Phosphatidylinositol 3-kinase Pathway Research Articles

20(S)-ginsenoside Rh2 induces caspase-dependent promyelocytic leukemia-retinoic acid receptor A degradation in NB4 cells via Akt/Bax/caspase9 and TNF-α/caspase8 signaling cascades

BackgroundAcute promyelocytic leukemia (APL) is a hematopoietic malignancy driven by promyelocytic leukemia–retinoic acid receptor A (PML-RARA) fusion gene. The therapeutic drugs currently used to treat APL have adverse effects. 20(S)-ginsenoside Rh2 (GRh2) is an anticancer medicine with high effectiveness and low toxicity. However, the underlying anticancer mechanisms of GRh2-induced PML-RARA degradation and apoptosis in human APL cell line (NB4 cells) remain unclear. MethodsApoptosis-related indicators and PML-RARA expression were determined to investigate the effect of GRh2 on NB4 cells. Z-VAD-FMK, LY294002, and C 87, as inhibitors of caspase, and the phosphatidylinositol 3-kinase (PI3K) and tumor necrosis factor-α (TNF-α ) pathways were used to clarify the relationship between GRh2-induced apoptosis and PML-RARA degradation. ResultsGRh2 dose- and time-dependently decreased NB4 cell viability. GRh2-induced apoptosis, cell cycle arrest, and caspase3, caspase8, and caspase9 activation in NB4 cells after a 12-hour treatment. GRh2-induced apoptosis in NB4 cells was accompanied by massive production of reactive oxygen species, mitochondrial damage and upregulated Bax/Bcl-2 expression. GRh2 also induced PML/PML-RARA degradation, PML nuclear bodies formation, and activation of the downstream p53 pathway in NB4 cells. Z-VAD-FMK inhibited caspase activation and significantly reversed GRh2-induced apoptosis and PML-RARA degradation. GRh2 also upregulated TNF-α expression and inhibited Akt phosphorylation. LY294002, an inhibitor of the PI3K pathway, enhanced the antitumor effects of GRh2, and C 87, an inhibitor of the TNF-α pathway, reversed NB4 cell viability, and GRh2-mediated apoptosis in a caspase-8-dependent manner. ConclusionGRh2 induced caspase-dependent PML-RARA degradation and apoptosis in NB4 cells via the Akt/Bax/caspase9 and TNF-α/caspase8 pathways.

OR20-01 AMH Preserves Fertility in Chemotherapy-Treated Mice Through a Mechanism Involving Autophagy

Chemotherapy-induced ovarian failure and infertility is an important concern in female cancer patients. The follicular ovarian reserve is established early in life then keeps declining regularly along reproductive life. This reserve is constituted by dormant primordial follicles (PMF) which are necessary to maintain female reproductive function. There is a continuous repression of PMF activation in early growing follicle through the balance between factors activating the initiation of follicular growth, mainly actors of the phosphatidyl-inositol-3-kinase (PI3K) signaling pathway, and inhibiting factors such as Anti-Müllerian Hormone (AMH). Any alteration of this equilibrium may induce early follicle depletion and subsequent infertility. Cyclophosphamide (Cy) one of the alkylating agents commonly used for treating breast cancer is able to trigger PMF activation further leading to premature ovarian insufficiency. Preventing chemotherapy-induced ovarian dysfunction might represent an option for preserving optimal chances of natural or medically assisted conceptions after healing.We showed in a model of Cy-treated pubertal mice, that AMH administration was able to restrain PMF depletion by counting the total PMF number within mouse ovaries. Moreover, the PI3K signaling pathway was evaluated following Cy administration with and without AMH injection. We showed that AMH decreased the phosphorylation of FOXO3A, a transcription factor of PMF activation and induced its nuclear translocation. Altogether, the results support a protective role of AMH against Cy-induced follicular loss. To better understand AMH action in the ovary, we investigated the molecular mechanism to explain the protective effect of this hormone on the PMF pool. It has been reported that autophagy, a lysosomal degradative ubiquitous process implicated in cellular homeostasis, was involved in both ovarian follicular death and survival mostly by PI3K pathway (Gawriluk et al. Reproduction 2011 141, 759–765). We show in mice that Cy inhibits autophagy in the ovary while AMH induces autophagy. In vivo analysis of autophagic flux is currently in progress to dissect this process more finely. Interestingly, FOXO3A was shown to be related to autophagy activation. To investigate the role of FOXO3A in AMH-induced autophagy further, we analyzed mRNA and protein expression of autophagy-related genes controlled by FOXO3A, including BECLIN-1, ATG12, ULK1, BNIP3, GABARAP, and LC3B. These findings establish a close relationship between AMH and autophagy to protect PMF stockpile and to limit follicular depletion induced by Cy.

