Delayed Akt suppression in the lipopolysaccharide-induced acute lung injury promotes resolution that is associated with enhanced effector regulatory T cells.

The adaptive immune response could play a major role in the resolution of lung injury. Although regulatory T cells (Tregs) have been implicated in promoting the resolution of lung injury, therapeutic strategies to enhance Treg quantity and activity at the site of injury need further exploration. In the current study, Akt inhibition using triciribine (TCBN), given 48 h after lipopolysaccharide (LPS) administration, increased Tregs-promoted resolution of acute lung injury (ALI). TCBN treatment enhanced the resolution of LPS-induced ALI on day 7 by reducing pulmonary edema and neutrophil activity associated with an increased number of CD4+/FoxP3+/CD103+ and CTLA4+ effector Tregs, specifically in the injured lungs and not in the spleen. Treatment of EL-4 T-lymphocytes with two Akt inhibitors (TCBN and MK-2206) for 72 h resulted in increased FoxP3 expression in vitro. On the other end, Treg-specific PTEN knockout (PTENTreg KO) mice that have a higher Akt activity in its Tregs exhibited a significant impairment in ALI resolution, increased edema, and neutrophil activity associated with a reduced number of CD4+/FoxP3+/CD103+ and CTLA4+ effector Tregs as compared with the control group. In conclusion, our study identifies a potential target for the treatment of late-stage ALI by promoting resolution through effector Treg-mediated suppression of inflammation.

Patients with acute respiratory distress syndrome exhibit increased stromelysin1 activity in the blood samples.

Enzyme activity analyses in the blood are expected to be reliable, non-invasive diagnostic as well as prognostic markers to reflect disease progression in acute lung injury (ALI). The objective of the current study was to evaluate the enzymatic activity of stromelysin1 (matrix metalloprotease-3) in the plasma/serum samples collected from ALI patients compared to the samples collected from healthy controls. Gene expression omnibus (GEO) database analysis indicated a correlation between increased stromelysin1 gene expression and the incidence of ALI in various animal models. Our analysis of patient plasma/serum samples from healthy controls and ALI patients revealed a significant, 3-fold increase in stromelysin1 activity in ALI plasma/serum compared to healthy subjects with no difference in stromelysin1 activity between the serum and plasma samples. Interestingly, no significant difference in stromelysin1 activity between non-smoking and smoking subjects was observed. These findings provide fundamental information on the potential reliability of stromelysin1 activity analysis, combined with other biomarkers in development, in blood samples for the early detection of ALI.

Endothelial stromelysin1 regulation by the forkhead box-O transcription factors is crucial in the exudative phase of acute lung injury.

Enhanced vascular permeability is associated with inflammation and edema in alveoli during the exudative phase of acute respiratory distress syndrome (ARDS). Mechanisms leading to the endothelial contribution on the early exudative stage of ARDS are not precise. We hypothesized that modulation of endothelial stromelysin1 expression and activity by Akt1-forkhead box-O transcription factors 1/3a (FoxO1/3a) pathway could play a significant role in regulating pulmonary edema during the initial stages of acute lung injury (ALI). We utilized lipopolysaccharide (LPS)-induced mouse ALI model in vivo and endothelial barrier resistance measurements in vitro to determine the specific role of the endothelial Akt1-FoxO1/3a-stromelysin1 pathway in ALI. LPS treatment of human pulmonary endothelial cells resulted in increased stromelysin1 and reduced tight junction claudin5 involving FoxO1/3a, associated with decreased trans-endothelial barrier resistance as determined by electric cell-substrate impedance sensing technology. In vivo, LPS-induced lung edema was significantly higher in endothelial Akt1 knockdown (EC-Akt1-/-) compared to wild-type mice, which was reversed upon treatment with FoxO inhibitor (AS1842856), stromelysin1 inhibitor (UK356618) or with shRNA-mediated FoxO1/3a depletion in the mouse lungs. Overall, our study provides the hope that targeting FoxO and styromelysin1 could be beneficial in the treatment of ALI.

