Phosphoinositol 3-kinase Pathway Research Articles

Impact of PIK3CA gain and PTEN loss on mantle cell lymphoma biology and sensitivity to targeted therapies

AbstractBesides many other mutations in known cancer driver genes, mantle cell lymphoma (MCL) is characterized by recurrent genetic alterations of important regulators of the phosphoinositol-3-kinase (PI3K) cascade including PIK3CA gains and PTEN losses. To evaluate the biological and functional consequences of these aberrations in MCL, we have introduced transgenic expression of PIK3CA (PIK3CA UP) and performed knockout/knockdown of PTEN gene (PTEN KO/KD) in 5 MCL cell lines. The modified cell lines were tested for associated phenotypes including dependence on upstream B-cell receptor (BCR) signaling (by an additional BCR knockout). PIK3CA overexpression decreased the dependence of the tested MCL on prosurvival signaling from BCR, decreased levels of oxidative phosphorylation, and increased resistance to 2-deoxy-glucose, a glycolysis inhibitor. Unchanged protein kinase B (AKT) phosphorylation status and unchanged sensitivity to a battery of PI3K inhibitors suggested that PIK3CA gain might affect MCL cells in AKT-independent manner. PTEN KO was associated with a more distinct phenotype: AKT hyperphosphorylation and overactivation, increased resistance to multiple inhibitors (most of the tested PI3K inhibitors, Bruton tyrosine kinase inhibitor ibrutinib, and BCL2 inhibitor venetoclax), increased glycolytic rates with resistance to 2-deoxy-glucose, and significantly decreased dependence on prosurvival BCR signaling. Our results suggest that the frequent aberrations of the PI3K pathway may rewire associated signaling with lower dependence on BCR signaling, better metabolic and hypoxic adaptation, and targeted therapy resistance in MCL.

PIK3R3 is a candidate regulator of platelet count in people of Bangladeshi ancestry

BackgroundBlood platelets are mediators of atherothrombotic disease and are regulated by complex sets of genes. Association studies in European ancestry populations have already detected informative platelet regulatory loci. Studies in other ancestries can potentially reveal new associations because of different allele frequencies, linkage structures, and variant effects. ObjectivesTo reveal new regulatory genes for platelet count (PLT). MethodsGenome-wide association studies (GWAS) were performed in 20,218 Bangladeshi and 9198 Pakistani individuals from the Genes & Health study. Loci significantly associated with PLT underwent fine-mapping to identify candidate genes. ResultsOf 1588 significantly associated variants (P < 5 × 10−8) at 20 loci in the Bangladeshi analysis, most replicated findings in prior transancestry GWAS and in the Pakistani analysis. However, the Bangladeshi locus defined by rs946528 (chr1:46019890) did not associate with PLT in the Pakistani analysis but was in the same linkage disequilibrium block (r2 ≥ 0.5) as PLT-associated variants in prior East Asian GWAS. The single independent association signal was refined to a 95% credible set of 343 variants spanning 8 coding genes. Functional annotation, mapping to megakaryocyte regulatory regions, and colocalization with blood expression quantitative trait loci identified the likely mediator of the PLT phenotype to be PIK3R3 encoding a regulator of phosphoinositol 3-kinase (PI3K). ConclusionAbnormal PI3K activity in the vessel wall is already implicated in the pathogenesis of atherothrombosis. Our identification of a new association between PIK3R3 and PLT provides further mechanistic insights into the contribution of the PI3K pathway to platelet biology.

Unveiling novel 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl)quinolines as GPCR ligands via PI3-kinase/PAR-1 antagonism and platelet aggregation valuations; development of a new class of anticancer drugs with thrombolytic effects

In the present study, novel 2-cyclopropyl-3-ethynyl-4-(4-fluorophenyl) quinolines (4a-l) were recognized and evaluated as G-Protein Coupled Receptor (GPCR) ligands through molecular evaluations. Thrombin mediates adhesion of mast cell, a type of cell abundantly found in connective tissue and releasing histamine and other substances during inflammatory and allergic reactions, through phosphoinositol 3-kinase pathway. With this background, as preliminary, 4a-l are resolute to be potential leads, designated from their effective phosphoinositol 3-kinase (PI3-Kinase) inhibition potentials, best-docked scores, comparative ligand efficiency, and significant structural attributes evaluated by ab initio simulations. Since thrombin is one of the main reason for various cancer invasion in association with PI3Kinase, a thrombolytic potential of the compounds also analyzed. The experimental in vitro studies confirmed the significant enhancement as PI3Kinase inhibitors and appreciable enhancement in MTT assay of breast and skin cancer cell lines. Significantly, acetophenone substituent in the quinoline scaffold could be coherent to note the significant binding affinity to all the evaluated drug targets.

