Deficiency of the Deubiquitinase UCHL1 Attenuates Pulmonary Arterial Hypertension.

BACKGROUND: The ubiquitin-proteasome system regulates protein degradation and the development of pulmonary arterial hypertension (PAH), but knowledge about the role of deubiquitinating enzymes in this process is limited. UCHL1 (ubiquitin carboxyl-terminal hydrolase 1), a deubiquitinase, has been shown to reduce AKT1 (AKT serine/threonine kinase 1) degradation, resulting in higher levels. Given that AKT1 is pathological in pulmonary hypertension, we hypothesized that UCHL1 deficiency attenuates PAH development by means of reductions in AKT1. METHODS: Tissues from animal pulmonary hypertension models as well as human pulmonary artery endothelial cells from patients with PAH exhibited increased vascular UCHL1 staining and protein expression. Exposure to LDN57444, a UCHL1-specific inhibitor, reduced human pulmonary artery endothelial cell and smooth muscle cell proliferation. Across 3 preclinical PAH models, LDN57444-exposed animals, Uchl1 knockout rats (Uchl1-/-), and conditional Uchl1 knockout mice (Tie2Cre-Uchl1fl/fl) demonstrated reduced right ventricular hypertrophy, right ventricular systolic pressures, and obliterative vascular remodeling. Lungs and pulmonary artery endothelial cells isolated from Uchl1-/- animals exhibited reduced total and activated Akt with increased ubiquitinated Akt levels. UCHL1-silenced human pulmonary artery endothelial cells displayed reduced lysine(K)63-linked and increased K48-linked AKT1 levels. RESULTS: Supporting experimental data, we found that rs9321, a variant in a GC-enriched region of the UCHL1 gene, is associated with reduced methylation (n=5133), increased UCHL1 gene expression in lungs (n=815), and reduced cardiac index in patients (n=796). In addition, Gadd45α (an established demethylating gene) knockout mice (Gadd45α-/-) exhibited reduced lung vascular UCHL1 and AKT1 expression along with attenuated hypoxic pulmonary hypertension. CONCLUSIONS: Our findings suggest that UCHL1 deficiency results in PAH attenuation by means of reduced AKT1, highlighting a novel therapeutic pathway in PAH.

PIM1 targeted degradation prevents the emergence of chemoresistance in prostate cancer.

PIM kinases have important pro-tumorigenic roles and mediate several oncogenic traits, including cell proliferation, survival, and chemotherapeutic resistance. As a result, multiple PIM inhibitors have been pursued as investigational new drugs in cancer; however, response to PIM inhibitors in solid tumors has fallen short of expectations. We found that inhibition of PIM kinase activity stabilizes protein levels of all three PIM isoforms (PIM1/2/3), and this can promote resistance to PIM inhibitors and chemotherapy. To overcome this effect, we designed PIM proteolysis targeting chimeras (PROTACs) to target PIM for degradation. PIM PROTACs effectively downmodulated PIM levels through the ubiquitin-proteasome pathway. Importantly, degradation of PIM kinases was more potent than inhibition of catalytic activity at inducing apoptosis in prostate cancer cell line models. In conclusion, we provide evidence of the advantages of degrading PIM kinases versus inhibiting their catalytic activity to target the oncogenic functions of PIM kinases.

PIM1 drives lipid droplet accumulation to promote proliferation and survival in prostate cancer.

Lipid droplets (LDs) are dynamic organelles with a neutral lipid core surrounded by a phospholipid monolayer. Solid tumors exhibit LD accumulation, and it is believed that LDs promote cell survival by providing an energy source during energy deprivation. However, the precise mechanisms controlling LD accumulation and utilization in prostate cancer are not well known. Here, we show peroxisome proliferator-activated receptor α (PPARα) acts downstream of PIM1 kinase to accelerate LD accumulation and promote cell proliferation in prostate cancer. Mechanistically, PIM1 inactivates glycogen synthase kinase 3 beta (GSK3ß) via serine 9 phosphorylation. GSK3ß inhibition stabilizes PPARα and enhances the transcription of genes linked to peroxisomal biogenesis (PEX3 and PEX5) and LD growth (Tip47). The effects of PIM1 on LD accumulation are abrogated with GW6471, a specific inhibitor for PPARα. Notably, LD accumulation downstream of PIM1 provides a significant survival advantage for prostate cancer cells during nutrient stress, such as glucose depletion. Inhibiting PIM reduces LD accumulation in vivo alongside slow tumor growth and proliferation. Furthermore, TKO mice, lacking PIM isoforms, exhibit suppression in circulating triglycerides. Overall, our findings establish PIM1 as an important regulator of LD accumulation through GSK3ß-PPARα signaling axis to promote cell proliferation and survival during nutrient stress.