Tep1 Regulates Yki Activity in Neural Stem Cells in Drosophila Glioma Model.

Glioblastoma Multiforme (GBM) is the most common form of malignant brain tumor with poor prognosis. Amplification of Epidermal Growth Factor Receptor (EGFR), and mutations leading to activation of Phosphatidyl-Inositol-3 Kinase (PI3K) pathway are commonly associated with GBM. Using a previously published Drosophila glioma model generated by coactivation of PI3K and EGFR pathways [by downregulation of Pten and overexpression of oncogenic Ras] in glial cells, we showed that the Drosophila Tep1 gene (ortholog of human CD109) regulates Yki (the Drosophila ortholog of human YAP/TAZ) via an evolutionarily conserved mechanism. Oncogenic signaling by the YAP/TAZ pathway occurs in cells that acquire CD109 expression in response to the inflammatory environment induced by radiation in clinically relevant models. Further, downregulation of Tep1 caused a reduction in Yki activity and reduced glioma growth. A key function of Yki in larval CNS is stem cell renewal and formation of neuroblasts. Other reports suggest different upstream regulators of Yki activity in the optic lobe versus the central brain regions of the larval CNS. We hypothesized that Tep1 interacts with the Hippo pathway effector Yki to regulate neuroblast numbers. We tested if Tep1 acts through Yki to affect glioma growth, and if in normal cells Tep1 affects neuroblast number and proliferation. Our data suggests that Tep1 affects Yki mediated stem cell renewal in glioma, as reduction of Tep significantly decreases the number of neuroblasts in glioma. Thus, we identify Tep1-Yki interaction in the larval CNS that plays a key role in glioma growth and progression.

Investigation of the effects of indoxyl sulfate, a uremic toxin, on the intracellular oxidation level and phagocytic activity using an HL-60-differentiated human macrophage cell model

Indoxyl sulfate (IS), a uremic toxin, is a sulfate-conjugated metabolite originated from tryptophan. Accumulating uremic toxins may worsen renal diseases and further complicate related disorders including impaired immune functions under oxidative stress conditions. However, it has remained unclear whether or not IS can directly cause the cellular immune dysfunction. We investigated the effects of IS on the intracellular oxidation level and phagocytic activity in a HL-60-differantiated human macrophage cell model. Incubation of the cells in the presence of IS resulted in increasing intracellular oxidation level and decreasing phagocytic activity. In addition to inhibitors for NADH oxidase (NOX), organic anion transporting polypeptide2B1 (OATP2B1), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K), a representative antioxidant Trolox, was also shown to significantly relieve the IS-induced oxidation and restore weakened phagocytosis. Collectively, IS may directly down-regulate the phagocytic immune function of macrophages through the oxidation mechanisms including OATP2B1, PKC, PI3K, and NOX pathways. Abbreviations: CKD: Chronic kidney disease; IS: Indoxyl sulfate; ROS: Reactive oxygen species; NOX: NADH oxidase; OATP2B1: Organic anion transporting polypeptide2B1; PKC: Protein kinase C; PI3K: Phosphoinositide 3-kinase; 2-APT: 2-acetylphenothiazine.

Immunohistochemical assessment of HRAS Q61R mutations in breast adenomyoepitheliomas.