Identification of NRAS isoform 2 overexpression as a mechanism facilitating BRAF inhibitor resistance in malignant melanoma

Activating mutations in BRAF are found in 50% of melanomas and although treatment with BRAF inhibitors (BRAFi) is effective, resistance often develops. We now show that recently discovered NRAS isoform 2 is up-regulated in the setting of BRAF inhibitor resistance in melanoma, in both cell lines and patient tumor tissues. When isoform 2 was overexpressed in BRAF mutant melanoma cell lines, melanoma cell proliferation and in vivo tumor growth were significantly increased in the presence of BRAFi treatment. shRNA-mediated knockdown of isoform 2 in BRAFi resistant cells restored sensitivity to BRAFi compared with controls. Signaling analysis indicated decreased mitogen-activated protein kinase (MAPK) pathway signaling and increased phosphoinositol-3-kinase (PI3K) pathway signaling in isoform 2 overexpressing cells compared with isoform 1 overexpressing cells. Immunoprecipitation of isoform 2 validated a binding affinity of this isoform to both PI3K and BRAF/RAF1. The addition of an AKT inhibitor to BRAFi treatment resulted in a partial restoration of BRAFi sensitivity in cells expressing high levels of isoform 2. NRAS isoform 2 may contribute to resistance to BRAFi by facilitating PI3K pathway activation.

Abstract 1780: Identifying preclinical predictive biomarkers to taselisib in breast cancer cell lines

Abstract The phosphoinositol-3-kinase (PI3K) signaling pathway is one of the most frequently activated pathways in cancer, and controls critical cellular processes such as proliferation, transcription and survival. Taselisib (GDC0032) is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. Previously published data demonstrated that taselisib has increased activity against PIK3CA mutant cancer cell lines (Ndubaku CO et al, J Med Chem, 2013). A panel of 50 breast cancer cell lines were profiled for sensitivity to taselisib using viability assays and subsequently correlated with PIK3CA mutations and expression of ER, PR, HER2 and PTEN. The PIK3CA mutant cell lines displayed an average of 30-fold greater sensitivity in IC50 compared to the PIK3CA wild-type cell lines. PTEN null cell lines were largely refractory to taselisib. Within the PIK3CA wild-type cell lines, the HER2 amplified cells showed an average of 18-fold greater sensitivity in IC50 over the HER2 wild-type cell lines. No differences in taselisib sensitivity were seen between the kinase, helical, or the C2 domain of the PIK3CA gene within the PIK3CA mutant cell lines. In HER2 amplified PIK3CA wild-type cell lines, taselisib did not fully suppress pAKT and pS6 signaling, however full suppression was observed in combination with the HER2 inhibitor, lapatinib. Increased apoptosis was observed with the combination of taselisib and lapatinib in the HER2 amplified, PIK3CA mutant cells, but not in HER2 amplified, PIK3CA wild-type cells. In PIK3CA mutant luminal cell lines, taselisib induced ESR1 transcriptional activity, but not in PIK3CA wild-type luminal lines, as assessed using PR, GREB1 and IGFBP4 gene expression. This induction was ER- and estradiol-dependent, and was suppressed using the estrogen degrader, fulvestrant. Lastly, as up to 40% of ER+, second line PIK3CA mutant metastatic tumors have been shown to harbor a mutation in the ESR1 gene (Spoerke JM et al., Nat Commun, 2016), we assessed the crosstalk between the PI3K and ER pathways in PIK3CA mutant MCF-7 cells that overexpress two ESR1 hotspot mutations. These ESR1 mutant cell lines displayed increased ER transcriptional activity, which was further activated by taselisib, an observation that was reversed with fulvestrant co-treatment. These data suggest that the combination of taselisib and an estrogen degrader can suppress crosstalk between PI3K and ER in both an ESR1 mutant and wild-type background, and supports the ongoing SANDPIPER study of taselisib in combination with fulvestrant in metastatic ER+, PIK3CA mutant cancers that have progressed following an aromatase inhibitor therapy (NCT02340221). Citation Format: Heidi M. Savage, Heather M. Moore, Carol L. O'Brien, Wei Zhou, Ciara Metcalfe, Mark R. Lackner, Timothy R. Wilson. Identifying preclinical predictive biomarkers to taselisib in breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1780. doi:10.1158/1538-7445.AM2017-1780

Abstract 372: Taselisib enhances the potency of ERBB inhibitors in biomarker-defined subsets of head and neck squamous carcinoma cell lines

