Increased Akt Activity Research Articles

FoxO1/Rictor axis induces a non-genetic adaptation to Ibrutinib via Akt activation in chronic lymphocytic leukemia.

BTK inhibitor therapy induces peripheral blood lymphocytosis in chronic lymphocytic leukemia (CLL) lasting for several months. It remains unclear whether non-genetic adaptation mechanisms exist, allowing CLL cells' survival during BTK inhibitor-induced lymphocytosis and/or playing a role in therapy resistance. We show that in approximately 70 % of CLL cases, ibrutinib treatment in vivo increases Akt activity above pre-therapy levels within several weeks, leading to compensatory CLL cell survival and a more prominent lymphocytosis on therapy. Ibrutinib-induced Akt phosphorylation (pAktS473) is caused by the upregulation of FoxO1 transcription factor, which induces expression of Rictor, an assembly protein for mTORC2 protein complex that directly phosphorylates Akt at serine 473 (S473). Knock-out or inhibition of FoxO1 or Rictor led to a dramatic decrease in Akt phosphorylation and growth disadvantage for malignant B cells in the presence of ibrutinib (or PI3K inhibitor idelalisib) in vitro and in vivo. FoxO1/Rictor/pAktS473 axis represents an early non-genetic adaptation to BCR inhibitor therapy not requiring PI3Kδ or BTK kinase activity. We further demonstrate that FoxO1 can be targeted therapeutically, and its inhibition induces CLL cells' apoptosis alone or in combination with BTK inhibitors (ibrutinib, acalabrutinib, pirtobrutinib) and blocks their proliferation triggered by T-cell factors (CD40L, IL-4, and IL-21).

IL-10 Enhances the Inhibitory Effect of Adipose-Derived Stromal Cells on Insulin Resistance/Liver Gluconeogenesis by Treg Cell Induction.

The modulation of cellular phenotypes within adipose tissue provides a potential means for therapeutic intervention for diabetes. Endogenous interleukin-10 (IL-10) protects against diet-induced insulin resistance. We examined the effects and mechanisms of action of IL-10-treated adipose-derived stromal cells on diabetes-induced insulin resistance and liver gluconeogenesis. We harvested stromal vascular fractions (SVFs) from the adipose tissue of diabetic (Leprdb/db) mice and treated them with IL-10 in vitro. SVFs treated with 10 or 100 ng of IL-10 were injected into the inguinal adipose tissue of Leprdb/db mice. IL-10 treatment suppressed the mRNA expression of IL-6, IL-33, CCL2, TNF-α, and IL-1β. Additionally, it suppressed the protein expression of IL-6, pmTOR, pJNK, and pNF-κB but enhanced Foxp3 mRNA expression in SVFs from diabetic mice. Meanwhile, IL-10 treatment repressed CCL2 and PDGFRα expression in adipose tissue macrophages (ATMs) and IL-6 expression in non-ATMs but increased the Foxp3 and IL-10 mRNA expression of ATMs from diabetic mice. Injection of IL-10-treated SVFs decreased the IL-6, IL-33, CCL2, IL-1β, and CCL2 but enhanced the Foxp3 and IL-10 mRNA expression of adipose tissue from Leprdb/db mice. Furthermore, injection of IL-10-treated SVFs increased CD4+ regulatory T cells (Tregs) in SVFs and adipose IL-10 levels and suppressed plasma adiponectin levels and DPP4 activity in diabetic mice. Injection of IL-10-treated SVFs decreased hepatic G6PC and PCK1 mRNA expression and increased Akt activation, STAT3 phosphorylation in the liver, and glucose tolerance in diabetic mice. Our data suggest that IL-10 treatment decreases inflammation in adipose SVFs of diabetic mice. Injection of IL-10-treated SVFs into the adipose tissue decreased diabetes-induced gluconeogenesis gene expression, DPP4 activity, and insulin resistance by enhancing Treg cells in diabetic mice. These data suggest that IL-10-treated adipose stromal vascular cells could be a promising therapeutic strategy for diabetes mellitus.

Loss of Cavin-2 destabilizes phosphatase and tensin homologue and enhances Akt signalling pathway in cardiomyocytes.