PIM1 phosphorylates ABI2 to enhance actin dynamics and promote tumor invasion.

Distinguishing key factors that drive the switch from indolent to invasive disease will make a significant impact on guiding the treatment of prostate cancer (PCa) patients. Here, we identify a novel signaling pathway linking hypoxia and PIM1 kinase to the actin cytoskeleton and cell motility. An unbiased proteomic screen identified Abl-interactor 2 (ABI2), an integral member of the wave regulatory complex (WRC), as a PIM1 substrate. Phosphorylation of ABI2 at Ser183 by PIM1 increased ABI2 protein levels and enhanced WRC formation, resulting in increased protrusive activity and cell motility. Cell protrusion induced by hypoxia and/or PIM1 was dependent on ABI2. In vivo smooth muscle invasion assays showed that overexpression of PIM1 significantly increased the depth of tumor cell invasion, and treatment with PIM inhibitors significantly reduced intramuscular PCa invasion. This research uncovers a HIF-1-independent signaling axis that is critical for hypoxia-induced invasion and establishes a novel role for PIM1 as a key regulator of the actin cytoskeleton.

Targeting PIM Kinases to Improve the Efficacy of Immunotherapy.

The Proviral Integration site for Moloney murine leukemia virus (PIM) kinases is a family of serine/threonine kinases that regulates numerous signaling networks that promote cell growth, proliferation, and survival. PIM kinases are commonly upregulated in both solid tumors and hematological malignancies. Recent studies have demonstrated that PIM facilitates immune evasion in cancer by promoting an immunosuppressive tumor microenvironment that suppresses the innate anti-tumor response. The role of PIM in immune evasion has sparked interest in examining the effect of PIM inhibition in combination with immunotherapy. This review focuses on the role of PIM kinases in regulating immune cell populations, how PIM modulates the immune tumor microenvironment to promote immune evasion, and how PIM inhibitors may be used to enhance the efficacy of immunotherapy.

Optimized in vitro three-dimensional invasion assay for quantifying a wide range of cancer cell invasive behavior.

We describe a three-dimensional (3D) in vitro assay for quantifying cancer cell invasion into a 3D microenvironment with defined biochemical and biophysical properties. Researchers can quantify invasion dynamics (e.g., cell motility and directionality) and examine morphological changes during invasion, using live-cell and confocal imaging techniques. Together, these advantages over existing in vitro invasion assays, such as transwell-based assays, provide researchers with a valuable tool to gain insight into the mechanisms regulating cancer cell invasion. For complete details on the use and execution of this protocol, please refer to Padilla-Rodriguez et al. (2018) and Watson et al. (2021).

GLUT3/SLC2A3 Is an Endogenous Marker of Hypoxia in Prostate Cancer Cell Lines and Patient-Derived Xenograft Tumors.