Abstract The phosphoinositol-3-kinase (PI3K) signaling pathway is one of the most frequently activated pathways in oncogenesis, and controls critical cellular processes such as proliferation, transcription and survival. Taselisib (GDC 0032) is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30 fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. The gene that encodes the p110 alpha isoform of PI3K, PIK3CA, is frequently mutated in breast, colorectal and endometrial cancers. Previously published data demonstrated that taselisib has increased activity against PIK3CA mutant cancer cell lines (Ndubaku CO et al, J Med Chem, 2013). To determine if there are additional predictive biomarkers outside of PIK3CA mutations for sensitivity to taselisib, we profiled over 550 cell lines, encompassing 13 of the main tissue types, to increasing concentrations of taselisib. As expected, a small percentage of all tumor types responded to taselisib and correlated strongly with PIK3CA mutations. Intriguingly, the majority of head and neck squamous cell cancer (HNSCC) cell lines showed an IC50 concentration of less than 0.5uM, suggesting that HNSCC cell lines may be particularly susceptible to PI3K inhibition. Interestingly, the majority of HNSCC cell lines displayed an activated PI3K pathway as evidenced by phosphorylation of key proteins, such as AKT, S6 and PRAS40 implicating that downstream phospho-protein analysis of the PI3K pathway may not predict for sensitivity to taselisib. Mutational profiling identified that only 3 out of the tested 31 HNSCC cell lines harbored a mutation within PIK3CA, suggesting that additional genotypes may explain the sensitivity to taselisib. Additional molecular analysis assessing both gene expression and copy number levels of genes relevant to the PI3K pathway found that many of the taselisib sensitive cell lines had an activated ERBB signaling node through low level amplification of ERBB receptors (e.g. EGFR, FGFR1) or via a NRG1:ERBB3 autocrine mechanism. As many ERBB receptors heterodimerize with ERBB3, which contains p85 binding sites and potentially activates the PI3K pathway, we next tested whether taselisib would enhance the potency of ERBB receptor inhibitors in biomarker-defined subset of HNSCC cell lines. Combination screens were performed in cell lines with amplified EGFR, ERBB2, and NRG1:ERBB3 autocrine signaling with taselisib + tarceva or lapatinib. We found that combination effects assessed using the BLISS excess method showed a synergistic interaction. These results suggest that taselisib may have therapeutic potential for the treatment of HNSCC. Citation Format: Heidi M. Savage, Carol O’Brien, Heather Moore, Mark R. Lackner, Robert Yauch, Jeffrey Settleman, Timothy R. Wilson. Taselisib enhances the potency of ERBB inhibitors in biomarker-defined subsets of head and neck squamous carcinoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 372.

Hormone receptor positive, HER2 negative metastatic breast cancer: A systematic review of the current treatment landscape.

Endocrine therapy for the treatment of hormone receptor positive, HER2 negative, metastatic breast cancer is continually evolving. We systematically reviewed phase 2 and 3 randomized controlled trials (RCTs) of agents used in this setting to assess the effectiveness and safety of these agents for postmenopausal women. Across the 32 studies in more than 10,000 patients, the greatest improvement in progression-free survival (PFS) was seen with the addition of a cyclin-dependent kinase (CDK)4/6 inhibitor to standard endocrine therapy. Treatment with a mammalian target of rapamycin (mTOR) inhibitor, phosphoinositol-3-kinase (Pi3K) inhibitor, vascular endothelial growth factor (VEGF) inhibitor and with a selective estrogen receptor degrader (SERD) also showed benefit in PFS for selected trials. Overall survival (OS) improved with the use of mTOR inhibitors and a SERD; however, studies were not powered for an OS endpoint. Encouraging results from early studies of histone deacetylase (HDAC) and B-cell lymphoma (BCL2) inhibitors are yet to be confirmed in phase III clinical trials. Study discontinuation rates and toxicity-related deaths were highest with VEGF inhibitors in combination with endocrine therapy, limiting their use in hormone receptor positive breast cancer. CDK4/6 inhibitors and mTOR inhibitors appeared to have activity in both first and second line settings, but required additional monitoring for common toxicities. The activity of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors was limited to the first-line setting and treatment discontinuation rates were higher than with mTOR inhibitors and SERDs. Overall, PFS benefit appears to be greatest when agents acting on CDK4/6, mTOR and Pi3K pathways, and SERDs are added to standard endocrine therapy. If these early results persist in further studies, these data are likely to change the way we treat hormone receptor positive, HER2 negative metastatic breast cancer. In the follow-up article to this review, we will consider the potential future treatment options for these patients.

Phospho-PRAS40Thr246 predicts trastuzumab response in patients with HER2-positive metastatic breast cancer.