Specific cavins and caveolins, known as caveola-related proteins, have been implicated in cardiac hypertrophy and myocardial injury. Cavin-2 forms complexes with other caveola-related proteins, but the role of Cavin-2 in cardiomyocytes (CMs) is poorly understood. Here, we investigated an unknown function of Cavin-2 in CMs. Under cardiac stress-free conditions, systemic Cavin-2 knockout (KO) induced mild and significant CM hypertrophy. Cavin-2 KO suppressed phosphatase and tensin homologue (PTEN) associated with Akt signalling, whereas there was no difference in Akt activity between the hearts of the wild-type and the Cavin-2 KO mice under cardiac stress-free conditions. However, after swim training, CM hypertrophy was more facilitated with enhanced phosphoinositide 3-kinase (PI3K)-Akt activity in the hearts of Cavin-2 KO mice. Cavin-2 knockdown neonatal rat CMs (NRCMs) using adenovirus expressing Cavin-2 short hairpin RNA were hypertrophied and resistant to hypoxia and H2O2-induced apoptosis. Cavin-2 knockdown increased Akt phosphorylation in NRCMs, and an Akt inhibitor inhibited Cavin-2 knockdown-induced anti-apoptotic responses in a dose-dependent manner. Cavin-2 knockdown increased phosphatidylinositol-3,4,5-triphosphate production and attenuated PTEN at the membrane fraction of NRCMs. Immunostaining and immunoprecipitation showed that Cavin-2 was associated with PTEN at the plasma membrane of NRCMs. A protein stability assay showed that Cavin-2 knockdown promoted PTEN destabilization in NRCMs. In an Angiotensin II (2-week continuous infusion)-induced pathological cardiac hypertrophy model, CM hypertrophy and CM apoptosis were suppressed in CM-specific Cavin-2 conditional KO (Cavin-2 cKO) mice. Because Cavin-2 cKO mouse hearts showed increased Akt activity but not decreased extracellular signal-regulated kinase activity, suppression of pathological hypertrophy by Cavin-2 loss may be due to increased survival of healthy CMs. Cavin-2 plays a negative regulator in the PI3K-Akt signalling in CMs through interaction with PTEN. Loss of Cavin-2 enhances Akt activity by promoting PTEN destabilization, which promotes physiological CM hypertrophy and may enhance Akt-mediated cardioprotective effects against pathological CM hypertrophy.

BMPR2 Loss Activates AKT by Disrupting DLL4/NOTCH1 and PPARγ Signaling in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by pathologic vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in pulmonary artery endothelial cells (PAECs) activated AKT and suppressed the expression of DLL4. Consistent with these in vitro findings, increased AKT activation and reduced DLL4 expression was found in the small pulmonary arteries of patients with PAH. Increased NOTCH1 activation through exogenous DLL4 blocked AKT activation, decreased proliferation and reversed EndoMT. Exogenous and overexpression of DLL4 induced BMPR2 and PPRE promoter activity, and BMPR2 and PPARG mRNA in idiopathic PAH (IPAH) ECs. PPARγ, a nuclear receptor associated with EC homeostasis, suppressed by BMPR2 loss was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH ECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Directly blocking AKT or restoring DLL4/NOTCH1/PPARγ signaling may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.

Abstract 6631: Aging influences the pancreatic ductal adenocarcinoma tumor immune microenvironment to promote features of immunosuppression

Abstract Advancing age is the strongest independent risk factor for the development of pancreatic ductal adenocarcinoma (PDAC) and a negative prognostic factor. PDACs are typically immunologically “cold” tumors and do not often respond to currently approved immunotherapy treatment approaches. How aging may alter non-malignant cells, such as fibroblasts and immune cell populations, in the PDAC tumor microenvironment (TME) to decrease anti-tumor immune responses is not understood. We identified that aged microenvironments significantly alter the growth and progression of PDAC tumors. Orthotopic KPC and Panc02 mouse PDAC cell line injections into the pancreata of aged (>64-week-old) compared to young (6-8-week-old) C57BL/6 mice result in more rapid tumor growth and increased frequency of liver metastases in vivo. Tumors from aged mice have high expression of the TGFβ family member GDF-15 and hyperactivation of AKT signaling driven by the release of secreted factors from aged pancreatic fibroblasts. We found that in aged microenvironments there is a marked reduction in the number of CD8+ T cells and an increase in FoxP3+ regulatory T cells in primary orthotopic pancreatic tumors using the KPC and Panc02 models. In addition, we performed portal vein injections to establish liver metastases to study the differences in immune cell infiltration in metastases from young vs. aged microenvironments. Aged mice form more and larger liver metastases compared to young mice, and metastatic lesions in aged mice have decreased CD8+ T cell infiltration when compared to young mice. Since tumors in aged microenvironments have increased AKT activation compared to young microenvironments, we next tested if AKT signaling was sufficient to alter immune cell infiltration in PDAC tumors. In young mice, treatment with recombinant GDF-15, which activates AKT signaling, reduced CD8+ T cell infiltration into tumors. Conversely, in KPC tumors grown in aged mice treated with the AKT inhibitor MK-2206, there was a trend toward increased CD8+ T cell infiltration and a significant reduction in immunosuppressive FoxP3+ Tregs. Together, these data highlight age-dependent differences in the immune infiltration in PDAC tumors. They suggest that activation of AKT signaling can drive features of immunosuppression in PDAC, providing the rationale for targeting AKT signaling to improve antitumor immune responses in PDAC. Citation Format: Emma Kartalia, James M. Leatherman, Jae W. Lee, Elizabeth M. Jaffee, Ashani T. Weeraratna, Daniel J. Zabransky. Aging influences the pancreatic ductal adenocarcinoma tumor immune microenvironment to promote features of immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6631.

CASK Mediates Oxidative Stress-Induced Microglial Apoptosis-Inducing Factor-Independent Parthanatos Cell Death via Promoting PARP-1 Hyperactivation and Mitochondrial Dysfunction.

