and myostatin together with decreased Akt/mTOR/FoxO signaling are associated with diabetic muscle atrophy. The increased REDD1 and decreased Akt/mTOR/FoxO signaling followed a similar time course and thus may be explained, in part, by increased expression of genes in DNA damage/repair and possibly also decrease in ATP-production pathways.

Abstract C58: Causal Network™ Modeling identifies common and unique mechanisms for sensitivity to the PI3K inhibitor GSK1059615 and the AKT inhibitor GSK690693

Abstract Background: Aberrant PI3K and AKT signaling play an important role in the development of some human tumors by promoting cell proliferation and survival. Inhibitors that target the kinase activity of PI3K or AKT may constitute an effective treatment for human malignancies that rely on these molecules for their growth. GlaxoSmithKline has developed a PI3K inhibitor, GSK1059615, and a pan-AKT inhibitor, GSK690693. Since AKT is activated by PI3K via generation of PIP3, and is a key effector of PI3K signaling, a high degree of overlap is expected in the effects of a PI3K and an AKT inhibitor. To determine whether the effects of the PI3K inhibitor are mediated solely through the AKT pathway, we compared the molecular response of BT474 human breast cancer cells to both GSK1059615 and GSK690693 as well as the sensitivity profile of 16 other human cancer cell lines. Methods: The mechanisms of action of the PI3K inhibitor GSK1059615 and the AKT inhibitor GSK690693 were evaluated in BT474 human breast cancer cells using RNA expression and phosphoproteomic data. Causal Network™ Modeling was used to characterize these data sets to identify processes that contribute to the inhibition of proliferation observed in cells sensitive to these inhibitors, and processes that could explain differences in sensitivity to the two inhibitors. The sensitivity/resistance profile of 16 human cancer cell lines to GSK1059615 and GSK690693 was also determined. Results: Four common processes were found in BT474 cells treated with either GSK690693 or GSK1059615: RB1-Mediated Cell Cycle Arrest Increased FOXO Signaling Decreased MYC and TFRC Signaling Decreased Metabolism We identified one process specific to the PI3K inhibitor GSK1059615-treated cells: Decreased MAPK Signaling Conclusions: In BT474 cells, GSK690693 and GSK1059615 inhibit cell proliferation by increasing RB1-mediated cell cycle arrest, increasing FOXO signaling, decreasing MYC signaling, and decreasing cellular metabolism, as identified by Causal Network™ Modeling. In addition to these four shared mechanisms, the PI3K inhibitor GSK1059615 attenuates MAPK signaling. Five of 16 cell lines tested for sensitivity to the inhibitors were sensitive to GSK1059615 treatment only, and these five cell lines were the only ones that contained mutations activating MAPK signaling. Thus decreased MAPK signaling is likely an additional mechanism by which GSK1059615 inhibits cell proliferation, and may confer sensitivity to this PI3K inhibitor in cells resistant to the AKT inhibitor. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C58.

Mutations inProkineticin 2andProkineticin receptor 2genes in Human Gonadotrophin-Releasing Hormone Deficiency: Molecular Genetics and Clinical Spectrum

Mice deficient in prokineticin 2(PROK2) and prokineticin receptor2 (PROKR2) exhibit variable olfactory bulb dysgenesis and GnRH neuronal migration defects reminiscent of human GnRH deficiency. We aimed to screen a large cohort of patients with Kallmann syndrome (KS) and normosmic idiopathic hypogonadotropic hypogonadism (IHH) for mutations in PROK2/PROKR2, evaluate their prevalence, define the genotype/phenotype relationship, and assess the functionality of these mutant alleles in vitro. Sequencing of the PROK2 and PROKR2 genes was performed in 170 KS patients and 154 nIHH. Mutations were examined using early growth response 1-luciferase assays in HEK 293 cells and aequorin assays in Chinese hamster ovary cells. Four heterozygous and one homozygous PROK2 mutation (p.A24P, p.C34Y, p.I50M, p.R73C, and p.I55fsX1) were identified in five probands. Four probands had KS and one nIHH, and all had absent puberty. Each mutant peptide impaired receptor signaling in vitro except the I50M. There were 11 patients who carried a heterozygous PROKR2 mutation (p.R85C, p.Y113H, p.V115M, p.R164Q, p.L173R, p.W178S, p.S188L, p.R248Q, p.V331M, and p.R357W). Among them, six had KS, four nIHH, and one KS proband carried both a PROKR2 (p.V115M) and PROK2 (p.A24P) mutation. Reproductive phenotypes ranged from absent to partial puberty to complete reversal of GnRH deficiency after discontinuation of therapy. All mutant alleles appear to decrease intracellular calcium mobilization; seven exhibited decreased MAPK signaling, and six displayed decreased receptor expression. Nonreproductive phenotypes included fibrous dysplasia, sleep disorder, synkinesia, and epilepsy. Finally, considerable variability was evident in family members with the same mutation, including asymptomatic carriers. Loss-of-function mutations in PROK2 and PROKR2 underlie both KS and nIHH.