The microenvironment of solid tumors is dynamic and frequently contains pockets of low oxygen levels (hypoxia) surrounded by oxygenated tissue. Indeed, a compromised vasculature is a hallmark of the tumor microenvironment, creating both spatial gradients and temporal variability in oxygen availability. Notably, hypoxia associates with increased metastasis and poor survival in patients. Therefore, to aid therapeutic decisions and better understand hypoxia's role in cancer progression, it is critical to identify endogenous biomarkers of hypoxia to spatially phenotype oncogenic lesions in human tissue, whether precancerous, benign, or malignant. Here, we characterize the glucose transporter GLUT3/SLC2A3 as a biomarker of hypoxic prostate epithelial cells and prostate tumors. Transcriptomic analyses of non-tumorigenic, immortalized prostate epithelial cells revealed a highly significant increase in GLUT3 expression under hypoxia. Additionally, GLUT3 protein increased 2.4-fold in cultured hypoxic prostate cell lines and was upregulated within hypoxic regions of xenograft tumors, including two patient-derived xenografts (PDX). Finally, GLUT3 out-performs other established hypoxia markers; GLUT3 staining in PDX specimens detects 2.6-8.3 times more tumor area compared to a mixture of GLUT1 and CA9 antibodies. Therefore, given the heterogeneous nature of tumors, we propose adding GLUT3 to immunostaining panels when trying to detect hypoxic regions in prostate samples.

Stabilization of PIM Kinases in Hypoxia Is Mediated by the Deubiquitinase USP28.

Proviral integration sites for Moloney murine leukemia virus (PIM) kinases are upregulated at the protein level in response to hypoxia and have multiple protumorigenic functions, promoting cell growth, survival, and angiogenesis. However, the mechanism responsible for the induction of PIM in hypoxia remains unknown. Here, we examined factors affecting PIM kinase stability in normoxia and hypoxia. We found that PIM kinases were upregulated in hypoxia at the protein level but not at the mRNA level, confirming that PIMs were upregulated in hypoxia in a hypoxia inducible factor 1-independent manner. PIM kinases were less ubiquitinated in hypoxia than in normoxia, indicating that hypoxia reduced their proteasomal degradation. We identified the deubiquitinase ubiquitin-specific protease 28 (USP28) as a key regulator of PIM1 and PIM2 stability. The overexpression of USP28 increased PIM protein stability and total levels in both normoxia and hypoxia, and USP28-knockdown significantly increased the ubiquitination of PIM1 and PIM2. Interestingly, coimmunoprecipitation assays showed an increased interaction between PIM1/2 and USP28 in response to hypoxia, which correlated with reduced ubiquitination and increased protein stability. In a xenograft model, USP28-knockdown tumors grew more slowly than control tumors and showed significantly lower levels of PIM1 in vivo. In conclusion, USP28 blocked the ubiquitination and increased the stability of PIM1/2, particularly in hypoxia. These data provide the first insight into proteins responsible for controlling PIM protein degradation and identify USP28 as an important upstream regulator of this hypoxia-induced, protumorigenic signaling pathway.

Digital image analysis using video microscopy of human-derived prostate cancer vs normal prostate organoids to assess migratory behavior on extracellular matrix proteins.

The advent of perpetuating living organoids derived from patient tissue is a promising avenue for cancer research but is limited by difficulties with precise characterization. In this brief communication, we demonstrate via time-lapse imaging distinct phenotypes of prostate organoids derived from patient material- without confirmation of cellular identity. We show that organoids derived from histologically normal tissue more readily spread on a physiologic extracellular matrix (ECM) than on pathologic ECM (p<0.0001), while tumor-derived organoids spread equally on either substrate (p=0.2406). This study is an important proof-of-concept to defer precise characterization of organoids and still glean information into disease pathology.

The long noncoding RNA H19 regulates tumor plasticity in neuroendocrine prostate cancer.

Neuroendocrine (NE) prostate cancer (NEPC) is a lethal subtype of castration-resistant prostate cancer (PCa) arising either de novo or from transdifferentiated prostate adenocarcinoma following androgen deprivation therapy (ADT). Extensive computational analysis has identified a high degree of association between the long noncoding RNA (lncRNA) H19 and NEPC, with the longest isoform highly expressed in NEPC. H19 regulates PCa lineage plasticity by driving a bidirectional cell identity of NE phenotype (H19 overexpression) or luminal phenotype (H19 knockdown). It contributes to treatment resistance, with the knockdown of H19 re-sensitizing PCa to ADT. It is also essential for the proliferation and invasion of NEPC. H19 levels are negatively regulated by androgen signaling via androgen receptor (AR). When androgen is absent SOX2 levels increase, driving H19 transcription and facilitating transdifferentiation. H19 facilitates the PRC2 complex in regulating methylation changes at H3K27me3/H3K4me3 histone sites of AR-driven and NEPC-related genes. Additionally, this lncRNA induces alterations in genome-wide DNA methylation on CpG sites, further regulating genes associated with the NEPC phenotype. Our clinical data identify H19 as a candidate diagnostic marker and predictive marker of NEPC with elevated H19 levels associated with an increased probability of biochemical recurrence and metastatic disease in patients receiving ADT. Here we report H19 as an early upstream regulator of cell fate, plasticity, and treatment resistance in NEPC that can reverse/transform cells to a treatable form of PCa once therapeutically deactivated.