Calcium/calmodulin-dependent serine protein kinase (CASK) is a scaffold protein and plays critical roles in neuronal synaptic formation and brain development. Previously, CASK was shown to associate with EGFR to maintain the vulval cell differentiation in C. elegans. In this study, we explored the role of CASK in CHME3 microglial cells. We found that CASK silencing protects cells from H2O2-induced cell death by attenuating PARP-1 activation, mitochondrial membrane potential loss, reactive oxygen species production, and mitochondrial fission, but it increases oxidative phosphorylation. The PARP-1 inhibitor olaparib blocks H2O2-induced cell death, suggesting the death mode of parthanatos. CASK silencing also increases AKT activation but decreases AMPK activation under H2O2 treatment. Pharmacological data further indicate that both signaling changes contribute to cell protection. Different from the canonical parthanatos pathway, we did not observe the AIF translocation from mitochondria into the nucleus, suggesting a non-canonical AIF-independent parthanatos in H2O2-treated CHME3 cells. Moreover, we found that CASK silencing upregulates the EGFR gene and protein expression and increases H2O2-induced EGFR phosphorylation in CHME3 microglia. However, EGFR activation does not contribute to cell protection caused by CASK silencing. In conclusion, CASK plays a crucial role in microglial parthanatos upon H2O2 treatment via stimulation of PARP-1 and AMPK but the inhibition of AKT. These findings suggest that CASK might be an ideal therapeutic target for CNS disorders.

Long-term Rapamycin Treatment Inhibit AKT Activity and Lower Intracellular Calcium Expression in Organotypic Hippocampal Slice Cultures Model of Epilepsy

Despite the development of anti-epilepsy drugs, drug-refractory epilepsy still becomes a challenging problem along with an increased incidence of epilepsy. To face that challenge and increase patients’ quality of life, treatment of epilepsy must effectively prevent epileptogenesis, not only symptomatic treatment. AKT signaling pathway was proven to have important roles in epilepsy through its function in the synaptic plasticity, neurogenesis, axon guidance, modulation of the glutamate transporter, and activation of the Ca2+ channel. AKT also activated mTOR signaling pathway as activator of mTORC1 and also effector of mTORC2. Several studies showed the ability of long-term rapamycin treatment to inhibit mTORC2. This study used organotypic hippocampal slice cultures (OHSC) and long-term rapamycin treatment was administered for 3, 5, 8, and 10 days at a dose of 20 nM after induction of epilepsy by low-Mg2+ medium administration for 40 minutes. Low-Mg2+ medium administration induced seizure activity in OHSC showed by significant increase in intracellular Ca2+expressionand also significantly increase AKT activity. After administration of long-term rapamycin treatment AKT activity and intracellular Ca2+expression were significantly reduced. The longer the treatment of rapamycin, the lower the AKT activity and intracellular Ca2+expression. Long-term rapamycin treatment has the potential to become a novel epilepsy drug through its ability to attenuate AKT activity and suppress the seizures proven by lower intracellular Ca2+expression.

Neuron-secreted NLGN3 ameliorates ischemic brain injury via activating Gαi1/3-Akt signaling

We here tested the potential activity and the underlying mechanisms of neuroligin-3 (NLGN3) against ischemia-reperfusion-induced neuronal cell injury. In SH-SY5Y neuronal cells and primary murine cortical neurons, NLGN3 activated Akt-mTOR and Erk signalings, and inhibited oxygen and glucose deprivation (OGD)/re-oxygenation (OGD/R)-induced cytotoxicity. Akt activation was required for NLGN3-induced neuroprotection. Gαi1/3 mediated NLGN3-induced downstream signaling activation. NLGN3-induced Akt-S6K1 activation was largely inhibited by Gαi1/3 silencing or knockout. Significantly, NLGN3-induced neuroprotection against OGD/R was almost abolished by Gαi1/3 silencing or knockout. In vivo, the middle cerebral artery occlusion (MCAO) procedure induced NLGN3 cleavage and secretion, and increased its expression and Akt activation in mouse brain tissues. ADAM10 (A Disintegrin and Metalloproteinase 10) inhibition blocked MCAO-induced NLGN3 cleavage and secretion, exacerbating ischemic brain injury in mice. Neuronal silencing of NLGN3 or Gαi1/3 in mice also inhibited Akt activation and intensified MCAO-induced ischemic brain injury. Conversely, neuronal overexpression of NLGN3 increased Akt activation and alleviated MCAO-induced ischemic brain injury. Together, NLGN3 activates Gαi1/3-Akt signaling to protect neuronal cells from ischemia-reperfusion injury.