Endothelial Akt1 loss promotes prostate cancer metastasis via ß-catenin-regulated tight-junction protein turnover.

BACKGROUND: Cancer research, in general, is focused on targeting tumour cells to limit tumour growth. These studies, however, do not account for the specific effects of chemotherapy on tumour endothelium, in turn, affecting metastasis. METHODS: We determined how endothelial deletion of Akt1 promotes prostate cancer cell invasion in vitro and metastasis to the lungs in vivo in endothelial-specific Akt1 knockdown mice. RESULTS: Here we show that metastatic human PC3 and DU145 prostate cancer cells invade through Akt1-deficient human lung endothelial cell (HLEC) monolayer with higher efficiency compared to control HLEC. Although the endothelial Akt1 loss in mice had no significant effect on RM1 tumour xenograft growth in vivo, it promoted metastasis to the lungs compared to the wild-type mice. Mechanistically, Akt1-deficient endothelial cells exhibited increased phosphorylation and nuclear translocation of phosphorylated ß-catenin, and reduced expression of tight-junction proteins claudin-5, ZO-1 and ZO-2. Pharmacological inhibition of ß-catenin nuclear translocation using compounds ICG001 and IWR-1 restored HLEC tight-junction integrity and inhibited prostate cancer cell transendothelial migration in vitro and lung metastasis in vivo. CONCLUSIONS: Here we show for the first time that endothelial-specific loss of Akt1 promotes cancer metastasis in vivo involving ß-catenin pathway.

Inducible overexpression of endothelial proNGF as a mouse model to study microvascular dysfunction.

Impaired maturation of nerve growth factor precursor (proNGF) and its accumulation has been reported in several neurodegenerative diseases, myocardial infarction and diabetes. To elucidate the direct impact of proNGF accumulation identified the need to create a transgenic model that can express fully mutated cleavage-resistant proNGF. Using Cre-Lox technology, we developed an inducible endothelial-specific proNGF transgenic mouse (proNGFLoxp) that overexpresses GFP-conjugated cleavage-resistant proNGF123 when crossed with VE-cadherin-CreERT2 (Cre). Expression of proNGF, inflammatory mediators, NGF and VEGF was evaluated by PCR, Western blot and immunohistochemistry. EC-proNGF overexpression was confirmed using colocalization of anti-proNGF within retinal vasculature. EC-proNGF did not cause retinal neurotoxicity or marked glial activation at 4-weeks. Microvascular preparation from Cre-proNGF mice showed significant imbalance of proNGF/NGF ratio, enhanced expression of TNF-α and p75NTR, and tendency to impair TrkA phosphorylation compared to controls. EC-proNGF overexpression triggered mRNA expression of p75NTR and inflammatory mediators in both retina and renal cortex compared to controls. EC-proNGF expression induced vascular permeability including breakdown of BRB and albuminuria in the kidney without affecting VEGF level at 4-weeks. Histopathological changes were assessed after 8-weeks and the results showed that EC-proNGF triggered formation of occluded (acellular) capillaries, hall mark of retinal ischemia. EC-proNGF resulted in glomerular enlargement and kidney fibrosis, hall mark of renal dysfunction. We have successfully created an inducible mouse model that can dissect the contribution of autocrine direct action of cleavage-resistant proNGF on systemic microvascular abnormalities in both retina and kidney, major targets for microvascular complication.

Modulation of long-term endothelial-barrier integrity is conditional to the cross-talk between Akt and Src signaling.

