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Aggressive tumors of epithelial origin shed cells that intravasate and become circulating tumor cells (CTC). The CTCs that are able to survive the stresses encountered in the bloodstream can then seed metastases. We demonstrated previously that CTCs isolated from the blood of prostate cancer patients display specific nanomechanical phenotypes characteristic of cell endurance and invasiveness and patient sensitivity to androgen deprivation therapy. Here we report that patient-isolated CTCs are nanomechanically distinct from cells randomly shed from the tumor, with high adhesion as the most distinguishing biophysical marker. CTCs uniquely coisolated with macrophage-like cells bearing the markers of tumor-associated macrophages (TAM). The presence of these immune cells was indicative of a survival-promoting phenotype of “mechanical fitness” in CTCs based on high softness and high adhesion as determined by atomic force microscopy. Correlations between enumeration of macrophages and mechanical fitness of CTCs were strong in patients before the start of hormonal therapy. Single-cell proteomic analysis and nanomechanical phenotyping of tumor cell–macrophage cocultures revealed that macrophages promoted epithelial–mesenchymal plasticity in prostate cancer cells, manifesting in their mechanical fitness. The resulting softness and adhesiveness of the mechanically fit CTCs confer resistance to shear stress and enable protective cell clustering. These findings suggest that selected tumor cells are coached by TAMs and accompanied by them to acquire intermediate epithelial/mesenchymal status, thereby facilitating survival during the critical early stage leading to metastasis.Significance:The interaction between macrophages and circulating tumor cells increases the capacity of tumor cells to initiate metastasis and may constitute a new set of blood-based targets for pharmacologic intervention.

Supplementary Materials and Methods, Figures and Tables

The proteasome has key roles in neuronal proteostasis, including the removal of misfolded and oxidized proteins, presynaptic protein turnover, and synaptic efficacy and plasticity. Proteasome dysfunction is a prominent feature of Alzheimer’s disease (AD). We show that prevention of proteasome dysfunction by genetic manipulation delays mortality, cell death, and cognitive deficits in fly and cell culture AD models. We developed a transgenic mouse with neuronal-specific proteasome overexpression that, when crossed with an AD mouse model, showed reduced mortality and cognitive deficits. To establish translational relevance, we developed a set of TAT-based proteasome-activating peptidomimetics that stably penetrated the blood-brain barrier and enhanced 20 S /26 S proteasome activity. These agonists protected against cell death, cognitive decline, and mortality in cell culture, fly, and mouse AD models. The protective effects of proteasome overexpression appear to be driven, at least in part, by the proteasome’s increased turnover of the amyloid precursor protein along with the prevention of overall proteostatic dysfunction.

2-Methoxyestradiol (2-ME2) possesses anti-tumorigenic activities in multiple tumor models with acceptable tolerability profile in humans. Incomplete understanding of the mechanism has hindered its development as an anti-tumorigenic compound. We have identified for the first-time macrophage stimulatory protein 1 receptor (MST1R) as a potential target of 2-ME2 in prostate cancer cells. Human tissue validation studies show that MST1R (a.k.a RON) protein levels are significantly elevated in prostate cancer tissues compared to adjacent normal/benign glands. Serum levels of macrophage stimulatory protein (MSP), a ligand for RON, is not only associated with the risk of disease recurrence, but also significantly elevated in samples from African American patients. 2-ME2 treatment inhibited mechanical properties such as adhesion and elasticity that are associated with epithelial mesenchymal transition by downregulating mRNA expression and protein levels of MST1R in prostate cancer cell lines. Intervention with 2-ME2 significantly reduced tumor burden in mice. Notably, global metabolomic profiling studies identified significantly higher circulating levels of bile acids in castrated animals that were decreased with 2-ME2 intervention. In summary, findings presented in this manuscript identified MSP as a potential marker for predicting biochemical recurrence and suggest repurposing 2-ME2 to target RON signaling may be a potential therapeutic modality for prostate cancer.