Bifidobacterium bifidum and Lactobacillus paracasei alleviate sarcopenia and cognitive impairment in aged mice by regulating gut microbiota-mediated AKT, NF-κB, and FOXO3a signaling pathways

Sarcopenia is closely associated with gut dysbiosis. Probiotics alleviate gut dysbiosis. Therefore, we selected probiotics Lactobacillus paracasei P62 (Lp) and Bifidobacterium bifidum P61 (Bb), which suppressed muscle RING-finger protein-1 (MuRF1) expression and NF-κB activation in C2C12 cells, and examined their effects on muscle mass loss and dysfunction in aged mice. Oral administration of Lp, Bb, or their mix (LB) increased grip strength and treadmill running distance and time. They significantly increased muscle weight in aged mice. They also increased AKT activation, PGC1α, SIRT1, and myosin heavy chain (MyHC) expression, MyHC-positive cell population, and cell size in the gastrocnemius (GA) muscle, while FOXO3a and NF-κB activation, MuRF1, muscle atrophy F-box, and p16 expression, and NF-κB+CD11c+ cell population decreased. Furthermore, they reduced cognitive impairment-like behavior, IL-6 expression, FOXO3a activation, and NF-κB-positive cell population in the hippocampus, GA, and colon, while hippocampal brain-derived neurotropic factor expression increased. They shifted gut microbiota composition in aged mice: they increased Akkermansiaceae and Bacteroidaceae populations, which were positively correlated with total muscle weight and MyHC expression, and decreased Odoribacteraceae and Deferribacteriaceae populations, which were positively correlated with MuRF1 and IL-6 expression. LB alleviated sarcopenia- and cognitive impairment-like symptoms more potently than Lp or Bb alone. Based on these findings, probiotics, particularly Lp, Bb, and LB, can alleviate aging-dependent sarcopenia and cognitive impairment by regulating gut microbiota-mediated AKT, NF-κB, and/or FOXO3a signaling pathways.

A Novel Mouse Model of Combined Hepatocellular-Cholangiocarcinoma Induced by Diethylnitrosamine and Loss of Ppp2r5d.

Primary liver cancer (PLC) can be classified in hepatocellular (HCC), cholangiocarcinoma (CCA), and combined hepatocellular-cholangiocarcinoma (cHCC-CCA). The molecular mechanisms involved in PLC development and phenotype decision are still not well understood. Complete deletion of Ppp2r5d, encoding the B56δ subunit of Protein Phosphatase 2A (PP2A), results in spontaneous HCC development in mice via a c-MYC-dependent mechanism. In the present study, we aimed to examine the role of Ppp2r5d in an independent mouse model of diethylnitrosamine (DEN)-induced hepatocarcinogenesis. Ppp2r5d deletion (heterozygous and homozygous) accelerated HCC development, corroborating its tumor-suppressive function in liver and suggesting Ppp2r5d may be haploinsufficient. Ppp2r5d-deficient HCCs stained positively for c-MYC, consistent with increased AKT activation in pre-malignant and tumor tissues of Ppp2r5d-deficient mice. We also found increased YAP activation in Ppp2r5d-deficient tumors. Remarkably, in older mice, Ppp2r5d deletion resulted in cHCC-CCA development in this model, with the CCA component showing increased expression of progenitor markers (SOX9 and EpCAM). Finally, we observed an upregulation of Ppp2r5d in tumors from wildtype and heterozygous mice, revealing a tumor-specific control mechanism of Ppp2r5d expression, and suggestive of the involvement of Ppp2r5d in a negative feedback regulation restricting tumor growth. Our study highlights the tumor-suppressive role of mouse PP2A-B56δ in both HCC and cHCC-CCA, which may have important implications for human PLC development and targeted treatment.

MiR-21 alleviates renal tubular epithelial cells injury induced by ischemia by targeting TLR4

Renal ischemia is the initial stage of kidney damage, leading to mitochondrial metabolism disorders and cell necrosis. In this study, we aimed to investigate the biological functions and potential mechanisms of miR-21 in protecting renal tubular epithelial cells from oxidative stress and apoptosis following oxygen glucose deprivation (OGD). Following an OGD injury, miR-21 levels increased in HK-2 renal tubular epithelial cells. Overexpression of miR-21 decreased the protein expressions of cleaved caspase-3, BAX, P53, cell apoptosis and increased Bcl-2 expression in HK-2 cells with OGD injury. In vivo studies found that miR-21 agomir reduced renal tissue apoptosis, while miR-21 antagomir increased it. In addition, overexpression of miR-21 reduced levels of reactive oxygen species (ROS), malondialdehyde (MDA) and lactate dehydrogenase (LDH) in HK-2 cells with OGD injury. However, miR-21 inhibition exhibited the opposite effect. A dual-luciferase reporter assay demonstrated that miR-21 directly regulates Toll-like receptor 4 (TLR4) by targeting the 3′-UTR of TLR4 mRNA. Overexpression of miR-21 led to decreased TLR4 protein expression, and TLR4 knockdown was shown to greatly increase AKT activity in HK-2 cells by in vitro kinase assay. Additionally, TLR4 knockdown promoted AKT phosphorylation and hypoxia-inducible factor-1α (HIF-1α) expression, while TLR4 overexpression inhibited these processes. Furthermore, AKT activation abolished the effect of TLR4 on HIF-1α, while AKT inhibition decreased the expression of TLR4 on HIF-1α in TLR4 knockdown HK-2 cells. Further study revealed that HIF-1α inhibition abolished the protective effect of miR-21 overexpression on ROS, LDH levels and cell apoptosis in HK-2 cells after OGD injury, which is indicated by increased levels of ROS and LDH, as well as increased cell apoptosis after HIF-1α inhibition in miR-21-treated HK-2 cells. In conclusion, miR-21 defends OGD-induced HK-2 cell injury via the TLR4/AKT/HIF-1α axis.