Breast adenomyoepitheliomas (AMEs) are uncommon tumours. Most oestrogen receptor (ER)-positive AMEs have mutations in phosphoinositide 3-kinase (PI3K) pathway genes, whereas ER-negative AMEs usually harbour concurrent mutations affecting the HRAS Q61 hotspot and PI3K pathway genes. Here, we sought to determine the sensitivity and specificity of RAS Q61R immunohistochemical (IHC) analysis for detection of HRAS Q61R mutations in AMEs. Twenty-six AMEs (14 ER-positive; 12 ER-negative) previously subjected to massively parallel sequencing (n=21) or Sanger sequencing (n=5) of the HRAS Q61 hotspot locus were included in this study. All AMEs were subjected to IHC analysis with a monoclonal (SP174) RAS Q61R-specific antibody, in addition to detailed histopathological analysis. Nine ER-negative AMEs harboured HRAS mutations, including Q61R (n=7) and Q61K (n=2) mutations. Five of seven (71%) AMEs with HRAS Q61R mutations were immunohistochemically positive, whereas none of the AMEs lacking HRAS Q61R mutations (n=17) were immunoreactive. RAS Q61R immunoreactivity was restricted to the myoepithelium in 80% (4/5) of cases, whereas one case showed immunoreactivity in both the epithelial component and the myoepithelial component. RAS Q61R immunohistochemically positive AMEs were associated with infiltrative borders (P<0.001), necrosis (P<0.01) and mitotic index in the epithelial (P<0.05) and myoepithelial (P<0.01) components. RAS Q61R IHC assessment did not reveal Q61K mutations (0/2). IHC analysis of RAS Q61R shows high specificity (100%) and moderate sensitivity (71%) for detection of HRAS Q61R mutations in breast AMEs, and appears not to detect HRAS Q61K mutations. IHC analysis of RAS Q61R may constitute a useful technique in the diagnostic workup of ER-negative AMEs.

3O Mutation analysis of circulating tumour DNA from baseline and study discontinuation samples in SANDPIPER, a phase III study of taselisib or placebo with fulvestrant in oestrogen receptor-positive, human epidermal growth factor receptor 2-negative, PIK3CA-mutant advanced breast cancer

Activating mutations in the phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and AKT1 oncogenes, and mutations in the PTEN tumour suppressor gene, occur in 40–50% of oestrogen receptor (ER)-positive, human epidermal growth factor receptor 2 (HER2)-negative breast cancer (BC). We assessed phosphatidylinositol 3-kinase (PI3K) pathway mutations in circulating tumour DNA (ctDNA) from responding patients in SANDPIPER (NCT02340221), a phase III study of taselisib (PI3K inhibitor) or placebo with fulvestrant in ER-positive, HER2-negative, PIK3CA-mutant locally advanced or metastatic BC.

Osteopontin enhances cisplatin resistance of human A549 lung cancer cells via stimulating the PI3K signaling pathway and upregulating ERCC1 expression.

BackgroundTo investigate the regulatory role of osteopontin (OPN) in cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC).MethodsOPN over-expression and interference vectors were constructed. Human NSCLC A549 cells were divided into normal control, shRNA-NC, shRNA-OPN, OPN, negative control (NC), control+DDP, shRNA-NC+DDP, shRNA-OPN+DDP, OPN+DDP, and NC+DDP groups. Cell proliferation was measured by MTT assay. Cell apoptosis was detected by flow cytometry. PI3K (phosphoinositide 3-kinase), p-ERK1/2 (phospho-extracellular signal-regulated kinases) and ERCC1 (excision repair cross-complementation group 1) mRNA and protein expression was determined by real-time PCR and Western blot.ResultsOPN overexpression and interference vectors were successfully constructed. When compared with control group, OPN group had significantly stimulated cell viability, reduced cell apoptosis, and increased expression of PI3K, p-ERK1/2 and ERCC1 (all P<0.05), whereas all other groups had lower cell viability, higher cell apoptosis, and inhibited protein expression. Moreover, OPN+DDP group had significantly higher proliferation, lower cell apoptosis, and enhanced PI3K, p-ERK1/2 and ERCC1 mRNA and protein expression compared with shRNA-OPN+DDP (all P<0.05).ConclusionsOPN can promote tumor cell growth and reduce cell apoptosis by activating PI3K pathway, which may induce the resistance of NSCLC to cisplatin. Moreover, the stimulation of ERCC1 expression may also increase the cisplatin resistance of NSCLCs.

The Inhibition of Phosphatidylinositol-3-Kinase in activated T-cells by Hibiscone C derivatives

Abstract The phosphatidylinositol-3-kinase (PI3K) pathway is of significant interest due to its ability to regulate cell proliferation, growth, and migration in numerous contexts including in T cells. Furthermore, gain-of-function mutations in PI3K can lead to an upregulation of the pathway, resulting in tumorigenesis, autoimmunity, and leukemia. Specific furanosesquiterpenoids, such as wortmannin, have been known to inhibit the PI3K pathway in T cells. However, wortmannin has unfavorable characteristics as a chemotherapeutic or immunomodulatory drug due to its insolubility in neutral buffers, instability, and high toxicity in vivo. Hibiscone C, another furanosesquiterpenoid, lacks many of the functional groups as wortmannin, but contains the same diacyl furan ring that is critical for interacting with the ATP binding pocket of PI3K. Via analysis of phosphorylation of the downstream effector molecule Akt in activated T cells, we demonstrate that Hibiscone C also can irreversibly inhibit PI3K activity, however not as effectively as wortmannin. These results led us to test other derivatives of hibiscone C, to see if modifications to the molecule’s structure would affect potency of the molecule.

CD28 regulates T cell metabolic reprogramming through ARS2-dependent effects on alternative splicing

Abstract An adaptive immune response requires activation of naïve T cells via T cell receptor (TCR) ligation and engagement of costimulatory molecules such as CD28, which enables cell growth, rapid proliferation, and effector differentiation. To accomplish such bioenergetically demanding processes, T cells must dynamically regulate their metabolism upon activation. After initial TCR triggering of aerobic glycolysis, CD28 co-stimulation maintains uptake and catabolism of glucose required for CD8+ T cells to attain full effector status. Despite evidence that CD28 signaling through the PI3-kinase pathway induces glucose uptake via increased GLUT1 surface expression, knockin T cells expressing PI3-kinase signaling-deficient CD28 are functional in vivo and displayed only transient defects in glycolysis. In contrast, knockin T cells expressing Grb2/Vav signaling-deficient CD28 lacked in vivo function and displayed sustained defects in glycolysis. Here we identify a novel link between CD28-Grb2/Vav signaling and T cell glycolytic reprogramming through the RNA binding protein ARS2, a protein central to co-transcriptional processing of nascent RNA polymerase II transcripts. Data show that ARS2 is a critical downstream mediator of CD28-Grb2/Vav signaling that supports alternative splicing (AS) of the rate-limiting glycolytic enzyme pyruvate kinase M from the M1 to M2 isoform. Mechanistically, ARS2 binds known Pkm splicing factors and promotes their interaction with Pkm mRNA. Consistent with this, ARS2 knockout T cells displayed reduced Pkm2 AS, defective glycolytic reprogramming and lacked the ability to control in vivo tumor growth, suggesting a novel mechanism by which co-stimulation drives T cell metabolic reprogramming.

PI3K Inhibitors and Their Role as Novel Agents for Targeted Therapy in Lymphoma.

Phosphatidylinositol 3-kinase (PI3K) inhibitors represent a novel class of agents targeting the key cellular regulatory PI3K/AKT/mTOR pathway involved in crucial functions such as cellular proliferation, cell cycle regulation, protein synthesis, and cell motility. This review starts with an overview of the PI3K pathway and the rationale for its targeting in lymphoma and potential on-target side effects of PI3K inhibition. With three agents now FDA approved for the treatment of relapsed and refractory (R/R) indolent non-Hodgkin lymphoma (iNHL), idelalisib, copanlisib, and duvelisib, we aim to review the pivotal trials leading to their approval as well as their clinical applications according to lymphoma subtypes. Important treatment-related adverse events are also reviewed and a perspective on the clinical role of these agents is provided, as well as some practical guidance on how to prevent, monitor, and manage potential adverse events in the clinic. PI3K inhibitors have an established role in the management of R/R iNHL, but their use and development are hampered by adverse events, particularly when used in combination with other anti-lymphoma therapies. Finally, this review highlights areas in need of more research in order to optimally use these agents in the care of patients with lymphoma.