Endometriosis is an invasive disease, and a leading cause of pain, infertility and disability among women, with an incidence 10 fold that of cancer. A more complete understanding of disease pathogenesis is essential for the development of non-surgical diagnostic assays and non-hormonal therapeutics. Avoidance of immune clearance and implantation of endometrial tissue on peritoneal surfaces are features of endometriosis lesion formation that overlap with cancer metastasis. Connexins, and the gap junctions they form, have been implicated in cancer progression, and may be associated endometriosis pathophysiology. Single cell transcriptomic profiling of endometrial epithelial and stromal cells from women with endometriosis reveals a striking and progressive shift in expression of connexins and related regulatory and junctional genes. We demonstrate that gap junction coupling between endometrial cells and the peritoneal mesothelium is dramatically induced, specifically in endometriosis patients, and is required for invasion by inducing breakdown of the mesothelial barrier function.

The NAD+-dependent deacetylase, Sirtuin 1 (SIRT1) is involved in prostate cancer pathogenesis. However, the actual contribution is unclear as some reports propose a protective role while others suggest it is harmful. We provide evidence for a contextual role for SIRT1 in prostate cancer. Our data show that (i) mice orthotopically implanted with SIRT1-silenced LNCaP cells produced smaller tumors; (ii) SIRT1 suppression mimicked AR inhibitory effects in hormone responsive LNCaP cells; and (iii) caused significant reduction in gene signatures associated with E2F and MYC targets in AR-null PC-3 and E2F and mTORC1 signaling in castrate-resistant ARv7 positive 22Rv1 cells. Our findings further show increased nuclear SIRT1 (nSIRT1) protein under androgen-depleted relative to androgen-replete conditions in prostate cancer cell lines. Silencing SIRT1 resulted in decreased recruitment of AR to PSA enhancer selectively under androgen-deprivation conditions. Prostate cancer outcome data show that patients with higher levels of nSIRT1 progress to advanced disease relative to patients with low nSIRT1 levels. Collectively, we demonstrate that lowering SIRT1 levels potentially provides new avenues to effectively prevent prostate cancer recurrence.

Aggressive tumors of epithelial origin shed cells that intravasate and become circulating tumor cells (CTC). The CTCs that are able to survive the stresses encountered in the bloodstream can then seed metastases. We demonstrated previously that CTCs isolated from the blood of prostate cancer patients display specific nanomechanical phenotypes characteristic of cell endurance and invasiveness and patient sensitivity to androgen deprivation therapy. Here we report that patient-isolated CTCs are nanomechanically distinct from cells randomly shed from the tumor, with high adhesion as the most distinguishing biophysical marker. CTCs uniquely coisolated with macrophage-like cells bearing the markers of tumor-associated macrophages (TAM). The presence of these immune cells was indicative of a survival-promoting phenotype of “mechanical fitness” in CTCs based on high softness and high adhesion as determined by atomic force microscopy. Correlations between enumeration of macrophages and mechanical fitness of CTCs were strong in patients before the start of hormonal therapy. Single-cell proteomic analysis and nanomechanical phenotyping of tumor cell–macrophage cocultures revealed that macrophages promoted epithelial–mesenchymal plasticity in prostate cancer cells, manifesting in their mechanical fitness. The resulting softness and adhesiveness of the mechanically fit CTCs confer resistance to shear stress and enable protective cell clustering. These findings suggest that selected tumor cells are coached by TAMs and accompanied by them to acquire intermediate epithelial/mesenchymal status, thereby facilitating survival during the critical early stage leading to metastasis. Significance: The interaction between macrophages and circulating tumor cells increases the capacity of tumor cells to initiate metastasis and may constitute a new set of blood-based targets for pharmacologic intervention.