Concurrent induction of apoptosis and necroptosis inapigenin‑treated malignant mesothelioma cells: Reversal ofWarburg effect through Akt inhibition and p53 upregulation.

A high dependence on aerobic glycolysis, known as the Warburg effect, is one of the metabolic features exhibited by tumor cells. Therefore, targeting glycolysis is becoming a very promising strategy for the development of anticancer drugs. In the present study, it was investigated whether pre‑adaptation of malignant mesothelioma (MM) cells to an acidic environment was associated with a metabolic shift to the Warburg phenotype in energy production, and whether apigenin targets acidosis‑driven metabolic reprogramming. Cell viability, glycolytic activity, Annexin V‑PE binding activity, reactive oxygen species (ROS) levels, mitochondrial membrane potential, ATP content, western blot analysis and spheroid viability were assessed in the present study. MM cells pre‑adapted to lactic acid were resistant to the anticancer drug gemcitabine, increased Akt activation, downregulated p53 expression, and upregulated rate‑limiting enzymes in glucose metabolism compared with their parental cells. Apigenin treatment increased cytotoxicity, Akt inactivation and p53 upregulation. Apigenin also reduced glucose uptake along with downregulation of key regulatory enzymes in glycolysis, increased ROS levels with loss of mitochondrial membrane potential, and downregulated the levels of complexes I, III and IV in the mitochondrial electron transport chain with intracellular ATP depletion, resulting in upregulation of molecules mediating apoptosis and necroptosis. Apigenin‑induced alterations of cellular responses were similar to those of Akt inactivation by Ly294002. Overall, the present results provide mechanistic evidence supporting the anti‑glycolytic and cytotoxic role of apigenin via inhibition of the PI3K/Akt signaling pathway and p53 upregulation.

Reply.

We thank the authors of Hu et al. for their comments on our study.1 We understand the complexity of signaling pathways that can make the current study complicated for some to understand but are confident of its relevance and reproducibility. With regards to concern 1, relationship between hepatic mechanistic target of rapamycin complex 1 (mTORC1) and β‐catenin was shown in four ways. First, we identified compensatory activation of mTORC1 and its downstream effectors in β‐cateninΔHC mice after hepatectomy. Second, we showed demise of RaptorΔHC‐β‐cateninΔHC mice by 12 h and of RaptorΔHC mice by 48 h posthepatectomy. Third, RaptorΔHC mice without any challenge died at 40 days and showed early proliferative β‐catenin activation. RaptorΔHC‐β‐cateninΔHC mice showed demise by 30 days. Lastly, pharmacological inhibition of mechanistic target of rapamycin led to impaired liver regeneration (LR) and demise of β‐cateninΔHC by 14 days postsurgery. Collectively, our data demonstrate compensatory interplay between mTORC1 and β‐catenin signaling. For point 2, AAV8‐TBG–mediated gene deletion in our model should not disrupt pancreatic mTORC1 signaling.2 Decreased serum glucose level in Raptor knockouts has been reported due to compensatory AKT activation.3 Deleting Akt2 in the Raptor knockouts reversed changes in serum glucose and may be mediated by AKT‐driven glycogen synthase kinase 3 beta (GSK3β) phosphorylation and inactivation.3 We also observed increased AKT activation and GSK3β inactivation in RaptorΔHC mice. Thorough understanding of the intricacies of LR is important to appreciate the sequence of experimentation in our study. Also, because of the numbers of mice and time points, it is impossible to show every piece of data, but appropriate statistics were always included. Analysis of known mitogenic and developmental pathways during LR in β‐cateninΔHC mice was inconsequential. We identified insulin‐mTORC1 signaling as one possible mechanism driving LR in these mice. We did not address the role of Wnt‐dependent stabilization of proteins signaling, which may be agnostically contributing to cell division in LR.4 It is unlikely that Wnt/mTORC1 pathway signaling is playing a role in LR in β‐cateninΔHC mice because we did not see any difference in LDL related protein 6 phosphorylation.5 Eventually, we agree that, as a responsible researcher, it is pertinent to be cautious in interpreting every scientific study because there often are multiple explanations underlying an observation.

Abstract PD5-08: PD5-08 Drug target activation mapping of matched triple negative primary and axillary lymph node metastases identifies and links the ER-AR-GR steroid hormone receptor axis with downstream AKT activation