Resistance to trastuzumab is frequently observed during the treatment of patients with human epidermal growth factor 2 (HER2)-positive metastatic breast cancers. The aim of the present study was to determine if the phosphorylated proline-rich Akt substrate of 40 kDa (phospho-PRAS40Thr246), a novel biomarker for phosphoinositol-3 kinase (PI3K) pathway activation, could predict the response of HER2-positive metastatic breast cancers to treatment with trastuzumab. Formalin-fixed, paraffin-embedded tumor tissue samples were retrospectively collected from 55 trastuzumab-treated patients. Next, the expression of phospho-PRAS40Thr246 and phosphatase and tensin homolog (PTEN) was assessed by immunohistochemistry. In total, five common phosphoinositol-3 kinase α catalytic subunit mutations, namely E542K, E545K, E545D, H1047R and H1047L, were identified by the amplification-refractory mutation system, using the allele-specific polymerase chain reaction. The activation of the PI3K pathway, as determined by low PTEN expression or the presence of oncogenic PIK3CA mutations, was observed in 49.1% (27 cases) of the 55 HER2-positive metastatic breast cancer tissues. In total, 40% of the tumors were defined as being phospho-PRAS40Thr246-positive. Furthermore, the results revealed that phospho-PRAS40Thr246 expression was associated with the PI3K pathway activation status and an increased risk of tumor progression in HER2-positive metastatic breast cancer patients who had received trastuzumab-based therapy. Therefore, phospho-PRAS40Thr246 expression levels may reflect the PI3K pathway activation status and act as a biomarker for HER2-amplified breast cancer patients who are unlikely to respond to trastuzumab-based therapy.

Abstract 915: Expanded biomarker results from a phase I dose escalation study of GDC-0032, a beta isoform-sparing PI3K inhibitor

Abstract The phosphoinositol-3-kinase (PI3K) signaling pathway is one of the most frequently activated pathways in oncogenesis, and controls critical cellular processes such as proliferation, transcription and survival. GDC 0032 is an orally bioavailable, potent, and selective inhibitor of Class I PI3K alpha, delta, and gamma isoforms, with 30 fold less inhibition of the PI3K beta isoform relative to the PI3K alpha isoform. The gene that encodes the p110 alpha isoform of PI3K, PIK3CA, is frequently mutated in breast, colorectal and endometrial cancers. Preclinical data indicate that GDC-0032 has increased activity against PIK3CA mutant cancer cell lines. As presented by Juric et al., 34 patients were enrolled in this study and dose escalation has been completed (Juric D. et al. AACR 2013, Abstract LB-64). Metabolic partial responses via FDG-PET were observed in 6 out of 13 patients assessed, and clinical partial responses (PRs) were observed in 6 patients, with 5 of these patients having PIK3CA mutant cancers. Tumor tissue was obtained from 30 out of 34 patients enrolled onto the study. The most frequently recruited cancer type was breast cancer (41%) followed by colorectal (15%) and lung cancer (15%). Fourteen patients enrolled onto the study had PIK3CA mutant tumors (41%) as determined using a TaqMan-based PCR assay. Three patients had total loss of PTEN expression, as assessed by immunohistochemical staining, and a further 3 patients had reduced PTEN expression based on a H-score assessment. Out of the 6 patients that had reduced PTEN expression, 2 patients had coexisting mutations with PIK3CA. Where known, 4 out of 5 patients that had a PR showed intact PTEN expression, with the fifth patient having reduced PTEN expression. Samples were further analyzed using an in-house developed PCR-based multiplexed assay that detects activating mutations within an additional ten oncogenes. PIK3CA mutations were largely mutually exclusive with mutations in the Ras pathway, however 3 out of the 14 PIK3CA mutant patients had a coexisting mutation within KRas. A further 2 patients had KRas mutations, 1 patient had a NRas mutation and 2 patients had EGFR mutations. Preliminary analysis suggests lack of benefit in patients with KRas mutations treated with GDC-0032 single agent. Optional on-study biopsies were collected from 2 patients and demonstrated pharmacodynamic inhibition of the PI3K pathway as assessed by reverse phase protein array for approximately 45 endpoints, including 1 patient at the lowest dose. In conclusion, our preliminary data indicates that GDC-0032 demonstrates single agent activity in patients with PIK3CA mutations tumors with unaltered PTEN or MAP-kinase pathways. Citation Format: Timothy R. Wilson, Heidi Savage, Carol O'Brien, Sandra Sanabria, Ray S. Lin, Marie-Claire Wagle, Yibing Yan, Mark R. Lackner, Hema Parmar, Jerry Y. Hsu, Dejan Juric, Ian E. Krop, Ramesh K. Ramanathan, Daniel D. Von Hoff, Jose Baselga. Expanded biomarker results from a phase I dose escalation study of GDC-0032, a beta isoform-sparing PI3K inhibitor. [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 915. doi:10.1158/1538-7445.AM2014-915