The prostate-expressed sulfotransferase SULT2B1b (SULT2B) regulates intracrine androgen homeostasis by mediating 3β-sulfation of DHEA, thus reducing the precursor pool in the androgen biosynthesis pathway. We explored how loss of SULT2B might influence prostate cancer progression. Results show that SULT2B ablation in castration-resistant prostate cancer (CRPC) cells, generated by stable RNA interference or gene knockout, led to robust activation of the ERK1/2 Map kinase survival signal and induction of epithelial to mesenchymal transition (EMT). EMT activation was concluded on the basis of increased levels of vimentin (a mesenchymal protein) and the EMT-activating transcription factors SNAI1 (Snail) and TWIST1, shown by Western blotting, mass spectrometry and single-cell mass cytometry. Loss of SULT2B was associated with enhanced motility and invasive activity of CRPC cells in vitro and their growth escalation in vivo as xenografts. Higher invasion and metastasis potential of SULT2B-ablated CRPC cells was further indicated by results that these cells are less adhesive (i.e. easily detachable) and less stiff (i.e. more pliable) based on atomic force microscopy analysis of individual cells. Notably, AKR1C3, an aldo-keto reductase, which is elevated frequently in advanced prostate cancer, showed marked upregulation in SULT2B-deficient cells. AKR1C3 regulates androgen receptor (AR) signaling by promoting androgen biosynthesis and functioning as an AR-selective coactivator. While levels of AR and DHT did not change, AR activity was elevated, since PSA and FKBP5 mRNA induction by DHT-activated AR was several fold higher in SULT2B-silenced cells. The DHT-metabolizing AKR1C2 aldo-keto reductase was also upregulated, which likely accounts for a steady-state androgen level despite elevated AKR1C3 expression. Phosphorylation of ERK decreased in AKR1C3-silenced cells, signifying a causal link between AKR1C3 upregulation and ERK1/2 activation. SULT2B was undetectable immunohistochemically in tissue microarrays of clinical CRPC metastases, while SULT2B-negative samples showed AKR1C3-positive immunostaining. Primary prostate cancer exhibited variable, Gleason score independent SULT2B levels -- varying from strong positive to significantly reduced or undetectable. The reciprocal expression pattern for SULT2B and AKR1C3 in clinical CRPC suggests that AKR1C3 upregulation, ERK1/2 activation and increased aggressive traits of SULT2B-ablated cells, observed in vitro in cell models, may be clinically significant. Pathways regulating the inhibitory SULT2B-AKR1C3 axis may inform new avenue(s) for delaying disease progression in SULT2B-deficient prostate cancer.Funding Support: 1I01BX000280, VA (BC); W81XWH-14-1-0606, DOD (BC); IK6 BX004207, VA (BC); P50 CA97186, NIH & W81XWH-12-1-0208, DOD (EAM)

Emerging evidence indicates that adipose stromal cells (ASC) are recruited to enhance cancer development. In this study, we examined the role these adipocyte progenitors play relating to intercellular communication in obesity-associated endometrial cancer. This is particularly relevant given that gap junctions have been implicated in tumor suppression. Examining the effects of ASCs on the transcriptome of endometrial epithelial cells (EEC) in an in vitro coculture system revealed transcriptional repression of GJA1 (encoding the gap junction protein Cx43) and other genes related to intercellular communication. This repression was recapitulated in an obesity mouse model of endometrial cancer. Furthermore, inhibition of plasminogen activator inhibitor 1 (PAI-1), which was the most abundant ASC adipokine, led to reversal of cellular distribution associated with the GJA1 repression profile, suggesting that PAI-1 may mediate actions of ASC on transcriptional regulation in EEC. In an endometrial cancer cohort (n = 141), DNA hypermethylation of GJA1 and related loci TJP2 and PRKCA was observed in primary endometrial endometrioid tumors and was associated with obesity. Pharmacologic reversal of DNA methylation enhanced gap-junction intercellular communication and cell–cell interactions in vitro. Restoring Cx43 expression in endometrial cancer cells reduced cellular migration; conversely, depletion of Cx43 increased cell migration in immortalized normal EEC. Our data suggest that persistent repression by ASC adipokines leads to promoter hypermethylation of GJA1 and related genes in the endometrium, triggering long-term silencing of these loci in endometrial tumors of obese patients. Significance: Studies reveal that adipose-derived stem cells in endometrial cancer pathogenesis influence epigenetic repression of gap junction loci, which suggests targeting of gap junction activity as a preventive strategy for obesity-associated endometrial cancer.