Abstract Title: Drug target activation mapping of matched triple negative primary and axillary LN metastases identifies and links the ER-AR-GR steroid hormone receptor axis with downstream AKT activation Background: Triple negative breast cancer (TNBC) is comprised of heterogeneous subtypes that arise from various molecular alterations that result in TNBC having the poorest overall prognosis. There is growing interest in more sensitive ways to measure estrogen receptor (ER) and HER2 levels in the ER “negative” and HER2 “low/negative” settings, as there are patients with TNBC whose tumors are actually ER and/or HER2 “expressing/active”, and who may benefit from ER- and/or HER2-targeted therapies. Given the limitations of IHC in reliably quantitating ER and HER2 in low (0-1+) expressing tumors, we utilized highly sensitive reverse phase protein array (RPPA) to quantitate expression of these therapeutic targets along with downstream signaling activation mapping in a unique pilot set of patient-matched primary (P) and axillary lymph node (LN) metastases obtained synchronously. The quantitative expression of steroid hormone receptor expression/activation (ER, glucocorticoid receptor (GR), androgen receptor (AR)) along with their signaling architecture and dynamics were analyzed. Methods: A 12 P-LN paired tumor set was chosen for analysis (N=24). Manual macrodissection was used to enrich for tumor epithelium prior to RPPA analysis that quantitatively measured 155 proteins/phosphoprotein drug targets in key signaling pathways known to be involved in steroid hormone receptor biology and tumorigenesis. Statistical analysis using paired sample t-testing or Wilcoxon rank testing was performed (p< 0.05 uncorrected). Results: Protein pathway activation mapping and unsupervised clustering analysis revealed a subset of TNBCs from 3 of the 12 (25%) patients (N=4: 1 LN and 1 P, and one patient-matched LN and P) that was characterized by high relative levels of total ER. Interestingly, these same tumors also had amongst the highest relative levels of both total AR and activated (phosphorylated) GR compared to the rest of the 20 tumor samples. Downstream signaling analysis found significant (p< 0.05) activation/phosphorylation of AKT (S473 and/or T308) associated with the ER-AR-GR/steroid hormone activated/”high” TNBC signature compared to the 20 tumors that were steroid hormone “low”. Conclusions: Functional protein pathway activation mapping of a unique study set of synchronously obtained TNBC primary and LN metastasis revealed a steroid hormone receptor expression/activation signature of co-incidentally activated/expressed ER, GR and AR, that was also characterized by increased AKT activation (p< 0.05). This steroid hormone receptor “activated” signature was not observed to be significantly differentially expressed in LN compared to P tissues in cohort or matched pair analysis, suggesting that this signature may be present as an intrinsic event in the P, and not acquired in LN metastases. Recent data from stage I-III TNBC patients treated in the neoadjuvant setting with an AKT inhibitor1 revealed that responding TNBC patients had pre-treatment tumor biopsies that were enriched for both high AR expression and high phosphoAKT (S473 and/or T308) levels. If these observations that a subset of TNBCs are steroid hormone receptor “activated” despite being ER “negative” are confirmed in larger study sets, we hypothesize that this subgroup, identified by RPPA analysis of both P and LN metastases, could be potentially sensitive to AKT inhibition in combination with agents that target steroid hormone receptors. 1Shi Z, et al. Functional Mapping of AKT Signaling and Biomarkers of Response from the FAIRLANE Trial of Neoadjuvant Ipatasertib plus Paclitaxel for Triple-Negative Breast Cancer. Clin Cancer Res. 2022 Mar 1;28(5):993-1003 Citation Format: Julia Wulfkuhle, Page Blas, Heather Williams, Claudius Mueller, Rosa I. Gallagher, Mariaelena Pierobon, Joyce O’Shaughnessy, Emanuel F. Petricoin. PD5-08 Drug target activation mapping of matched triple negative primary and axillary lymph node metastases identifies and links the ER-AR-GR steroid hormone receptor axis with downstream AKT activation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD5-08.

TRIB2 safeguards naive T cell homeostasis during aging

Naive CD4+ Tcells are more resistant to age-related loss than naive CD8+ Tcells, suggesting mechanisms that preferentially protect naive CD4+ Tcells during aging. Here, we show that TRIB2 is more abundant in naive CD4+ than CD8+ Tcells and counteracts quiescence exit by suppressing AKT activation. TRIB2 deficiency increases AKT activity and accelerates proliferation and differentiation in response to interleukin-7 (IL-7) in humans and during lymphopenia in mice. TRIB2 transcription is controlled by the lineage-determining transcription factors ThPOK and RUNX3. Ablation of Zbtb7b (encoding ThPOK) and Cbfb (obligatory RUNT cofactor) attenuates the difference in lymphopenia-induced proliferation between naive CD4+ and CD8+ cells. In older adults, ThPOK and TRIB2 expression wanes in naive CD4+ Tcells, causing loss of naivety. These findings assign TRIB2 a key role in regulating Tcell homeostasis and provide a model to explain the lesser resilience of CD8+ Tcells to undergo changes with age.

Targeting hormone-resistant breast cancer cells with docetaxel: a look inside the resistance.