Reversal of Murine Epidermal Atrophy by Topical Modulation of Calcium Signaling

Cytosolic Ca(2+) signals are performed by Ca(2+) releases from the endoplasmic reticulum and Ca(2+) influx from the extracellular medium. Releases rely on the refilling of the intracellular Ca(2+) stores by the Ca(2+) influx "Store-Operated Calcium Entry" (SOCE) via the channel Orai1. Here we show that Orai1 expression, SOCE amplitude, and epidermal proliferation are decreased in the epidermis of patients with skin fragility when compared with aged nonatrophic skin. Epidermal atrophy was induced in mice by the inhibition of Orai1 with small interfering RNA and the topical application of a SOCE blocker BTP2. The inhibition of Orai1 impaired the heparin-binding epidermal growth factor (HB-EGF)-induced Ca(2+) influxes and fully prevented the mitogen effect of HB-EGF in primary human keratinocytes. Importantly, epidermal proliferation correlated with Orai1 expression in mice. Conversely, the topical application of an Orai1 activator, the benzohydroquinone (BHQ), increased the epidermal thickness and proliferation, whereas the pro-proliferative effect of BHQ was prevented by the inhibition of Orai1. Finally, the topical application of BHQ reversed the epidermal atrophy induced by corticosteroids in mice. The topical modulation of Ca(2+) signals may thus be a promising therapeutic strategy in dermatology.

P62/SQSTM1 accumulation in squamous cell carcinoma of head and neck predicts sensitivity to phosphatidylinositol 3-kinase pathway inhibitors.

The phosphoinositol-3 kinase (PI3K) pathway is highly dysregulated in squamous cell carcinoma of the head and neck (SCCHN). While inhibitors of the PI3K/AKT pathway are being developed in cancer, their efficacy does not appear to be related to the presence of mutations or amplification in pathway genes. The PI3K pathway is a major regulator of macro-autophagy, an evolutionarily conserved catabolic process that degrades cellular materials to promote cellular homeostasis and survival under stress. Employing a panel of SCCHN cell lines, we observed a significant correlation between the activity of PI3K/AKT inhibitors and their ability to induce autophagy. More specifically, resistance to these inhibitors was associated with accumulation of p62/SQSTM1, a pleotropic protein that is consumed during autophagy, while loss of autophagy was, for the first time, found to be due to silencing of an essential autophagy gene, ATG7. Moreover, modulating ATG7 and p62/SQSTM1 could regulate sensitivity to PI3K/AKT inhibitors, underscoring a mechanistic link between autophagy and drug sensitivity. Analysis of human tissues revealed progressive accumulation of p62/SQSTM1 in a significant proportion of cancer samples compared to normal tissue, suggesting that defective autophagy has relevance to SCCHN. These findings are further validated by analysis of TCGA data confirming homozygous deletion and mRNA down-regulation of ATG7 in 10.0% of SCCHN samples. Taken together, these data indicate that p62/SQSTM1 levels modulate sensitivity to PI3K/AKT inhibitors; cancers vary in their capacity to undergo autophagy through epigenetic modification and, when deficient, accumulate p62/SQSTM1; and expression of autophagy-related proteins may serve as markers for resistance to PI3K/AKT inhibitors in SCCHN.

Induction of M2 Regulatory Macrophages through the β2-Adrenergic Receptor with Protection during Endotoxemia and Acute Lung Injury

The main drivers of acute inflammation are macrophages, which are known to have receptors for catecholamines. Based on their function, macrophages are broadly categorized as having either M1 (proinflammatory) or M2 phenotypes (anti-inflammatory). In this study, we investigated catecholamine-induced alterations in the phenotype of activated macrophages. In the presence of lipopolysaccharide (LPS), mouse peritoneal macrophages acquired an M1 phenotype. However, the copresence of LPS and either epinephrine or norepinephrine resulted in a strong M2 phenotype including high levels of arginase-1 and interleukin-10, and a reduced expression of M1 markers. Furthermore, epinephrine enhanced macrophage phagocytosis and promoted type 2 T-cell responses in vitro, which are known features of M2 macrophages. Analysis of M2 subtype-specific markers indicated that LPS and catecholamine-cotreated macrophages were not alternatively activated but were rather of the regulatory macrophage subtype. Interestingly, catecholamines signaled through the β₂-adrenergic receptor but not the canonical cAMP/protein kinase A signaling pathway. Instead, the M2 pathway required an intact phosphoinositol 3-kinase pathway. Blockade of the β₂-adrenergic receptor reduced survival and enhanced injury in mouse models of endotoxemia and LPS-induced acute lung injury, respectively. These results demonstrate a role for the β₂-adrenergic receptor in promoting the M2 macrophage phenotype.

Pten loss in the bone marrow leads to G-CSF–mediated HSC mobilization

The phosphatase and tumor suppressor PTEN inhibits the phosphoinositol-3-kinase (PI3K) signaling pathway and plays a key role in cell growth, proliferation, survival, and migration. Pten conditional deletion using MxCre or Scl-CreER(T) leads to splenomegaly and leukemia formation, which occurs after the relocation of normal hematopoietic stem cells (HSCs) from the bone marrow to the spleen. Unexpectedly, dormant HSCs in the bone marrow do not enter the cell cycle upon Pten loss, they do not lose self-renewal activity, and they are not exhausted. Instead, Pten deficiency causes an up-regulation of the PI3K pathway in myeloid cells, but not in HSCs. Strikingly, myeloid cells secrete high levels of G-CSF upon Pten loss, leading to the mobilization of HSCs from the bone marrow and accumulation in the spleen. After deletion of Pten in mice lacking G-CSF, the splenomegaly, myeloproliferative disease, and splenic HSC accumulation are rescued. Our data show that although PTEN has little if any role in normal HSCs, it is essential to prevent overt G-CSF production by myeloid and stromal cells which otherwise causes HSCs to relocate to the spleen followed by lethal leukemia initiation.