Although numerous studies have implicated Akt and Src kinases in vascular endothelial growth factor (VEGF) and Angiopoietin-1 (Ang-1)-induced endothelial-barrier regulation, a link between these two pathways has never been demonstrated. We determined the long-term effects of Akt inhibition on Src activity and vice versa, and in turn, on the human microvascular endothelial cell (HMEC) barrier integrity at the basal level, and in response to growth factors. Our data showed that Akt1 gene knockdown increases gap formation in HMEC monolayer at the basal level. Pharmacological inhibition of Akt, but not Src resulted in exacerbated VEGF-induced vascular leakage and impaired Ang-1-induced HMEC-barrier protection in vitro at 24 hr. Whereas inhibition of Akt had no effect on VEGF-induced HMEC gap formation in the short term, inhibition of Src blunted this process. In contrast, inhibition of Akt disrupted the VEGF and Ang-1 stabilized barrier integrity in the long-term while inhibition of Src did not. Interestingly, both long-term Akt inhibition and Akt1 gene knockdown in HMECs resulted in increased Tyr416 phosphorylation of Src. Treatment of HMECs with transforming growth factor-ß1 (TGFß1) that inhibited Akt Ser473 phosphorylation in the long-term, activated Src through increased Tyr416 phosphorylation and decreased HMEC-barrier resistance. The effect of TGFß1 on endothelial-barrier breakdown was blunted in Akt1 deficient HMEC monolayers, where endothelial-barrier resistance was already impaired compared to the control. To our knowledge, this is the first report demonstrating a direct cross-talk between Akt and Src in endothelial-barrier regulation.

Akt1 promotes stimuli-induced endothelial-barrier protection through FoxO-mediated tight-junction protein turnover.

Vascular permeability regulated by the vascular endothelial growth factor (VEGF) through endothelial-barrier junctions is essential for inflammation. Mechanisms regulating vascular permeability remain elusive. Although 'Akt' and 'Src' have been implicated in the endothelial-barrier regulation, it is puzzling how both agents that protect and disrupt the endothelial-barrier activate these kinases to reciprocally regulate vascular permeability. To delineate the role of Akt1 in endothelial-barrier regulation, we created endothelial-specific, tamoxifen-inducible Akt1 knockout mice and stable ShRNA-mediated Akt1 knockdown in human microvascular endothelial cells. Akt1 loss leads to decreased basal and angiopoietin1-induced endothelial-barrier resistance, and enhanced VEGF-induced endothelial-barrier breakdown. Endothelial Akt1 deficiency resulted in enhanced VEGF-induced vascular leakage in mice ears, which was rescued upon re-expression with Adeno-myrAkt1. Furthermore, co-treatment with angiopoietin1 reversed VEGF-induced vascular leakage in an Akt1-dependent manner. Mechanistically, our study revealed that while VEGF-induced short-term vascular permeability is independent of Akt1, its recovery is reliant on Akt1 and FoxO-mediated claudin expression. Pharmacological inhibition of FoxO transcription factors rescued the defective endothelial barrier due to Akt1 deficiency. Here we provide novel insights on the endothelial-barrier protective role of VEGF in the long term and the importance of Akt1-FoxO signaling on tight-junction stabilization and prevention of vascular leakage through claudin expression.

Renin-angiotensin system as a potential therapeutic target in stroke and retinopathy: experimental and clinical evidence.

As our knowledge expands, it is now clear that the renin-angiotensin (Ang) system (RAS) mediates functions other than regulating blood pressure (BP). The RAS plays a central role in the pathophysiology of different neurovascular unit disorders including stroke and retinopathy. Moreover, the beneficial actions of RAS modulation in brain and retina have been documented in experimental research, but not yet exploited clinically. The RAS is a complex system with distinct yet interconnected components. Understanding the different RAS components and their functions under brain and retinal pathological conditions is crucial to reap their benefits. The aim of the present review is to provide an experimental and clinical update on the role of RAS in the pathophysiology and treatment of stroke and retinopathy. Combining the evidence from both these disorders allows a unique opportunity to move both fields forward.

TNFα induces inflammatory stress response in microvascular endothelial cells via Akt- and P38 MAP kinase-mediated thrombospondin-1 expression.