Direct phosphorylation and stabilization of HIF-1α by PIM1 kinase drives angiogenesis in solid tumors.

Angiogenesis is essential for the sustained growth of solid tumors. Hypoxia-inducible factor 1 (HIF-1) is a master regulator of angiogenesis and constitutive activation of HIF-1 is frequently observed in human cancers. Therefore, understanding the mechanisms governing the activation of HIF-1 is critical for successful therapeutic targeting of tumor angiogenesis. Herein, we establish a new regulatory mechanism responsible for the constitutive activation of HIF-1α in cancer, irrespective of oxygen tension. PIM1 kinase directly phosphorylates HIF-1α at threonine 455, a previously uncharacterized site within its oxygen-dependent degradation domain. This phosphorylation event disrupts the ability of prolyl hydroxylases to bind and hydroxylate HIF-1α, interrupting its canonical degradation pathway and promoting constitutive transcription of HIF-1 target genes. Moreover, phosphorylation of the analogous site in HIF-2α (S435) stabilizes the protein through the same mechanism, indicating post-translational modification within the oxygen-dependent degradation domain as a mechanism of regulating the HIF-α subunits. In vitro and in vivo models demonstrate that expression of PIM1 is sufficient to stabilize HIF-1α and HIF-2α in normoxia and stimulate angiogenesis in a HIF-1-dependent manner. CRISPR mutants of HIF-1α (Thr455D) promoted increased tumor growth, proliferation, and angiogenesis. Moreover, HIF-1α-T455D xenograft tumors were refractory to the anti-angiogenic and cytotoxic effects of PIM inhibitors. These data identify a new signaling axis responsible for hypoxia-independent activation of HIF-1 and expand our understanding of the tumorigenic role of PIM1 in solid tumors.

Targeting PIM Kinases to Overcome Therapeutic Resistance in Cancer.

Cancer progression and the onset of therapeutic resistance are often the results of uncontrolled activation of survival kinases. The proviral integration for the Moloney murine leukemia virus (PIM) kinases are oncogenic serine/threonine kinases that regulate tumorigenesis by phosphorylating a wide range of substrates that control cellular metabolism, proliferation, and survival. Because of their broad impact on cellular processes that facilitate progression and metastasis in many cancer types, it has become clear that the activation of PIM kinases is a significant driver of resistance to various types of anticancer therapies. As a result, efforts to target PIM kinases for anticancer therapy have intensified in recent years. Clinical and preclinical studies indicate that pharmacologic inhibition of PIM has the potential to significantly improve the efficacy of standard and targeted therapies. This review focuses on the signaling pathways through which PIM kinases promote cancer progression and resistance to therapy, as well as highlights biological contexts and promising strategies to exploit PIM as a therapeutic target in cancer.

PIM kinases alter mitochondrial dynamics and chemosensitivity in lung cancer.