Aim: The study aims to analyze the effect of long-term incubation of ERα-positive MCF7 breast cancer cells with 4-hydroxytamoxifen (HT) on their sensitivity to tubulin polymerization inhibitor docetaxel. Methods: The analysis of cell viability was performed by the MTT method. The expression of signaling proteins was analyzed by immunoblotting and flow cytometry. ERα activity was evaluated by gene reporter assay. To establish hormone-resistant subline MCF7, breast cancer cells were treated with 4-hydroxytamoxifen for 12 months. Results: The developed MCF7/HT subline has lost sensitivity to 4-hydroxytamoxifen, and the resistance index was 2. Increased Akt activity (2.2-fold) and decreased ERα expression (1.5-fold) were revealed in MCF7/HT cells. The activity of the estrogen receptor α was reduced (1.5-fold) in MCF7/HT. Evaluation of class III β-tubulin expression (TUBB3), a marker associated with metastasis, revealed the following trends: higher expression of TUBB3 was detected in triple-negative breast cancer MDA-MB-231 cells compared to hormone-responsive MCF7 cells (P < 0.05). The lowest expression of TUBB3 was found in hormone-resistant MCF7/HT cells (MCF7/HT < MCF7 < MDA-MB-231, approximately 1:2:4). High TUBB3 expression strongly correlated with docetaxel resistance: IC50 value of docetaxel for MDA-MB-231 cells was greater than that for MCF7 cells, whereas resistant MCF7/HT cells were the most sensitive to the drug. The accumulation of cleaved PARP (a 1.6-fold increase) and Bcl-2 downregulation (1.8-fold) were more pronounced in docetaxel-treated resistant cells (P < 0.05). The expression of cyclin D1 decreased (2.8-fold) only in resistant cells after 4 nM docetaxel treatment, while this marker was unchanged in parental MCF7 breast cancer cells. Conclusion: Further development of taxane-based chemotherapy for hormone-resistant cancer looks highly promising, especially for cancers with low TUBB3 expression.

Cystathionine γ-lyase and hydrogen sulfide modulates glucose transporter Glut1 expression via NF-κB and PI3k/Akt in macrophages during inflammation.

Macrophages play a crucial role in inflammation, a defense mechanism of the innate immune system. Metabolic function powered by glucose transporter isoform 1 (Glut1) is necessary for macrophage activity during inflammation. The present study investigated the roles of cystathionine-γ-lyase (CSE) and its byproduct, hydrogen sulfide (H2S), in macrophage glucose metabolism to explore the mechanism by which H2S acts as an inflammatory regulator in lipopolysaccharide- (LPS) induced macrophages. Our results demonstrated that LPS-treated macrophages increased Glut1 expression. LPS-induced Glut1 expression is regulated via nuclear factor (NF)-κB activation and is associated with phosphatidylinositol-3-kinase PI3k activation. Small interfering (si) RNA-mediated silencing of CSE decreased the LPS-induced NF-κB activation and Glut1 expression, suggesting a role for H2S in metabolic function in macrophages during pro-inflammatory response. Confoundingly, treatment with GYY4137, an H2S-donor molecule, also displayed inhibitory effects upon LPS-induced NF-κB activation and Glut1 expression. Moreover, GYY4137 treatment increased Akt activation, suggesting a role in promoting resolution of inflammation. Our study provides evidence that the source of H2S, either endogenous (via CSE) or exogenous (via GYY4137), supports or inhibits the LPS-induced NF-κB activity and Glut1 expression, respectively. Therefore, H2S may influence metabolic programming in immune cells to alter glucose substrate availability that impacts the immune response.

Role of Nivolumab in the Modulation of PD-1 and PD-L1 Expression in Papillary and Clear Cell Renal Carcinoma (RCC).

The expression and cellular mechanisms of programmed cell death-1 protein (PD-1) and its ligands (PD-L1 and PD-L2) in renal cancer cells are not well known. Here, we aimed to investigate the response of renal carcinoma subtypes to the immune checkpoint inhibitor nivolumab and its impact on related signaling pathways. All cell lines analyzed (clear cell (cc)RCC (Caki-1, RCC31) and papillary (p)RCC (ACHN, RCC30)) expressed PD-1 and both ccRCC cell lines, and RCC30 expressed PD-L1. Nivolumab treatment at increasing doses led to increased PD-1 levels in analyzed cells and resulted in aggressive behavior of pRCC but diminished this behavior in ccRCC. The analysis of PD-1/PD-L1-associated signaling pathways demonstrated increased AKT activity in Caki-1 and RCC30 cells but decreased activity in ACHN and RCC31 cells, while ribosomal protein S6 remained largely unchanged. Androgen receptors are related to RCC and were predominantly increased in RCC30 cells, which were the only cells that formed nivolumab-dependent spheroids. Finally, all cell lines exhibited a complex response to nivolumab treatment. Since the pRCC cells responded with increased tumorigenicity and PD-1/PD-L1 levels while ccRCC tumorigenicity was diminished, further studies are needed to improve nivolumab-based therapy for renal carcinoma subtypes, especially the identification of response-involved molecular pathways.

Overexpression of KiSS1 Induces the Proliferation of Hepatocarcinoma and Increases Metastatic Potential by Increasing Migratory Ability and Angiogenic Capacity.