Women 50 Years Or Younger With Endometrial Cancer: The Argument for Universal Mismatch Repair Screening and Potential for Targeted Therapeutics

To identify young patients with endometrial cancer with potential Lynch-related DNA mismatch repair (MMR) protein expression defects and stathmin (STMN1) expression (part of the phosphoinositol 3-kinase pathway) and to correlate clinical data. This retrospective study included women with endometrial cancer who were 50 years or younger at diagnosis. Clinical data were abstracted from chart review. Immunohistochemistry for MMR protein expression, STMN1, and pSTMN1 was performed and univariate analyses performed. The mean age of 111 patients was 43 years, and the mean body mass index was 39.6 kg/m². The majority of the endometrial cancers were endometrioid histology (87.4%), International Federation of Gynecology and Obstetrics stage I (73%) and grade 1 (58.6%). Loss of at least one MMR protein on immunohistochemistry was identified in 26% to 41% of patients depending on stringency. Women with loss of MMR protein expression were compared to women with intact tumor protein expression and were less likely to be stage I (58.6% vs 78.0%; P = 0.043), more likely to have grade 3 tumors (32.1% vs 13.9%; P = 0.034), had larger tumors (6.2 vs 3.7 cm; P < 0.001), had positive lymph nodes more often (24.1% vs 3.7%; P < 0.001), and more often reported a first-degree relative with colon cancer (17.2% vs 1.2%; P < 0.001). There were no significant differences in age, weight, body mass index, medical comorbidities, recurrence, or survival. Women with high STMN1 staining had significantly more grade 3 tumors (56.3% vs 15.8%; P = 0.001), more stage III/IV disease (37.5% vs 15.8%; P = 0.04), had higher mean percentage of myometrial invasion (38.9% vs 16.7%; P = 0.003), and more lymphovascular space invasion (43.8% vs 13.7%; P = 0.004). Clinical factors failed to differentiate between patients with intact or missing MMR protein expression, which supports universal screening for Lynch-associated protein defects in young women with endometrial cancer. Additionally, STMN1 staining may identify more aggressive tumors, which might benefit from more aggressive treatments or targeted treatment options.

Catecholamines promote an M2 macrophage activation phenotype (P1299)

Abstract Based on their function, activated macrophages are categorized as having M1 or M2 phenotypes. M1 macrophages are pro-inflammatory and mediate host defenses against pathogens. M2 macrophages promote immunosuppression and wound healing. Here, we report significant alterations in activated macrophage phenotypes by catecholamines. Epinephrine and norepinephrine dramatically reduced TNF production by LPS-stimulated mouse macrophages in vitro, with IC50s of 5.5 and 700 nM, respectively. Co-treatment of macrophages with LPS and either catecholamine promoted an anti-inflammatory phenotype, including increased IL-10 mRNA and protein production. Interestingly, in LPS-stimulated macrophages, co-presence of either catecholamine reduced iNOS (a marker for M1 macrophages) mRNA by 75-81%, while Arginase (M2 marker) mRNA expression was enhanced by 8.3 to 13.5-fold. Therefore, catecholamines promoted an M2-like macrophage activation phenotype. This effect was determined to be mediated through the beta-2 adrenergic receptor and the phosphoinositol 3-kinase pathway. Catecholamines modulated both MyD88-dependent and MyD88-independent signaling through several different TLRs. In rodent models of acute lung injury, co-administration of a beta-2 adrenergic receptor agonist reduced inflammatory mediator production and prevented injury. These data have significant clinical implications for diseases ranging from allergy and asthma to sepsis.

Abstract 235: Oncogene addiction switch from NOTCH to PI3K/AKT requires simultaneous targeting of NOTCH and PI3K pathway inhibition in glioblastoma.

