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

Strange Bedfellows: Nuclear Factor, Erythroid 2-Like 2 (Nrf2) and Hypoxia-Inducible Factor 1 (HIF-1) in Tumor Hypoxia

The importance of the tumor microenvironment for cancer progression and therapeutic resistance is an emerging focus of cancer biology. Hypoxia, or low oxygen, is a hallmark of solid tumors that promotes metastasis and represents a significant obstacle to successful cancer therapy. In response to hypoxia, cancer cells activate a transcriptional program that allows them to survive and thrive in this harsh microenvironment. Hypoxia-inducible factor 1 (HIF-1) is considered the main effector of the cellular response to hypoxia, stimulating the transcription of genes involved in promoting angiogenesis and altering cellular metabolism. However, growing evidence suggests that the cellular response to hypoxia is much more complex, involving coordinated signaling through stress response pathways. One key signaling molecule that is activated in response to hypoxia is nuclear factor, erythroid 2 like-2 (Nrf2). Nrf2 is a transcription factor that controls the expression of antioxidant-response genes, allowing the cell to regulate reactive oxygen species. Nrf2 is also activated in various cancer types due to genetic and epigenetic alterations, and is associated with poor survival and resistance to therapy. Emerging evidence suggests that coordinated signaling through Nrf2 and HIF-1 is critical for tumor survival and progression. In this review, we discuss the distinct and overlapping roles of HIF-1 and Nrf2 in the cellular response to hypoxia, with a focus on how targeting Nrf2 could provide novel chemotherapeutic modalities for treating solid tumors

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

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

Insulin receptor substrate 1 is a substrate of the Pim protein kinases

The Pim family of serine/threonine protein kinases (Pim 1, 2, and 3) contribute to cellular transformation by regulating glucose metabolism, protein synthesis, and mitochondrial oxidative phosphorylation. Drugs targeting the Pim protein kinases are being tested in phase I/II clinical trials for the treatment of hematopoietic malignancies. The goal of these studies was to identify Pim substrate(s) that could help define the pathway regulated by these enzymes and potentially serve as a biomarker of Pim activity. To identify novel substrates, bioinformatics analysis was carried out to identify proteins containing a consensus Pim phosphorylation site. This analysis identified the insulin receptor substrate 1 and 2 (IRS1/2) as potential Pim substrates. Experiments were carried out in tissue culture, animals, and human samples from phase I trials to validate this observation and define the biologic readout of this phosphorylation. Our study demonstrates in both malignant and normal cells using either genetic or pharmacological inhibition of the Pim kinases or overexpression of this family of enzymes that human IRS1S1101 and IRS2S1149 are Pim substrates. In xenograft tumor experiments and in a human phase I clinical trial, a pan-Pim inhibitor administered in vivo to animals or humans decreased IRS1S1101 phosphorylation in tumor tissues. This phosphorylation was shown to have effects on the half-life of the IRS family of proteins, suggesting a role in insulin or IGF signaling. These results demonstrate that IRS1S1101 is a novel substrate for the Pim kinases and provide a novel marker for evaluation of Pim inhibitor therapy

Identification of a Highly Effective Rapamycin Schedule that Markedly Reduces the Size, Multiplicity, and Phenotypic Progression of Tobacco Carcinogen-Induced Murine Lung Tumors

PURPOSE: Human and murine preneoplastic lung lesions induced by tobacco exposure are characterized by increased activation of the Akt/mammalian target of rapamycin (mTOR) pathway, suggesting a role for this pathway in lung cancer development. To test this, we did studies with rapamycin, an inhibitor of mTOR, in A/J mice that had been exposed to the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). EXPERIMENTAL DESIGN: Tumorigenesis was induced by i.p. injection of NNK, and rapamycin was administered 1 or 26 weeks after NNK administration. Biomarkers associated with mTOR inhibition were assessed in lung and/or surrogate tissues using immunohistochemistry and immunoblotting. Rapamycin levels were measured using mass spectroscopy. RESULTS: Rapamycin was administered on a daily (5 of 7 days) regimen beginning 26 weeks after NNK decreased tumor size, proliferative rate, and mTOR activity. Multiplicity was not affected. Comparing this regimen with an every-other-day (qod) regimen revealed that rapamycin levels were better maintained with qod administration, reaching a nadir of 16.4 ng/mL, a level relevant in humans. When begun 1 week after NNK, this regimen was well tolerated and decreased tumor multiplicity by 90%. Tumors that did develop showed decreased phenotypic progression and a 74% decrease in size that correlated with decreased proliferation and inhibition of mTOR. CONCLUSIONS: Tobacco carcinogen-induced lung tumors in A/J mice are dependent upon mTOR activity because rapamycin markedly reduced the development and growth of tumors. Combined with the Food and Drug Administration approval of rapamycin and broad clinical experience, these studies provide a rationale to assess rapamycin in trials with smokers at high risk to develop lung cancer

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.University of Arizona Cancer Center support grant [NIH P30CA023074]; NIH [R01CA173200]; DOD [W81XWH-12-1-0560]; American Lung Association Award [LCD-504131]12 month embargo; published OnlineFirst September 6, 2018This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Hypoxia-induced PIM kinase and laminin-activated integrin alpha 6 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 α6β1-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 α6β1 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.NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [CA154835, P30CA023074]; Accelerate for Success Award from the TRIF initiative at University of Arizona; Van Andel Research Institute; American Cancer SocietyAmerican Cancer Society [RSG-16-159-01-CDD]; Department of defense PCRPUnited States Department of Defense [W81XWH-19-1-0455]; PNW Prostate Cancer SPORE [P50CA09-7186, P01CA163227]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]