Tumor necrosis factor-α (TNFα) and thrombospondin-1 (TSP-1) are well-known mediators of inflammation. However, a causal relationship between TNFα stimuli and TSP-1 expression in endothelial cell stress, and the underlying mechanisms has not yet been investigated. In our study, human microvascular endothelial cells (hMEC) were treated with TNFα and analyzed for endothelial dysfunction, TSP-1 expression, and associated mechanisms. TNFα treatment induced a dose-dependent increase in TSP-1 expression in hMEC associated with increased endothelial permeability, apoptosis, and reduced proliferation. Whereas TNFα activated Akt, ERK, and P38 mitogen-activated protein kinase (P38 MAPK) simultaneously in hMEC, inhibitors of Akt and P38 MAPK, but not ERK blunted TNFα-induced TSP-1 expression. Silencing of NFκB gene had no significant effect on TNFα-induced TSP-1 expression. Our study demonstrates the novel role of TNFα in inducing inflammatory stress response in hMEC through Akt- and P38 MAPK-mediated expression of TSP-1, independent of NFκB signaling.

UDP-6-glucose dehydrogenase in hormonally responsive breast cancers.

Survival for metastatic breast cancer is low and thus, continued efforts to treat and prevent metastatic progression are critical. Estrogen is shown to promote aggressive phenotypes in multiple cancer models irrespective of estrogen receptor (ER) status. Similarly, UDP-Glucose 6-dehydrogenase (UGDH) a ubiquitously expressed enzyme involved in extracellular matrix precursors, as well as hormone processing increases migratory and invasive properties in cancer models. While the role of UGDH in cellular migration is defined, how it intersects with and impacts hormone signaling pathways associated with tumor progression in metastatic breast cancer has not been explored. Here we demonstrate that UGDH knockdown blunts estrogen-induced tumorigenic phenotypes (migration and colony formation) in ER+ and ER- breast cancer in vitro. Knockdown of UGDH also inhibits extravasation of ER- breast cancer ex vivo, primary tumor growth and animal survival in vivo in both ER+ and ER- breast cancer. We also use single cell RNA-sequencing to demonstrate that our findings translate to a human breast cancer clinical specimen. Our findings support the role of estrogen and UGDH in breast cancer progression provide a foundation for future studies to evaluate the role of UGDH in therapeutic resistance to improve outcomes and survival for breast cancer patients.

Eosinophilia in cancer and its regulation by sex hormones.

Gender differences in the functionality of the immune system have been attributed, in part, to direct and indirect effects of sex steroids, especially estrogens, on immune cell repertoire and activity. Notable are studies that have defined roles for estrogens in the regulation of the biology of dendritic cells (DCs), macrophages, T cells and natural killer (NK) cells. Although estrogens can modulate eosinophil function, the mechanisms by which this occurs and how it contributes to the pathobiology of different diseases remains underexplored. Furthermore, although the importance of eosinophils in infection is well established, it remains unclear as to how these innate immune cells, which are present in different tumors, impact the biology of cancer cells and/or response to therapeutics. The observation that eosinophilia influences the efficacy of immune checkpoint blockers (ICBs) is significant considering the role of estrogens as regulators of eosinophil function and recent studies suggesting that response to ICBs is impacted by gender. Thus, in this review, we consider what is known about the roles of estrogen(s) in regulating tissue eosinophilia/eosinophil function and how this influences the pathobiology of breast cancer (in particular). This information provides the context for a discussion of how estrogens/the estrogen receptor (ER) signaling axis can be targeted in eosinophils and how this would be expected to influence the activity of standard-of-care interventions and contemporary immunotherapy regimens in cancer(s).

Increased CaMKK2 Expression Is an Adaptive Response That Maintains the Fitness of Tumor-Infiltrating Natural Killer Cells.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a key regulator of energy homeostasis in several cell types. Expression of this enzyme in tumor cells promotes proliferation and migration, and expression in tumor-associated immune cells facilitates M2 macrophage polarization and the development of myeloid-derived suppressor cells. Thus, there has been interest in developing CaMKK2 inhibitors as potential anticancer therapeutics. One impediment to clinical development of these agents is that the roles of CaMKK2 in other cellular compartments within the tumor immune microenvironment remain to be established. We report herein that CaMKK2 is expressed at low basal levels in natural killer (NK) cells but is upregulated in tumor-infiltrating NK cells where it suppresses apoptosis and promotes proliferation. NK cell-intrinsic deletion of CaMKK2 increased metastatic progression in several murine models, establishing a critical role for this enzyme in NK cell-mediated antitumor immunity. Ablation of the CaMKK2 protein, but not inhibition of its kinase activity, resulted in decreased NK-cell survival. These results indicate an important scaffolding function for CaMKK2 in NK cells and suggest that competitive CaMKK2 inhibitors and ligand-directed degraders (LDD) are likely to have distinct therapeutic utilities. Finally, we determined that intracellular lactic acid is a key driver of CaMKK2 expression, suggesting that upregulated expression of this enzyme is an adaptive mechanism by which tumor-infiltrating NK cells mitigate the deleterious effects of a lactic acid-rich tumor microenvironment. The findings of this study should inform strategies to manipulate the CaMKK2-signaling axis as a therapeutic approach in cancer.