Liver cancer has a high prevalence, with majority of the cases presenting as hepatocellular carcinoma (HCC). The prognosis of metastatic HCC has hardly improved over the past decade, highlighting the necessity for liver cancer research. Studies have reported the ability of the KiSS1 gene to inhibit the growth or metastasis of liver cancer, but contradictory research results are also emerging. We, therefore, sought to investigate the effects of KiSS1 on growth and migration in human HCC cells. HepG2 human HCC cells were infected with lentivirus particles containing KiSS1. The overexpression of KiSS1 resulted in an increased proliferation rate of HCC cells. Quantitative polymerase chain reaction and immunoblotting revealed increased Akt activity, and downregulation of the G1/S phase cell cycle inhibitors. A significant increase in tumor spheroid formation with upregulation of β-catenin and CD133 was also observed. KiSS1 overexpression promoted the migratory, invasive ability, and metastatic capacity of the hepatocarcinoma cell line, and these effects were associated with changes in the expressions of epithelial mesenchymal transition (EMT)-related genes such as E-cadherin, N-cadherin, and slug. KiSS1 overexpression also resulted in dramatically increased tumor growth in the xenograft mouse model, and upregulation of proliferating cell nuclear antigen (PCNA) and Ki-67 in the HCC tumors. Furthermore, KiSS1 increased the angiogenic capacity by upregulation of the vascular endothelial growth factor A (VEGF-A) and CD31. Based on these observations, we infer that KiSS1 not only induces HCC proliferation, but also increases the metastatic potential by increasing the migratory ability and angiogenic capacity.

Combinatorial PI3K/AKT Pathway Inhibition As a Therapeutic Approach in Diffuse Large B-Cell Lymphoma

Background. Diffuse large B-cell lymphoma (DLBCL) is the most frequent type of non-Hodgkin lymphoma (NHL, ~ 25-30% of NHL cases). Currently, DLBCL treatment is based on non-specific chemotherapy combination (cyclophosphamide, doxorubicin, vincristine, and prednisone) with anti-CD20 monoclonal antibody. Although the DLBCL survival dramatically improved during last decades, approximately 40 % of DLBCL patients could not be cured with the currently used therapeutic approaches. Novel approaches are, therefore, needed to improve DLBCL survival and to decrease treatment related toxicity. Since many DLBCL tumors depend on phosphatidylinositol 3-kinase /protein kinase B (PI3K/AKT) signaling pathway, it represents one of the possible novel therapeutic targets. Despite promising results in various cancers, PI3K/AKT pathway inhibitors show only partial efficacy, low specificity, and resistance development in lymphomas. Since the PI3K/AKT activity is highly regulated, we hypothesize that the PI3K/AKT pathway needs to be inhibited at multiple levels simultaneously to achieve its full and lasting inhibition. Methods. To provide a proof of principle of multilevel vertical PI3K/AKT inhibition, we tested the effect of four inhibitors targeting the PI3K/AKT pathway at four different levels: PI3Kδ inhibitor idelalisib, PDPK1 inhibitor GSK2334470, AKT inhibitor ipatasertib, and mTOR inhibitor rapamycin on a spectrum of commonly used DLBCL model cell lines. Precise measurement of AKT activity in living cells was done using our recently published genetically encoded Förster Resonance Energy Transferred (FRET) based AKT activity reporter including novel methodology for flow cytometry-based FRET measurement (PMID: 34128311) allowing medium throughput and measurement in kinetics setting. Results. First, we measured how single inhibitors affect the AKT activity. The AKT activity IC50 concentrations were in 100 nM range except for rapamycin that had no AKT inhibitory effect. These concentrations were approximately 10 times lower in comparison to IC50 values calculated from net cell growth inhibition assessing the overall inhibitor toxicity. This suggests compensatory mechanisms and/or off target effects of PI3K/AKT inhibitors. Precise measurement of AKT activity allowed us to identify the minimal concentration of each inhibitor at which AKT activity was fully inhibited. Kinetics and short time course experiments showed that maximal AKT inhibition is achieved within hours of inhibitors treatment initiation. Importantly, pharmacological inhibition of one or two members of PI3K/AKT signaling cascade did not lead to lasting AKT activity inhibition. Cellular contra-regulatory mechanisms compensated for the decrease of AKT activity within 24 hours even in the presence of the inhibitors. Moreover, we detected a massive increase in AKT activity following release of the cells from media with PI3K/AKT pathway inhibitors. This further illustrates the powerful cellular contra-regulatory mechanisms and highlights the need for combinatorial approach. Combination of at least three inhibitors was necessary to achieve lasting AKT activity decrease (long time course experiment, Fig. 1A). Cross titration experiments further confirmed synergistic effect between tested PI3K/AKT pathway inhibitors on cell growth inhibition and allowed to determine the concentration ratios of their maximal synergism. Importantly, testing of single inhibitors and two, three, or all four inhibitors in fixed concentration combinations identified a substantial decrease in cell growth/toxicity IC50 concentrations of individual inhibitors (Fig. 1B). Conclusions. Concept of inhibiting more members of PI3K/AKT pathway was suggested before using combinations and/or dual inhibitors of AKT and mTOR, however, our data suggest that more than two inhibitors are needed for lasting PI3K/AKT inhibition. Moreover, we provided a proof of principle for further multilevel PI3K/AKT signaling inhibition testing, including in vivo cell line and/or patient derived xenograft models. Our data suggest that proposed combinations should allow significant decrease of inhibitors concentrations limiting off-target effect. This approach might overcome above mentioned issues of PI3K/AKT pathway inhibition and allow full therapeutical utilization of this pathway inhibition possibly applicable to other lymphoma types. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal