Lysosomal lipid switch sensitises to nutrient deprivation and mTOR targeting in pancreatic cancer.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with limited therapeutic options. However, metabolic adaptation to the harsh PDAC environment can expose liabilities useful for therapy. Targeting the key metabolic regulator mechanistic target of rapamycin complex 1 (mTORC1) and its downstream pathway shows efficacy only in subsets of patients but gene modifiers maximising response remain to be identified. DESIGN: Three independent cohorts of PDAC patients were studied to correlate PI3K-C2γ protein abundance with disease outcome. Mechanisms were then studied in mouse (KPC mice) and cellular models of PDAC, in presence or absence of PI3K-C2γ (WT or KO). PI3K-C2γ-dependent metabolic rewiring and its impact on mTORC1 regulation were assessed in conditions of limiting glutamine availability. Finally, effects of a combination therapy targeting mTORC1 and glutamine metabolism were studied in WT and KO PDAC cells and preclinical models. RESULTS: PI3K-C2γ expression was reduced in about 30% of PDAC cases and was associated with an aggressive phenotype. Similarly, loss of PI3K-C2γ in KPC mice enhanced tumour development and progression. The increased aggressiveness of tumours lacking PI3K-C2γ correlated with hyperactivation of mTORC1 pathway and glutamine metabolism rewiring to support lipid synthesis. PI3K-C2γ-KO tumours failed to adapt to metabolic stress induced by glutamine depletion, resulting in cell death. CONCLUSION: Loss of PI3K-C2γ prevents mTOR inactivation and triggers tumour vulnerability to RAD001 (mTOR inhibitor) and BPTES/CB-839 (glutaminase inhibitors). Therefore, these results might open the way to personalised treatments in PDAC with PI3K-C2γ loss.

Rab11 activity and PtdIns(3)P turnover removes recycling cargo from endosomes.

Directional transport of recycling cargo from early endosomes (EE) to the endocytic recycling compartment (ERC) relies on phosphatidylinositol 3-phosphate (PtdIns(3)P) hydrolysis and activation of the small GTPase Rab11. However, how these events are coordinated is yet unclear. By using a novel genetically-encoded FRET biosensor for Rab11, we report that generation of endosomal PtdIns(3)P by the clathrin-binding phosphoinositide 3-kinase class 2 alpha (PI3K-C2α) controls the activation of Rab11. Active Rab11, in turn, prompts the recruitment of the phosphatidylinositol 3-phosphatase myotubularin 1 (MTM1), eventually enabling the release of recycling cargo from the EE and its delivery toward the ERC. Our findings thus define that delivery of recycling cargo toward the ERC requires spatial and sequential coupling of Rab11 activity with PtdIns(3)P turnover.

Phosphoinositide 3-Kinase Gamma Inhibition Protects From Anthracycline Cardiotoxicity and Reduces Tumor Growth.

BACKGROUND: Anthracyclines, such as doxorubicin (DOX), are potent anticancer agents for the treatment of solid tumors and hematologic malignancies. However, their clinical use is hampered by cardiotoxicity. This study sought to investigate the role of phosphoinositide 3-kinase γ (PI3Kγ) in DOX-induced cardiotoxicity and the potential cardioprotective and anticancer effects of PI3Kγ inhibition. METHODS: Mice expressing a kinase-inactive PI3Kγ or receiving PI3Kγ-selective inhibitors were subjected to chronic DOX treatment. Cardiac function was analyzed by echocardiography, and DOX-mediated signaling was assessed in whole hearts or isolated cardiomyocytes. The dual cardioprotective and antitumor action of PI3Kγ inhibition was assessed in mouse mammary tumor models. RESULTS: PI3Kγ kinase-dead mice showed preserved cardiac function after chronic low-dose DOX treatment and were protected against DOX-induced cardiotoxicity. The beneficial effects of PI3Kγ inhibition were causally linked to enhanced autophagic disposal of DOX-damaged mitochondria. Consistently, either pharmacological or genetic blockade of autophagy in vivo abrogated the resistance of PI3Kγ kinase-dead mice to DOX cardiotoxicity. Mechanistically, PI3Kγ was triggered in DOX-treated hearts, downstream of Toll-like receptor 9, by the mitochondrial DNA released by injured organelles and contained in autolysosomes. This autolysosomal PI3Kγ/Akt/mTOR/Ulk1 signaling provided maladaptive feedback inhibition of autophagy. PI3Kγ blockade in models of mammary gland tumors prevented DOX-induced cardiac dysfunction and concomitantly synergized with the antitumor action of DOX by unleashing anticancer immunity. CONCLUSIONS: Blockade of PI3Kγ may provide a dual therapeutic advantage in cancer therapy by simultaneously preventing anthracyclines cardiotoxicity and reducing tumor growth.

Phosphoinositide 3-Kinase-C2α Regulates Polycystin-2 Ciliary Entry and Protects against Kidney Cyst Formation.

Signaling from the primary cilium regulates kidney tubule development and cyst formation. However, the mechanism controlling targeting of ciliary components necessary for cilium morphogenesis and signaling is largely unknown. Here, we studied the function of class II phosphoinositide 3-kinase-C2α (PI3K-C2α) in renal tubule-derived inner medullary collecting duct 3 cells and show that PI3K-C2α resides at the recycling endosome compartment in proximity to the primary cilium base. In this subcellular location, PI3K-C2α controlled the activation of Rab8, a key mediator of cargo protein targeting to the primary cilium. Consistently, partial reduction of PI3K-C2α was sufficient to impair elongation of the cilium and the ciliary transport of polycystin-2, as well as to alter proliferation signals linked to polycystin activity. In agreement, heterozygous deletion of PI3K-C2α in mice induced cilium elongation defects in kidney tubules and predisposed animals to cyst development, either in genetic models of polycystin-1/2 reduction or in response to ischemia/reperfusion-induced renal damage. These results indicate that PI3K-C2α is required for the transport of ciliary components such as polycystin-2, and partial loss of this enzyme is sufficient to exacerbate the pathogenesis of cystic kidney disease.

Exploiting pancreatic cancer metabolism: challenges and opportunities.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive form of pancreatic cancer, known for its challenging diagnosis and limited treatment options. The focus on metabolic reprogramming as a key factor in tumor initiation, progression, and therapy resistance has gained prominence. In this review we focus on the impact of metabolic changes on the interplay among stromal, immune, and tumor cells, as glutamine and branched-chain amino acids (BCAAs) emerge as pivotal players in modulating immune cell functions and tumor growth. We also discuss ongoing clinical trials that explore metabolic modulation for PDAC, targeting mitochondrial metabolism, asparagine and glutamine addiction, and autophagy inhibition. Overcoming challenges in understanding nutrient effects on immune-stromal-tumor interactions holds promise for innovative therapeutic strategies.

Novel Lymphocyte-Independent Antitumor Activity by PD-1 Blocking Antibody against PD-1+ Chemoresistant Lung Cancer Cells.

PURPOSE: Antibodies against the lymphocyte PD-1 (aPD-1) receptor are cornerstone agents for advanced non-small cell lung cancer (NSCLC), based on their ability to restore the exhausted antitumor immune response. Our study reports a novel, lymphocyte-independent, therapeutic activity of aPD-1 against NSCLC, blocking the tumor-intrinsic PD-1 receptors on chemoresistant cells. EXPERIMENTAL DESIGN: PD-1 in NSCLC cells was explored in vitro at baseline, including stem-like pneumospheres, and following treatment with cisplatin both at transcriptional and protein levels. PD-1 signaling and RNA sequencing were assessed. The lymphocyte-independent antitumor activity of aPD-1 was explored in vitro, by PD-1 blockade and stimulation with soluble ligand (PD-L1s), and in vivo within NSCLC xenograft models. RESULTS: We showed the existence of PD-1+ NSCLC cell subsets in cell lines and large in silico datasets (Cancer Cell Line Encyclopedia and The Cancer Genome Atlas). Cisplatin significantly increased PD-1 expression on chemo-surviving NSCLC cells (2.5-fold P = 0.0014), while the sequential treatment with anti-PD-1 Ab impaired their recovery after chemotherapy. PD-1 was found to be associated with tumor stemness features. PD-1 expression was enhanced in NSCLC stem-like pneumospheres (P < 0.0001), significantly promoted by stimulation with soluble PD-L1 (+27% ± 4, P < 0.0001) and inhibited by PD-1 blockade (-30% ± 3, P < 0.0001). The intravenous monotherapy with anti-PD-1 significantly inhibited tumor growth of NSCLC xenografts in immunodeficient mice, without the contribution of the immune system, and delayed the occurrence of chemoresistance when combined with cisplatin. CONCLUSIONS: We report first evidence of a novel lymphocyte-independent activity of anti-PD-1 antibodies in NSCLC, capable of inhibiting chemo-surviving NSCLC cells and exploitable to contrast disease relapses following chemotherapy. See related commentary by Augustin et al., p. 505.

Structural basis of phosphatidylinositol 3-kinase C2α function.

Phosphatidylinositol 3-kinase type 2α (PI3KC2α) is an essential member of the structurally unresolved class II PI3K family with crucial functions in lipid signaling, endocytosis, angiogenesis, viral replication, platelet formation and a role in mitosis. The molecular basis of these activities of PI3KC2α is poorly understood. Here, we report high-resolution crystal structures as well as a 4.4-Å cryogenic-electron microscopic (cryo-EM) structure of PI3KC2α in active and inactive conformations. We unravel a coincident mechanism of lipid-induced activation of PI3KC2α at membranes that involves large-scale repositioning of its Ras-binding and lipid-binding distal Phox-homology and C-C2 domains, and can serve as a model for the entire class II PI3K family. Moreover, we describe a PI3KC2α-specific helical bundle domain that underlies its scaffolding function at the mitotic spindle. Our results advance our understanding of PI3K biology and pave the way for the development of specific inhibitors of class II PI3K function with wide applications in biomedicine.

Phosphoinositide Conversion Inactivates R-RAS and Drives Metastases in Breast Cancer.

Breast cancer is the most prevalent cancer and a major cause of death in women worldwide. Although early diagnosis and therapeutic intervention significantly improve patient survival rate, metastasis still accounts for most deaths. Here it is reported that, in a cohort of more than 2000 patients with breast cancer, overexpression of PI3KC2α occurs in 52% of cases and correlates with high tumor grade as well as increased probability of distant metastatic events, irrespective of the subtype. Mechanistically, it is demonstrated that PI3KC2α synthetizes a pool of PI(3,4)P2 at focal adhesions that lowers their stability and directs breast cancer cell migration, invasion, and metastasis. PI(3,4)P2 locally produced by PI3KC2α at focal adhesions recruits the Ras GTPase activating protein 3 (RASA3), which inactivates R-RAS, leading to increased focal adhesion turnover, migration, and invasion both in vitro and in vivo. Proof-of-concept is eventually provided that inhibiting PI3KC2α or lowering RASA3 activity at focal adhesions significantly reduces the metastatic burden in PI3KC2α-overexpressing breast cancer, thereby suggesting a novel strategy for anti-breast cancer therapy.

PI(3,4)P2-mediated cytokinetic abscission prevents early senescence and cataract formation.

Cytokinetic membrane abscission is a spatially and temporally regulated process that requires ESCRT (endosomal sorting complexes required for transport)­dependent control of membrane remodeling at the midbody, a subcellular organelle that defines the cleavage site. Alteration of ESCRT function can lead to cataract, but the underlying mechanism and its relation to cytokinesis are unclear. We found a lens-specific cytokinetic process that required PI3K-C2α (phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2α), its lipid product PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate), and the PI(3,4)P2­binding ESCRT-II subunit VPS36 (vacuolar protein-sorting-associated protein 36). Loss of each of these components led to impaired cytokinesis, triggering premature senescence in the lens of fish, mice, and humans. Thus, an evolutionarily conserved pathway underlies the cell type­specific control of cytokinesis that helps to prevent early onset cataract by protecting from senescence.

PI(3,4)P2 Signaling in Cancer and Metabolism.

The phosphatidylinositide 3 kinases (PI3Ks) and their downstream mediators AKT and mammalian target of rapamycin (mTOR) are central regulators of glycolysis, cancer metabolism, and cancer cell proliferation. At the molecular level, PI3K signaling involves the generation of the second messenger lipids phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2]. There is increasing evidence that PI(3,4)P2 is not only the waste product for the removal of PI(3,4,5)P3 but can also act as a signaling molecule. The selective cellular functions for PI(3,4)P2 independent of PI(3,4,5)P3 have been recently described, including clathrin-mediated endocytosis and mTOR regulation. However, the specific spatiotemporal dynamics and signaling role of PI3K minor lipid messenger PI(3,4)P2 are not well-understood. This review aims at highlighting the biological functions of this lipid downstream of phosphoinositide kinases and phosphatases and its implication in cancer metabolism.

The PI3K/Akt/mTOR pathway in polycystic kidney disease: A complex interaction with polycystins and primary cilium.

Over-activation of the PI3K/Akt/mTOR network is a well-known pathogenic event that leads to hyper-proliferation. Pharmacological targeting of this pathway has been developed for the treatment of multiple diseases, including cancer. In polycystic kidney disease (PKD), the mTOR cascade promotes cyst growth by boosting proliferation, size and metabolism of kidney tubule epithelial cells. Therefore, mTOR inhibition has been tested in pre-clinical and clinical studies, but only the former showed positive results. This review reports recent discoveries describing the activity and molecular mechanisms of mTOR activation in tubule epithelial cells and cyst formation and discusses the evidence of an upstream regulation of mTOR by the PI3K/Akt axis. In particular, the complex interconnections of the PI3K/Akt/mTOR network with the principal signaling routes involved in the suppression of cyst formation are dissected. These interactions include the antagonism and the reciprocal negative regulation between mTOR complex 1 and the proteins whose deletion causes Autosomal Dominant PKD, the polycystins. In addition, the emerging role of phopshoinositides, membrane components modulated by PI3K, will be presented in the context of primary cilium signaling, cell polarization and protection from cyst formation. Overall, studies demonstrate that the activity of various members of the PI3K/Akt/mTOR network goes beyond the classical transduction of mitogenic signals and can impact several aspects of kidney tubule homeostasis and morphogenesis. These properties might be useful to guide the establishment of more effective treatment protocols to be tested in clinical trials.

A Novel Multiplex qRT-PCR Assay to Detect SARS-CoV-2 Infection: High Sensitivity and Increased Testing Capacity.

Rapid and sensitive screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential to limit the spread of the global pandemic we are facing. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is currently used for the clinical diagnosis of SARS-CoV-2 infection using nasopharyngeal swabs, tracheal aspirates, or bronchoalveolar lavage (BAL) samples. Despite the high sensitivity of the qRT-PCR method, false negative outcomes might occur, especially in patients with a low viral load. Here, we developed a multiplex qRT-PCR methodology for the simultaneous detection of SARS-CoV-2 genome (N gene) and of the human RNAse P gene as internal control. We found that multiplex qRT-PCR was effective in detecting SARS-Cov-2 infection in human specimens with 100% sensitivity. Notably, patients with few copies of SARS-CoV-2 RNA (<5 copies/reaction) were successfully detected by the novel multiplex qRT-PCR method. Finally, we assessed the efficacy of multiplex qRT-PCR on human nasopharyngeal swabs without RNA extraction. Collectively, our results provide evidence of a novel and reliable tool for SARS-CoV-2 RNA detection in human specimens, which allows the testing capacity to be expanded and the RNA extraction step to be bypassed.

Targeting PI3K signaling in cancer: Challenges and advances.

The key role of phosphoinositide 3-kinase (PI3K) pathway in different cellular processes and several disorders, together with the presence of targetable proteins, opened the way to promising studies for the development of small molecule inhibitors. Despite the high expectation, the shift of PI3K inhibitors to the clinic met several limitations due to the emergence of dose-limiting, on-target adverse effects. In this review, we will summarize the main issues and recent advances in PI3K inhibitors clinical trials. The effort to develop isoform-specific inhibitors, together with novel therapeutic strategies aimed at reducing the toxicity and adverse effects, opened a new promising era for PI3K inhibitors. In addition, we will focus on the recent emergence of class II and III PI3K inhibitors, which helped to define their class I non-redundant role.

Class II PI3K Functions in Cell Biology and Disease.

The phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that phosphorylate inositol phospholipids, thereby controlling membrane lipid composition and regulating a wide range of intracellular processes, including vesicular trafficking and signal transduction. Despite the vast knowledge on class I PI3Ks, recent studies are only now revealing the importance of class II PI3Ks in cell proliferation, survival, and migration. Increasing evidence suggests that the three class II PI3Ks isoforms (PI3K-C2α, PI3K-C2ß, and PI3K-C2γ) have distinct and non-overlapping cellular roles. Here, we focus on the cellular functions of class II PI3Ks in different cell systems and underline the emerging importance of these enzymes in various physiological and pathological contexts.

The turtle and the rabbit story in a modern (PI3)key.

Mitosis is a complex process controlling proper distribution of chromosomes and preventing genomic instability, a typical hallmark of cancer. We recently reported that that a class II isoform of Phosphoinositide 3-kinase (PI3K), PI3K-C2α, is involved in the organization of the mitotic spindle. Our study demonstrate that, differently to all other PI3K isoforms, PI3K-C2α shows an unexpected tumor suppressor function independent of its catalytic activity.

The role of metabolic adaptation to nutrient stress in pancreatic cancer.

Pancreatic cancer is the fourth most common cause of cancer-related mortality, with a dismal prognosis that has changed little over the past few decades. Despite extensive efforts in understanding the oncogenetics of this pathology, pancreatic cancer remained largely elusive. One of the main characteristics of pancreatic cancer is the reduced level of oxygen and nutrient perfusion, caused by the new matrix formation, through the activation of stromal cells (desmoplasia). This stromal reaction leads to metabolic adaptations in surviving tumor cells in order to cope with these challenging conditions. The oncogenic signaling driven by KRAS mutation is necessary to fuel pancreatic tumors by activating key metabolic processes, including enhanced glycolysis and glutamine consumption. Here we review our current understanding of the pancreatic cancer metabolism as well as discuss recent work pointing to the importance of various metabolic strategies as well as autophagy and macropinocytosis as critical nutrient supply pathways. The elucidation of these metabolic networks may highlight new opportunities to further develop novel therapeutic strategies.

Signaling Pathways Regulating Redox Balance in Cancer Metabolism.

The interplay between rewiring tumor metabolism and oncogenic driver mutations is only beginning to be appreciated. Metabolic deregulation has been described for decades as a bystander effect of genomic aberrations. However, for the biology of malignant cells, metabolic reprogramming is essential to tackle a harsh environment, including nutrient deprivation, reactive oxygen species production, and oxygen withdrawal. Besides the well-investigated glycolytic metabolism, it is emerging that several other metabolic fluxes are relevant for tumorigenesis in supporting redox balance, most notably pentose phosphate pathway, folate, and mitochondrial metabolism. The relationship between metabolic rewiring and mutant genes is still unclear and, therefore, we will discuss how metabolic needs and oncogene mutations influence each other to satisfy cancer cells' demands. Mutations in oncogenes, i.e., PI3K/AKT/mTOR, RAS pathway, and MYC, and tumor suppressors, i.e., p53 and liver kinase B1, result in metabolic flexibility and may influence response to therapy. Since metabolic rewiring is shaped by oncogenic driver mutations, understanding how specific alterations in signaling pathways affect different metabolic fluxes will be instrumental for the development of novel targeted therapies. In the era of personalized medicine, the combination of driver mutations, metabolite levels, and tissue of origins will pave the way to innovative therapeutic interventions.

PI3K Signaling in Tissue Hyper-Proliferation: From Overgrowth Syndromes to Kidney Cysts.

The members of the PhosphoInositide-3 Kinase (PI3K) protein family are well-known regulators of proliferative signals. By the generation of lipid second messengers, they mediate the activation of AKT/PKB (AKT) and mammalian Target Of Rapamycin (mTOR) pathways. Although mutations in the PI3K/AKT/mTOR pathway are highly characterized in cancer, recent evidence indicates that alterations in the proliferative signals are major drivers of other diseases such as overgrowth disorders and polycystic kidney disease. In this review, we briefly summarize the role of the PI3K/AKT/mTOR pathway in cell proliferation by comparing the effect of alterations in PI3K enzymes in different tissues. In particular, we discuss the most recent findings on how the same pathway may lead to different biological effects, due to the convergence and cooperation of different signaling cascades.

Mitotic Spindle Assembly and Genomic Stability in Breast Cancer Require PI3K-C2α Scaffolding Function.

Proper organization of the mitotic spindle is key to genetic stability, but molecular components of inter-microtubule bridges that crosslink kinetochore fibers (K-fibers) are still largely unknown. Here we identify a kinase-independent function of class II phosphoinositide 3-OH kinase α (PI3K-C2α) acting as limiting scaffold protein organizing clathrin and TACC3 complex crosslinking K-fibers. Downregulation of PI3K-C2α causes spindle alterations, delayed anaphase onset, and aneuploidy, indicating that PI3K-C2α expression is required for genomic stability. Reduced abundance of PI3K-C2α in breast cancer models initially impairs tumor growth but later leads to the convergent evolution of fast-growing clones with mitotic checkpoint defects. As a consequence of altered spindle, loss of PI3K-C2α increases sensitivity to taxane-based therapy in pre-clinical models and in neoadjuvant settings.

PI3K/AKT signaling pathway and cancer: an updated review.

Despite development of novel agents targeting oncogenic pathways, matching targeted therapies to the genetic status of individual tumors is proving to be a daunting task for clinicians. To improve the clinical efficacy and to reduce the toxic side effects of treatments, a deep characterization of genetic alterations in different tumors is required. The mutational profile often evidences a gain of function or hyperactivity of phosphoinositide 3-kinases (PI3Ks) in tumors. These enzymes are activated downstream tyrosine kinase receptors (RTKs) and/or G proteins coupled receptors (GPCRs) and, via AKT, are able to induce mammalian target of rapamycin (mTOR) stimulation. Here, we elucidate the impact of class I (p110α, ß, γ, and δ) catalytic subunit mutations on AKT-mediated cellular processes that control crucial mechanisms in tumor development. Moreover, the interrelation of PI3K signaling with mTOR, ERK, and RAS pathways will be discussed, exploiting the potential benefits of PI3K signaling inhibitors in clinical use.