Ca 2+ /Calmodulin Dependent Protein Kinase Kinase-2 (CaMKK2) promotes Protein Kinase G (PKG)-dependent actin cytoskeletal assembly to increase tumor metastasis.

Triple-negative breast cancers (TNBCs) tend to become highly invasive early during cancer development. Despite some successes in the initial treatment of patients diagnosed with early-stage localized TNBC, the rate of metastatic recurrence remains high with poor long-term survival outcomes. Here we show that elevated expression of the serine/threonine-kinase, Calcium/Calmodulin (CaM)-dependent protein kinase kinase-2 (CaMKK2), is highly correlated with tumor invasiveness. We determined that genetic disruption of CaMKK2 expression, or inhibition of its activity, disrupted spontaneous metastatic outgrowth from primary tumors in murine xenograft models of TNBC. High-grade serous ovarian cancer (HGSOC), a high-risk, poor-prognosis ovarian cancer subtype, shares many genetic features with TNBC, and importantly, CaMKK2 inhibition effectively blocked metastatic progression in a validated xenograft model of this disease. Probing the mechanistic links between CaMKK2 and metastasis we defined the elements of a new signaling pathway that impacts actin cytoskeletal dynamics in a manner which increases cell migration/invasion and metastasis. Notably, CaMKK2 increases the expression of the phosphodiesterase PDE1A which decreases the cGMP-dependent activity of protein kinase G1 (PKG1). This inhibition of PKG1 results in decreased phosphorylation of Vasodilator-Stimulated Phosphoprotein (VASP), which in its hypophosphorylated state binds to and regulates F-actin assembly to facilitate contraction/cell movement. Together, these data establish a targetable CaMKK2-PDE1A-PKG1-VASP signaling pathway that controls cancer cell motility and metastasis. Further, it credentials CaMKK2 as a therapeutic target that can be exploited in the discovery of agents for use in the neoadjuvant/adjuvant setting to restrict tumor invasiveness in patients diagnosed with early-stage TNBC or localized HGSOC.

Targeting CaMKK2 Inhibits Actin Cytoskeletal Assembly to Suppress Cancer Metastasis.

Triple-negative breast cancers (TNBC) tend to become invasive and metastatic at early stages in their development. Despite some treatment successes in early-stage localized TNBC, the rate of distant recurrence remains high, and long-term survival outcomes remain poor. In a search for new therapeutic targets for this disease, we observed that elevated expression of the serine/threonine kinase calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) is highly correlated with tumor invasiveness. In validation studies, genetic disruption of CaMKK2 expression or inhibition of its activity with small molecule inhibitors disrupted spontaneous metastatic outgrowth from primary tumors in murine xenograft models of TNBC. High-grade serous ovarian cancer (HGSOC), a high-risk, poor prognosis ovarian cancer subtype, shares many features with TNBC, and CaMKK2 inhibition effectively blocked metastatic progression in a validated xenograft model of this disease. Mechanistically, CaMKK2 increased the expression of the phosphodiesterase PDE1A, which hydrolyzed cyclic guanosine monophosphate (cGMP) to decrease the cGMP-dependent activity of protein kinase G1 (PKG1). Inhibition of PKG1 resulted in decreased phosphorylation of vasodilator-stimulated phosphoprotein (VASP), which in its hypophosphorylated state binds to and regulates F-actin assembly to facilitate cell movement. Together, these findings establish a targetable CaMKK2-PDE1A-PKG1-VASP signaling pathway that controls cancer cell motility and metastasis by impacting the actin cytoskeleton. Furthermore, it identifies CaMKK2 as a potential therapeutic target that can be exploited to restrict tumor invasiveness in patients diagnosed with early-stage TNBC or localized HGSOC. SIGNIFICANCE: CaMKK2 regulates actin cytoskeletal dynamics to promote tumor invasiveness and can be inhibited to suppress metastasis of breast and ovarian cancer, indicating CaMKK2 inhibition as a therapeutic strategy to arrest disease progression.

Regulation of Let-7a-5p and miR-199a-5p Expression by Akt1 Modulates Prostate Cancer Epithelial-to-Mesenchymal Transition via the Transforming Growth Factor-ß Pathway.

Akt1 suppression in advanced cancers has been indicated to promote metastasis. Our understanding of how Akt1 orchestrates this is incomplete. Using the NanoString®-based miRNA and mRNA profiling of PC3 and DU145 cells, and subsequent data analysis using the DIANA-mirPath, dbEMT, nCounter, and Ingenuity® databases, we identified the miRNAs and associated genes responsible for Akt1-mediated prostate cancer (PCa) epithelial-to-mesenchymal transition (EMT). Akt1 loss in PC3 and DU145 cells primarily induced changes in the miRNAs and mRNAs regulating EMT genes. These include increased miR-199a-5p and decreased let-7a-5p expression associated with increased TGFß-R1 expression. Treatment with locked nucleic acid (LNA) miR-199a-5p inhibitor and/or let-7a-5p mimic induced expression changes in EMT genes correlating to their anticipated effects on PC3 and DU145 cell motility, invasion, and TGFß-R1 expression. A correlation between increased miR-199a-5p and TGFß-R1 expression with reduced let-7a-5p was also observed in high Gleason score PCa patients in the cBioportal database analysis. Collectively, our studies show the effect of Akt1 suppression in advanced PCa on EMT modulating miRNA and mRNA expression changes and highlight the potential benefits of miR-199a-5p and let-7a-5p in therapy and/or early screening of mPCa.

A New Chemotype of Chemically Tractable Nonsteroidal Estrogens Based on a Thieno[2,3-d]pyrimidine Core.

Despite continued interest in the development of nonsteroidal estrogens and antiestrogens, there are only a few chemotypes of estrogen receptor ligands. Using targeted screening in a ligand sensing assay, we identified a phenolic thieno[2,3-d]pyrimidine with affinity for estrogen receptor α. An efficient three-step synthesis of the heterocyclic core and structure-guided optimization of the substituents resulted in a series of potent nonsteroidal estrogens. The chemical tractability of the thieno[2,3-d]pyrimidine chemotype will support the design of new estrogen receptor ligands as therapeutic hormones and antihormones.

ALK-1 to ALK-5 ratio dictated by the Akt1-ß-catenin pathway regulates TGFß-induced endothelial-to-mesenchymal transition.

Endothelial-to-mesenchymal transition (EndMT) indispensable in embryogenesis also occurs in several human pathologies. Although transforming growth factor-ß (TGFß) has been demonstrated to induce EndMT, the type-I receptors (ALK-1 and ALK-5) responsible for TGFß-induced EndMT is unclear. In the current study, we investigated the role of the Akt1 pathway in ALK1 and ALK5 expression regulation in response to TGFß1 and TGFß2 in human microvascular endothelial cells (HMECs). Whereas treatment with TGFß1 and TGFß2 or Akt1 gene silencing promoted EndMT accompanied by increased ALK5 expression and reduced ALK1 expression accompanied by increased expression of N-cadherin and reduced expression of eNOS in HMECs, treatment with ALK-5 inhibitor (SB431542) blunted these effects. Importantly, the inhibitor of ß-catenin (ICG-001) suppressed TGFß1- and TGFß2-induced ALK5 expression in both normal and Akt1 deficient HMECs indicating the integral role of Akt1-ß-catenin pathway in the regulation of ALK5 expression promoting EndMT.