9-Methyl-\u03b2-carboline inhibits monoamine oxidase activity and stimulates the expression of neurotrophic factors by astrocytes

β-Carbolines (BC) are pyridoindoles, which can be found in various exogenous and endogenous sources. Recent studies revealed neurostimulative, neuroprotective, neuroregenerative and anti-inflammatory effects of 9-methyl-BC (9-Me-BC). Additionally, 9-me-BC increased neurite outgrowth of dopaminergic neurons independent of dopamine uptake into these neurons. In this study, the role of astrocytes in neurostimulative, neuroregenerative and neuroprotective properties of 9-me-BC was further explored.9-Me-BC exerted anti-proliferative effects without toxic properties in dopaminergic midbrain and cortical astrocyte cultures. The organic cation transporter (OCT) but not the dopamine transporter seem to mediate at least part the effect of 9-me-BC on astrocytes. Remarkably, 9-me-BC stimulated the gene expression of several important neurotrophic factors for dopaminergic neurons like Artn, Bdnf, Egln1, Tgfb2 and Ncam1. These factors are well known to stimulate neurite outgrowth and to show neuroprotective and neuroregenerative properties to dopaminergic neurons against various toxins. Further, we show that effect of 9-me-BC is mediated through phosphatidylinositol 3-kinase (PI3K) pathway. Additionally, 9-me-BC showed inhibitory properties to monoamine oxidase (MAO) activity with an IC50 value of 1 µM for MAO-A and of 15.5 µM for MAO-B. The inhibition of MAO by 9-me-BC might contribute to the observed increased dopamine content and anti-apoptotic properties in cell culture after 9-me-BC treatment in recent studies. Thus, 9-me-BC have a plethora of beneficial effects on dopaminergic neurons warranting its exploration as a new multimodal anti-parkinsonian medication.

Mechanistic basis for PI3K inhibitor antitumor activity and adverse reactions in advanced breast cancer.

The phosphatidylinositol 3-kinase (PI3K) pathway is involved in several physiological processes, including glucose metabolism, cell proliferation, and cell growth. Hyperactivation of this signaling pathway has been associated with tumorigenesis and resistance to treatment in various cancer types. Mutations that activate PIK3CA, encoding the PI3K isoform p110α, are common in breast cancer, particularly in the hormone receptor-positive (HR+), human epidermal growth factor receptor-2-negative (HER2-) subtype. A number of PI3K inhibitors have been developed and evaluated for potential clinical use in combinations targeting multiple signaling pathways in cancer. The purpose of this review is to provide an overview of PI3K inhibitor mechanisms of action for antitumor activity and adverse events in advanced breast cancer (ABC). Published results from phase 3 trials evaluating the efficacy and safety of PI3K inhibitors in patients with ABC and relevant literature were reviewed. Although PI3K inhibitors have been shown to prolong progression-free survival (PFS), the therapeutic index is often unfavorable. Adverse events, such as hyperglycemia, rash, and diarrhea are frequently observed in these patients. In particular, hyperglycemia is intrinsically linked to the inhibition of PI3Kα, a key mediator of insulin signaling. Off-target effects, including mood disorders and liver toxicity, have also been associated with some PI3K inhibitors. Recent clinical trial results show that specifically targeting PI3Kα can improve PFS and clinical benefit. Broad inhibition of class I PI3Ks appears to result in an unfavorable safety profile due to off-target effects, limiting the clinical utility of the early PI3K inhibitors.