Resistance to chemotherapy represents a major obstacle to the successful treatment of non-small-cell lung cancer (NSCLC). The goal of this study was to determine how PIM kinases impact mitochondrial dynamics, ROS production, and response to chemotherapy in lung cancer. Live-cell imaging and microscopy were used to determine the effect of PIM loss or inhibition on mitochondrial phenotype and ROS. Inhibition of PIM kinases caused excessive mitochondrial fission and significant upregulation of mitochondrial superoxide, increasing intracellular ROS. Mechanistically, we define a signaling axis linking PIM1 to Drp1 and mitochondrial fission in lung cancer. PIM inhibition significantly increased the protein levels and mitochondrial localization of Drp1, causing marked fragmentation of mitochondria. An inverse correlation between PIM1 and Drp1 was confirmed in NSCLC patient samples. Inhibition of PIM sensitized NSCLC cells to chemotherapy and produced a synergistic antitumor response in vitro and in vivo. Immunohistochemistry and transmission electron microscopy verified that PIM inhibitors promote mitochondrial fission and apoptosis in vivo. These data improve our knowledge about how PIM1 regulates mitochondria and provide justification for combining PIM inhibition with chemotherapy in NSCLC.

Hypoxia-induced PIM kinase and laminin-activated integrin α6 mediate resistance to PI3K inhibitors in bone-metastatic CRPC.

Bone-metastatic castration-resistant prostate cancer (CRPC) is lethal due to inherent resistance to androgen deprivation therapy, chemotherapy, and targeted therapies. Despite the fact that a majority of CRPC patients (approximately 70%) harbor a constitutively active PI3K survival pathway, targeting the PI3K/mTOR pathway has failed to increase overall survival in clinical trials. Here, we identified two separate and independent survival pathways induced by the bone tumor microenvironment that are hyperactivated in CRPC and confer resistance to PI3K inhibitors. The first pathway involves integrin α6ß1-mediated adhesion to laminin and the second involves hypoxia-induced expression of PIM kinases. In vitro and in vivo models demonstrate that these pathways transduce parallel but independent signals that promote survival by reducing oxidative stress and preventing cell death. We further demonstrate that both pathways drive resistance to PI3K inhibitors in PTEN-negative tumors. These results provide preclinical evidence that combined inhibition of integrin α6ß1 and PIM kinase in CRPC is required to overcome tumor microenvironment-mediated resistance to PI3K inhibitors in PTEN-negative tumors within the hypoxic and laminin-rich bone microenvironment.

Gene Editing of α6 Integrin Inhibits Muscle Invasive Networks and Increases Cell-Cell Biophysical Properties in Prostate Cancer.

Human prostate cancer confined to the gland is indolent (low-risk), but tumors outside the capsule are aggressive (high-risk). Extracapsular extension requires invasion within and through a smooth muscle-structured environment. Because integrins respond to biomechanical cues, we used a gene editing approach to determine if a specific region of laminin-binding α6ß1 integrin was required for smooth muscle invasion both in vitro and in vivo. Human tissue specimens showed prostate cancer invasion through smooth muscle and tumor coexpression of α6 integrin and E-cadherin in a cell-cell location and α6 integrin in a cell-extracellular matrix (ECM) distribution. Prostate cancer cells expressing α6 integrin (DU145 α6WT) produced a 3D invasive network on laminin-containing Matrigel and invaded into smooth muscle both in vitro and in vivo. In contrast, cells without α6 integrin (DU145 α6KO) and cells expressing an integrin mutant (DU145 α6AA) did not produce invasive networks, could not invade muscle both in vitro and in vivo, and surprisingly formed 3D cohesive clusters. Using electric cell-substrate impedance testing, cohesive clusters had up to a 30-fold increase in normalized resistance at 400 Hz (cell-cell impedance) as compared with the DU145 α6WT cells. In contrast, measurements at 40,000 Hz (cell-ECM coverage) showed that DU145 α6AA cells were two-fold decreased in normalized resistance and were defective in restoring resistance after a 1 µmol/L S1P challenge as compared with the DU145 α6WT cells. The results suggest that gene editing of a specific α6 integrin extracellular region, not required for normal tissue function, can generate a new biophysical cancer phenotype unable to invade the muscle, presenting a new therapeutic strategy for metastasis prevention in prostate cancer. SIGNIFICANCE: This study shows an innovative strategy to block prostate cancer metastasis and invasion in the muscle through gene editing of a specific α6 integrin extracellular region.

The Tumor Microenvironments of Lethal Prostate Cancer.