Abstract Notch signaling pathway regulates normal stem cells in the brain and glioma stem cells (GSCs) with high Notch activity. However, blocking the proteolytic activation of NOTCH with γ-secretase inhibitors (GSIs) fails to alter the growth of some GSCs as GSIs seem to be active in only a fraction of GSCs lines with constitutive NOTCH activity and limiting the efficacy of this strategy. Linking the molecular aberrations of cancer to drug responses could guide treatment choice and identification of new therapeutic applications. Here we report loss of PTEN as a critical event leading to resistance to NOTCH inhibition, which causes the transfer of “oncogene addiction” from the NOTCH to the phosphoinositol-3 kinase (PI3K)/AKT pathway. This novel observation suggests the need to simultaneous inhibition of both pathways as a means to improve therapeutic efficacy in human GBMs. We investigated the effects of Notch pathway inhibition in GSC using GSI. Drug cytotoxicity test on 16 GSCs show differential growth response to GSI stratifying GSCs into two groups: responders (6 cell lines) vs non-responders (10 cell lines). Active Notch signaling seems to be important features for the GSC as Notch inhibition only affected GSCs defined as having increased endogenous Notch activity. However in the responder group GSCs with the PTEN mutation seems to be less sensitive to GSI treatment. Here we show that NOTCH regulates the expression of PTEN and the activity of the PI3K/AKT signaling pathway in GSCs since treatment with GSI attenuated Notch intracellular domain (NICD), Hes-1, Hes-3, and Hes-5 and increases PTEN expression. NOTCH regulates PTEN expression via Hes-1 as knockdown of either Notch or Hes1 led to increase expression of PTEN. In contrast, PTEN knockdown decreases the response of GSCs to GSI in PTEN wild type GSCs. Our data shows that PTEN is an important mediator of GSI induced attenuation of cell growth suggesting that there might be a regulatory circuit-linking Notch signaling with PTEN/PI3K/Akt pathway, providing the basis for the design of new therapeutic strategies and suggests the need to simultaneously inhibit both pathways as a means to improve therapeutic efficacy in human GBMs. Our preliminary data reveal the synergistic attenuation of cell growth using combination of GSI and PI3K inhibitors in PTEN mutant GSCs. Our results warrant further studies to test the efficacy of combined PI3K/AKT- and NOTCH-directed therapies in human cancer. Citation Format: Norihiko Saito, Jun Fu, Shuzhen Wang, Erik P. Sulman, Frederick F. Lang, Wk Alfred Yung, Dimpy Koul. Oncogene addiction switch from NOTCH to PI3K/AKT requires simultaneous targeting of NOTCH and PI3K pathway inhibition in glioblastoma. [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 235. doi:10.1158/1538-7445.AM2013-235

PTEN regulates the expressions of typeII collagen and aggrecan under oxidative stress in human chondrocytes

PTEN regulates the expressions of typeII collagen and aggrecan under oxidative stress in human chondrocytes

The Role of CD147 in the Invasiveness of Follicular Thyroid Carcinoma Cells

In patients without metastases, capsular and vascular invasion must be noted to make the diagnosis of follicular thyroid carcinoma (FTC). Some patients are initially diagnosed as follicular adenoma (FA) but develop metastases, indicating the original lesion was FTC. A diagnostic marker for FTCs that appear to be FAs by conventional histopathology is urgently needed. CD147 is a transmembrane glycoprotein that induces matrix metalloproteinases (MMPs) and participates in carcinoma invasion. The objective of this study was to determine whether CD147 is upregulated in FTC and if measures directed against it could reduce the invasive activity of FTC cells. The expression levels of CD147, MMP-1, MMP-2, MMP-3, MMP-7, and MMP-9 in surgical specimens of normal thyroid (n=8), FA (n=20), and FTC (n=9) was determined using immunoblot and immunohistochemical techniques. CD147 protein expression levels of epithelial growth factor stimulated FTC-133 cell lines was measured by immunoblotting with and without cell signaling inhibitors such as wortmannin, PD98059, SP600125, and SB203580. This was also done after exposure to short-hairpin interference RNA directed against CD147. Immunoblot analysis of thyroid tissues revealed significant increases in signals for CD147, MMP-3, MMP-7, and MMP-9 in FTC compared with FA or normal tissue, or both. Immunohistochemical analysis revealed colocalization of determinants of CD147 with those of all of MMPs studied, mainly in follicular cells in normal and neoplastic cells in FA and FTC; their immunoreactivities were to some extent more intense in the FTC than FA or normals. In FTC-133 cells, immunoreactive signals for CD147 were upregulated by epidermal growth factor (EGF), and the EGF-driven increases in CD147 were prevented by inhibitors against phosphoinositol-3 kinase (PI3K), extracellular signal-regulated protein kinase (ERK), or c-Jun N-terminal kinase (JNK) but not p38. RNA interference targeted against CD147 reduced the invasive activity of FTC-133 cells and was associated with downregulation of MMP-2, MMP-3, MMP-7, and MMP-9. These results provide in vivo evidence for CD147 upregulation in FTC and in vitro evidence for EGF-stimulated CD147 induction via the PI3K, ERK, and JNK pathways. They suggest the involvement of CD147 in the invasiveness of FTC cells via regulation of MMPs.

Genetic Evidence That Phosphoinositol 3 – Kinase (PI3K) is Required for the Epidermal Growth Factor (EGF) Induced Adaptive Response After Ileocecal Resection (ICR)

Background: Growth factors including EGF have emerged as potential therapeutic options for patients with surgical short gut syndrome. The potential augmentation of the adaptive response by EGF makes it an appealing therapy to promote intestinal autonomy. Crypt epithelium responds to small bowel (SB) resection with increased proliferation and fissioning. EGF augments the intestinal epithelial cell (IEC) responses to intestinal resection although its role in regulating β-catenin is unclear. To examine the potential that EGF-induced PI3K mediates the epithelial response to β-catenin activation in ICR, we utilized an inducible genetic model of intestinal epithelial specific deletion of Class IA PI3K p85α AhCre/pi3kr1 ). Our laboratory demonstrated cross-talk between PI3K and the Wnt/β-catenin signaling pathways in inflammatory bowel disease tissue. Previous data from Clevers and colleagues showed that the proliferative responses originate in intestinal stem cells (ISC) and are dependent on Wnt/β-catenin signaling. Here we test the notion that EGF stimulates IEC via β-catenin activation after ICR through PI3K pathway. Design/Methods: C57BL/6 wild type (WT) mice and AhCre/pi3kr1 animals underwent sham surgery, ICR, or no surgery. All animals were given 50 ug/kg/day of rEGF in osmotic pumps immediately after the surgery. Five days later, SB morphometrics, immunohistochemistry (IHC) for BrdU incorporation (2hr), phospho(p) AKT(Thr308), and pBcatenin(ser552) and western blots of purified IEC proteins including cleaved caspase-3 and nuclear total β-catenin were performed. Results: Compared to EGF-treated sham, EGF-treated ICR mice have increased crypt length (63.3 ± 7 vs 118.3 ±2 8.9 μm, p< 0.05), BrdU incorporation, pAKT(Thr308), pβ-catenin(ser552) by IHC, and nuclear total β-catenin (WB). Data on AhCre/pi3kr1 ICR mice revealed reduced crypt length (118.3±28.9 vs. 67.1 ± 15.5 μm, p< 0.05 ), incorporation of pBcatenin(ser552) (1.43 ± 0.58 vs. 0.85 ± 0.19 counts/crypt, p< 0.05), and a 90% reduction of nuclear total β-catenin by WB. Interestingly, BrdU incorporation in AhCre/pi3kr1 ICR mice was similar to EGF-treated ICR alone, whereas cleaved caspase-3 (WB) doubled, suggesting enhanced apoptosis attenuated the observed induction of IEC proliferation. Conclusions: These data indicate that the epithelial response to ICR in EGF-treated mice enhanced crypt length and IEC proliferation along with PI3K and β-catenin signaling. Furthermore, data indicate that PI3K enhanced IEC crypt lengths, β-catenin activation, and cell survival. Thus, we propose that enhanced IEC PI3K signaling in short bowel patients may facilitate the recovery of absorptive surface through epithelial restitution.

Loss of Phosphatase and Tensin Homolog or Phosphoinositol-3 Kinase Activation and Response to Trastuzumab or Lapatinib in Human Epidermal Growth Factor Receptor 2–Overexpressing Locally Advanced Breast Cancers

Phosphatase and tensin homolog (PTEN) loss or activating mutations of phosphoinositol-3 (PI3) kinase (PIK3CA) may be associated with trastuzumab resistance. Trastuzumab, the humanized human epidermal growth factor receptor 2 (HER2) monoclonal antibody, and lapatinib, an epidermal growth factor receptor/HER2 tyrosine kinase inhibitor, are both established treatments for HER2-overexpressing breast cancers. Understanding of the cellular response to HER2-targeted therapies is needed to tailor treatments and to identify patients less likely to benefit. We evaluated the effect of trastuzumab or lapatinib in three HER2-overexpressing cell lines. We confirmed the in vitro observations in two neoadjuvant clinical trials in patients with HER2 overexpression; 35 patients received trastuzumab as a single agent for the first 3 weeks, then docetaxel every 3 weeks for 12 weeks (trastuzumab regimen), whereas 49 patients received lapatinib as a single agent for 6 weeks, followed by trastuzumab/docetaxel for 12 weeks before primary surgery (lapatinib regimen). Apoptosis, Ki67, p-MAPK, p-AKT, and PTEN were assessed by immunohistochemistry. Genomic DNA was sequenced for PIK3CA mutations. Under low PTEN conditions, in vitro data indicate that lapatinib alone and in combination with trastuzumab was effective in decreasing p-MAPK and p-AKT levels, whereas trastuzumab was ineffective. In the clinical trials, we confirmed that low PTEN or activating mutation in PIK3CA conferred resistance to the trastuzumab regimen (P = .015), whereas low PTEN tumors were associated with a high pathologic complete response rate (P = .007). Activation of PI3 kinase pathway is associated with trastuzumab resistance, whereas low PTEN predicted for response to lapatinib. These observations support clinical trials with the combination of both agents.