Nrf2‐Activating Phytochemicals, Sulforaphane and Licochalcone A, Stimulate Cell Growth‐Regulating Kinases in HCT116 Human Colorectal Cancer Cells

Some phytochemicals have been reported to modulate phase II detoxifying/antioxidant enzyme expressions via both Keap1 (Kelch‐like ECH‐associated protein 1)‐dependent and independent regulations of a nuclear factor E2‐related factor 2/antioxidant response element (Nrf2/ARE) system. Based on multiple studies, various kinase signaling pathways are also responsible for regulation of Nrf2/ARE activity differently depending on cell types, microenvironmental factors, treatment doses, etc. Sulforaphane (SFN) and licochalcone A (LicA) are known as potent Nrf2/ARE inducers that can modulate several kinases including phosphoinositide 3‐kinase (PI3K)/Protein kinase B (Akt) and p38 mitogen‐activated protein kinase (MAPK) in propagating cancer cells. The purpose of the present study was to investigate the molecular mechanism through which SFN and LicA can activate Nrf2/ARE system and upregulate heme oxygenase‐1 (HO‐1) expression in HCT116 human colorectal cancer cells. Results revealed that SFN and LicA induced ARE‐transcriptional activity and subsequently increased HO‐1 expression through PI3K and p38 MAPK signaling pathways in HCT116 cells. In addition, HCT116 cell proliferation was significantly promoted when Nrf2/ARE activity was increased by either SFN‐induced PI3K activation or LicA‐induced p38 MAPK activation. These observations indicate that SFN and LicA regulated the Nrf2/ARE/HO‐1 system via phosphorylation of PI3K and p38 MAPK, respectively, in proliferation of HCT116 colorectal cancer cells. In conclusion, the present study highlights the working mechanism of potent Nrf2/ARE activators that possibly perturb the activities of various kinases in the growing cancer cells.Support or Funding InformationThis work was supported by the National Research Foundation of Korea grant (Grant No. 2017R1A2B4005087) funded by the Ministry of Science and ICT, Republic of Korea.

Regulation of PI3k-WNK Pathway by Ethyl Acetate Partition Fraction of Gardenia jasminoides var. radicans Makino in SHR, NRK52e Cells, and IMCD3 Cells

The antihypertensive mechanism was studied of the ethyl acetate fraction of the ethanol extract (EAPF) of Gardenia jasminoides var. radicans Makino (GJRM). GJRM is a fake product of Gardenia jasminoides Ellis ( G. jasminoides), but in China’s Henan province, the production of GJRM is much more than G. jasminoides’s, but its traditional usage is as a dye. Gardenia jasminoides can be used to reduce blood pressure and blood glucose levels. The chemical compositions of GJRM and G. jasminoides are similar, and we previously confirmed that GJRM can also reduce blood pressure. Here, we report that the EAPF of GJRM could activate the phosphoinositide 3-kinases (PI3K) pathway in the kidneys of spontaneously hypertensive rats, thus increasing the content of nitric oxide and bradykinin in sera and decreasing endothelin-1 content. EAPF can also decrease the levels of with-no-lysine kinase 1 (WNK1) expression, WNK4 and oxidative stress-responsive kinase 1 messenger ribonucleic acid (mRNA), and Na-K-2Cl cotransporter and sodium chloride cotransporters mRNA and phosphorylation. To investigate the antihypertensive effects of the EAPF of GJRM, 5 monoterpenoids isolated from EAPF were studied for their effects on NRK52e and IMCD3 cells. These compounds inhibited the PI3K-WNK signaling pathway to varying degrees under hypotonic conditions; 4-methoxyrehmapicrogenin had the best effect.

IPMK is Indispensable for Activation of AKT: Redefining PI3kinase pathway

Inositol polyphosphate multikinase (IPMK) can catalyze production of IP5 and PIP3. Deletion of IPMK abolish AKT phosphorylation at both T 308 and Ser 473 residues even when PI3kinase activity of classical PI3kinase is intact. Akt activation requires its membrane localization followed by phosphorylation by PDK1 and mTORC2. We found that classical PI3kinase exclusively mediates AKT localization to the membrane but dispensable for PDK1 membrane localization and activation of mTORC2. IPMK interacts with both PDK1 and mTORC2 complex. Generation of PIP3 by IPMK promotes membrane localization of PDK1 and also activation of mTORC2, however, shows no effect in AKT localization to the membrane. In summary, PIP3 generated by IPMK exclusively controls PDK1 and mTORC2 functioning, however, classical PI3Kinase only controls AKT localization to the membrane.Support or Funding InformationNIH grant 5R01MH018501‐48

Dihydromethysticin, a natural molecule from Kava, suppresses the growth of colorectal cancer via the NLRC3/PI3K pathway

Dihydromethysticin (DHM), a natural compound derived from Kava, has been reported to be effective against mental disorders and some malignant tumors. However, little is known about the inhibitory effect of DHM on colorectal cancer (CRC). First, we examined the impact of DHM on human colon cancer cell lines, which demonstrated that DHM inhibits proliferation, migration, and invasion and promotes apoptosis and cell cycle arrest in colon cancer cells in vitro. Using small hairpin RNA, we inhibited nucleotide-oligomerization domain-like receptor subfamily C3 (NLRC3)/phosphoinositide 3-kinase (PI3K) pathway to elucidate the partial signaling of DHM-mediated tumor suppression. Additionally, using an ectopic human CRC model, we verified whether DHM inhibits tumor growth and angiogenesis via the NLRC3/PI3K pathway in vivo. Overall, DHM showed an inhibitory effect on CRC by altering cell proliferation, migration, invasion, apoptosis, cell cycle, and angiogenesis, possibly via the NLRC3/PI3K pathway. Thus, DHM may be a promising candidate for CRC therapy.

Alterations in the PI3K Pathway Drive Resistance to MET Inhibitors in NSCLC Harboring MET Exon 14 Skipping Mutations.

Hepatocyte growth factor receptor (MET) tyrosine kinase inhibitors (MET TKIs) have been found to have efficacy against advanced NSCLC with mutations causing MET exon 14 skipping (METex14 mutations), but primary resistance seems frequent, as response rates are lower than those for targeted TKIs of other oncogene-addicted NSCLCs. Given the known interplay between MET and phosphoinositide 3-kinases (PI3K), we hypothesized that in METex14 NSCLC, PI3K pathway alterations might contribute to primary resistance to MET TKIs. We reviewed clinical data from 65 patients with METex14 NSCLC, assessing PI3K pathway alterations by targeted next-generation sequencing (mutations) and immunohistochemistry (loss of phosphatase and tensin homolog [PTEN]). Using a cell line derived from a patient with primary resistance to a MET TKI and cell lines harboring both a METex14 mutation and a PI3K pathway alteration, we assessed sensitivity to MET TKIs used alone or with a PI3K inhibitor and investigated relevant signaling pathways. We found a phosphatidylinositol 3-kinase catalytic subunit alpha (PIK3CA) mutation in two of 65 samples (3%) and loss of PTEN in six of 26 samples (23%). All three of the MET TKI-treated patients with a PI3K pathway alteration had been found to have progressive disease at first assessment. Likewise, MET TKIs had no effect on the proliferation of METex14-mutated cell lines with a PI3K pathway alteration, including the PTEN-lacking patient-derived cell line. Treatment combining a MET TKI with a PI3K inhibitor caused inhibition of both PI3K and MAPK signaling and restored sensitivity to MET TKIs. PI3K pathway alterations are common in METex14 NSCLC and may confer primary resistance to MET TKIs. In preclinical models, PI3K inhibition restores sensitivity to MET TKIs.

Angiotensin II represses Npr1 expression and receptor function by recruitment of transcription factors CREB and HSF-4a and activation of HDACs

The two vasoactive hormones, angiotensin II (ANG II; vasoconstrictive) and atrial natriuretic peptide (ANP; vasodilatory) antagonize the biological actions of each other. ANP acting through natriuretic peptide receptor-A (NPRA) lowers blood pressure and blood volume. We tested hypothesis that ANG II plays critical roles in the transcriptional repression of Npr1 (encoding NPRA) and receptor function. ANG II significantly decreased NPRA mRNA and protein levels and cGMP accumulation in cultured mesangial cells and attenuated ANP-mediated relaxation of aortic rings ex vivo. The transcription factors, cAMP-response element-binding protein (CREB) and heat-shock factor-4a (HSF-4a) facilitated the ANG II-mediated repressive effects on Npr1 transcription. Tyrosine kinase (TK) inhibitor, genistein and phosphatidylinositol 3-kinase (PI-3K) inhibitor, wortmannin reversed the ANG II-dependent repression of Npr1 transcription and receptor function. ANG II enhanced the activities of Class I histone deacetylases (HDACs 1/2), thereby decreased histone acetylation of H3K9/14ac and H4K8ac. The repressive effect of ANG II on Npr1 transcription and receptor signaling seems to be transduced by TK and PI-3K pathways and modulated by CREB, HSF-4a, HDACs, and modified histones. The current findings suggest that ANG II-mediated repressive mechanisms of Npr1 transcription and receptor function may provide new molecular targets for treatment and prevention of hypertension and cardiovascular diseases.

Insulin-PI3K signalling: an evolutionarily insulated metabolic driver of cancer.

Cancer is driven by incremental changes that accumulate, eventually leading to oncogenic transformation. Although genetic alterations dominate the way cancer biologists think about oncogenesis, growing evidence suggests that systemic factors (for example, insulin, oestrogen and inflammatory cytokines) and their intracellular pathways activate oncogenic signals and contribute to targetable phenotypes. Systemic factors can have a critical role in both tumour initiation and therapeutic responses as increasingly targeted and personalized therapeutic regimens are used to treat patients with cancer. The endocrine system controls cell growth and metabolism by providing extracellular cues that integrate systemic nutrient status with cellular activities such as proliferation and survival via the production of metabolites and hormones such as insulin. When insulin binds to its receptor, it initiates a sequence of phosphorylation events that lead to activation of the catalytic activity of phosphoinositide 3-kinase (PI3K), a lipid kinase that coordinates the intake and utilization of glucose, and mTOR, a kinase downstream of PI3K that stimulates transcription and translation. When chronically activated, the PI3K pathway can drive malignant transformation. Here, we discuss the insulin-PI3K signalling cascade and emphasize its roles in normal cells (including coordinating cell metabolism and growth), highlighting the features of this network that make it ideal for co-option by cancer cells. Furthermore, we discuss how this signalling network can affect therapeutic responses and how novel metabolic-based strategies might enhance treatment efficacy for cancer.

Mutations in long-lived epithelial stem cells and their clonal progeny in pre-malignant lesions and in oral squamous cell carcinoma.

Oral squamous cell carcinomas (OSCCs) are the most common cancers of the oral cavity, but the molecular mechanisms driving OSCC carcinogenesis remain unclear. Our group previously established a murine OSCC model based on a 10-week carcinogen [4-nitroquinoline 1-oxide (4-NQO)] treatment. Here we used K14CreERTAM;Rosa26LacZ mice to perform lineage tracing to delineate the mutational profiles in clonal cell populations resulting from single, long-lived epithelial stem cells, here called LacZ+ stem cell clones (LSCCs). Using laser-capture microdissection, we examined mutational changes in LSCCs immediately after the 10-week 4-NQO treatment and >17 weeks after 4-NQO treatment. We found a 1.8-fold ±0.4 (P = 0.009) increase in single-nucleotide variants and insertions/deletions (indels) in tumor compared with pre-neoplastic LSCCs. The percentages of indels and of loss of heterozygosity events were 1.3-fold±0.3 (P = 0.02) and 2.2-fold±0.7 (P = 0.08) higher in pre-neoplastic compared with tumor LSCCs. Mutations in cell adhesion- and development-associated genes occurred in 83% of the tumor LSCCs. Frequently mutated genes in tumor LSCCs were involved in planar cell polarity (Celsr1, Fat4) or development (Notch1). Chromosomal amplifications in 50% of the tumor LSCCs occurred in epidermal growth factor receptor, phosphoinositide 3-kinase and cell adhesion pathways. All pre-neoplastic and tumor LSCCs were characterized by key smoking-associated changes also observed in human OSCC, C>A and G>T. DeconstructSigs analysis identified smoking and head and neck cancer as the most frequent mutational signatures in pre-neoplastic and tumor LSCCs. Thus, this model recapitulates a smoking-associated mutational profile also observed in humans and illustrates the role of LSCCs in early carcinogenesis and OSCCs.