Localized prostate cancer (confined to the gland) generally is considered curable, with nearly a 100% 5-year-survival rate. When the tumor escapes the prostate capsule, leading to metastasis, there is a poorer prognosis and higher mortality rate, with 5-year survival dropping to less than 30%. A major research question has been to understand the transition from indolent (low risk) disease to aggressive (high risk) disease. In this chapter, we provide details of the changing tumor microenvironments during prostate cancer invasion and their role in the progression and metastasis of lethal prostate cancer. Four microenvironments covered here include the muscle stroma, perineural invasion, hypoxia, and the role of microvesicles in altering the extracellular matrix environment. The adaptability of prostate cancer to these varied microenvironments and the cues for phenotypic changes are currently understudied areas. Model systems for understanding smooth muscle invasion both in vitro and in vivo are highlighted. Invasive human needle biopsy tissue and mouse xenograft tumors both contain smooth muscle invasion. In combination, the models can be used in an iterative process to validate molecular events for smooth muscle invasion in human tissue. Understanding the complex and interacting microenvironments in the prostate holds the key to early detection of high-risk disease and preventing tumor invasion through escape from the prostate capsule.

Mechanisms Behind Resistance to PI3K Inhibitor Treatment Induced by the PIM Kinase.

Cancer resistance to PI3K inhibitor therapy can be in part mediated by increases in the PIM1 kinase. However, the exact mechanism by which PIM kinase promotes tumor cell resistance is unknown. Our study unveils the pivotal control of redox signaling by PIM kinases as a driver of this resistance mechanism. PIM1 kinase functions to decrease cellular ROS levels by enhancing nuclear factor erythroid 2-related factor 2 (NRF2)/antioxidant response element activity. PIM prevents cell death induced by PI3K-AKT-inhibitory drugs through a noncanonical mechanism of NRF2 ubiquitination and degradation and translational control of NRF2 protein levels through modulation of eIF4B and mTORC1 activity. Importantly, PIM also controls NAD(P)H production by increasing glucose flux through the pentose phosphate shunt decreasing ROS production, and thereby diminishing the cytotoxicity of PI3K-AKT inhibitors. Treatment with PIM kinase inhibitors reverses this resistance phenotype, making tumors increasingly susceptible to small-molecule therapeutics, which block the PI3K-AKT pathway.

Hypoxia-Inducible PIM Kinase Expression Promotes Resistance to Antiangiogenic Agents.

Purpose: Patients develop resistance to antiangiogenic drugs, secondary to changes in the tumor microenvironment, including hypoxia. PIM kinases are prosurvival kinases and their expression increases in hypoxia. The goal of this study was to determine whether targeting hypoxia-induced PIM kinase expression is effective in combination with VEGF-targeting agents. The rationale for this therapeutic approach is based on the fact that antiangiogenic drugs can make tumors hypoxic, and thus more sensitive to PIM inhibitors.Experimental Design: Xenograft and orthotopic models of prostate and colon cancer were used to assess the effect of PIM activation on the efficacy of VEGF-targeting agents. IHC and in vivo imaging were used to analyze angiogenesis, apoptosis, proliferation, and metastasis. Biochemical studies were performed to characterize the novel signaling pathway linking PIM and HIF1.Results: PIM was upregulated following treatment with anti-VEGF therapies, and PIM1 overexpression reduced the ability of these drugs to disrupt vasculature and block tumor growth. PIM inhibitors reduced HIF1 activity, opposing the shift to a pro-angiogenic gene signature associated with hypoxia. Combined inhibition of PIM and VEGF produced a synergistic antitumor response characterized by decreased proliferation, reduced tumor vasculature, and decreased metastasis.Conclusions: This study describes PIM kinase expression as a novel mechanism of resistance to antiangiogenic agents. Our data provide justification for combining PIM and VEGF inhibitors to treat solid tumors. The unique ability of PIM inhibitors to concomitantly target HIF1 and selectively kill hypoxic tumor cells addresses two major components of tumor progression and therapeutic resistance. Clin Cancer Res; 24(1); 169-80. ©2017 AACR.