Inhibition of estrogen signaling in myeloid cells increases tumor immunity in melanoma.

Immune checkpoint blockade (ICB) therapies have significantly prolonged patient survival across multiple tumor types, particularly in melanoma. Interestingly, sex-specific differences in response to ICB have been observed, with males receiving a greater benefit from ICB than females, although the mechanism or mechanisms underlying this difference are unknown. Mining published transcriptomic data sets, we determined that the response to ICBs is influenced by the functionality of intratumoral macrophages. This puts into context our observation that estrogens (E2) working through the estrogen receptor α (ERα) stimulated melanoma growth in murine models by skewing macrophage polarization toward an immune-suppressive state that promoted CD8+ T cell dysfunction and exhaustion and ICB resistance. This activity was not evident in mice harboring macrophage-specific depletion of ERα, confirming a direct role for estrogen signaling within myeloid cells in establishing an immunosuppressed state. Inhibition of ERα using fulvestrant, a selective estrogen receptor downregulator (SERD), decreased tumor growth, stimulated adaptive immunity, and increased the antitumor efficacy of ICBs. Further, a gene signature that determines ER activity in macrophages predicted survival in patients with melanoma treated with ICB. These results highlight the importance of E2/ER signaling as a regulator of intratumoral macrophage polarization, an activity that can be therapeutically targeted to reverse immune suppression and increase ICB efficacy.

PAK1 inhibitor IPA-3 mitigates metastatic prostate cancer-induced bone remodeling.

Metastatic prostate cancer (PCa) has high mortality and a poor 5-year survival rate primarily due to the lack of effective treatments. Bone is the primary site of PCa metastasis in humans and the development of reliable therapeutic options for bone metastatic PCa will make a huge impact in reducing the mortality among these patients. Although P21 activated kinases (PAKs) have been studied in the past for their role in cancer, the efficacy of targeting PAKs to treat lung and bone metastatic PCa has not been tested yet. In the current study, we report that targeting PAK1 using IPA-3, an allosteric inhibitor of PAK1 kinase activity, significantly inhibits the murine metastatic PCa (RM1) cell proliferation and motility in vitro, and metastasis to the lungs in vivo. More importantly, we demonstrate for the first time that treatment with IPA-3 can blunt metastatic PCa-induced bone remodeling in vivo as analyzed by the 3-dimensional microcomputer tomography analysis. Our study has identified IPA-3 as a potential drug to treat bone metastatic PCa.

The Dysregulated Pharmacology of Clinically Relevant ESR1 Mutants is Normalized by Ligand-activated WT Receptor.

The estrogen receptor (ER/ESR1) is expressed in a majority of breast cancers and drugs that inhibit ER signaling are the cornerstone of breast cancer pharmacotherapy. Currently, aromatase inhibitors are the frontline endocrine interventions of choice although their durability in metastatic disease is limited by activating point mutations within the ligand-binding domain of ESR1 that permit ligand-independent activation of the receptor. It has been suggested that the most commonly occurring ESR1 mutations would likely compromise the clinical activity of selective estrogen receptor downregulators and selective estrogen receptor modulators (SERMs) when used as second-line therapies. It was unclear, however, how these mutations, which are likely coexpressed in cells with ERWT, may impact response to ER ligands in a clinically meaningful manner. To address this issue, we dissected the molecular mechanism(s) underlying ESR1-mutant pharmacology in models relevant to metastatic disease. These studies revealed that the response of ESR1 mutations to ligands was dictated primarily by the relative coexpression of ERWT in cells. Specifically, dysregulated pharmacology was only evident in cells in which the mutants were overexpressed relative to ligand-activated ERWT; a finding that highlights the role of allelism in determining ER-mutant pharmacology. Importantly, we demonstrated that the antagonist activity of the SERM, lasofoxifene, was not impacted by mutant status; a finding that has led to its clinical evaluation as a treatment for patients with advanced ER-positive breast cancer whose tumors harbor ESR1 mutations.