Allosteric regulators of clinically important enzymes are gaining popularity as alternatives to competitive inhibitors. This is also the case for the proteasome, a major intracellular protease and a target of anti-cancer drugs. All clinically used proteasome inhibitors bind to the active sites in catalytic chamber and display a competitive mechanism. Unfortunately, inevitable resistance associated with this type of inhibition drives the search for non-competitive agents. The multisubunit and multicatalytic "proteolytic machine" such as the proteasome is occasionally found to be affected by agents with other primary targets. For example the immunosuppressive agent rapamycin has been shown to allosterically inhibit the proteasome albeit at levels far higher than its mTOR related efficacy. As part of an ongoing program to search for novel proteasome-targeting pharmacophores, we identified the binding domain of rapamycin as required for proteasome inhibition even without the macrocyclic context of the parent compound. By subsequent structure-activity relationship studies, we generated a pipecolic ester derivative compound 3 representing a new class of proteasome inhibitors. Compound 3 affects the core proteasome activities and proliferation of cancer cells with low micromolar/high nanomolar efficacy. Molecular modeling, atomic force microscopy imaging and biochemical data suggest that compound 3 binds into one of intersubunit pockets in the proteasomal α ring and destabilizes the α face and the gate. The α face is used as a docking area for proteasome-regulating protein modules and the gate is critical for controlling access to the catalytic chamber. Thus, the pipecolic ester template elicits a new and attractive mechanism for proteasome inhibition distinct from classical competitive drugs.

The proteasome is a pivotal element of controlled proteolysis, responsible for the catabolic arm of proteostasis. By inducing apoptosis, small molecule inhibitors of proteasome peptidolytic activities are successfully utilized in treatment of blood cancers. However, the clinical potential of proteasome activation remains relatively unexplored. In this work, we introduce short TAT peptides derived from HIV-1 Tat protein and modified with synthetic turn-stabilizing residues as proteasome agonists. Molecular docking and biochemical studies point to the &alpha, 1/&alpha, 2 pocket of the core proteasome &alpha, ring as the binding site of TAT peptides. We postulate that the TATs&rsquo, pharmacophore consists of an N-terminal basic pocket-docking &ldquo, activation anchor&rdquo, connected via a &beta, turn inducer to a C-terminal &ldquo, specificity clamp&rdquo, that binds on the proteasome &alpha, surface. By allosteric effects&mdash, including destabilization of the proteasomal gate&mdash, the compounds substantially augment activity of the core proteasome in vitro. Significantly, this activation is preserved in the lysates of cultured cells treated with the compounds. We propose that the proteasome-stimulating TAT pharmacophore provides an attractive lead for future clinical use.

The 20S proteasome is the main protease that directly targets intrinsically disordered proteins (IDPs) for proteolytic degradation. Mutations, oxidative stress, or aging can induce the buildup of IDPs resulting in incorrect signaling or aggregation, associated with the pathogenesis of many cancers and neurodegenerative diseases. Drugs that facilitate 20S-mediated proteolysis therefore have many potential therapeutic applications. We report herein the modulation of proteasome assembly by the small molecule TCH-165, resulting in an increase in 20S levels. The increase in the level of free 20S corresponds to enhanced proteolysis of IDPs, including α-synuclein, tau, ornithine decarboxylase, and c-Fos, but not structured proteins. Clearance of ubiquitinated protein was largely maintained by single capped proteasome complexes (19S–20S), but accumulation occurs when all 19S capped proteasome complexes are depleted. This study illustrates the first example of a small molecule capable of targeting disordered proteins for degradation by regulating the dynamic equilibrium between different proteasome complexes.

Summary Older adults universally suffer from sarcopenia and approximately 60–70% are diabetic or prediabetic. Nonetheless, the mechanisms underlying these aging‐related metabolic disorders are unknown. NFκB has been implicated in the pathogenesis of several aging‐related pathologies including sarcopenia and type 2 diabetes and has been proposed as a target against them. NFκB also is thought to mediate muscle wasting seen with disuse, denervation, and some systemic diseases (e.g., cancer, sepsis). We tested the hypothesis that lifelong inhibition of the classical NFκB pathway would protect against aging‐related sarcopenia and insulin resistance. Aged mice with muscle‐specific overexpression of a super‐repressor IκBα mutant (MISR) were protected from insulin resistance. However, MISR mice were not protected from sarcopenia; to the contrary, these mice had decreases in muscle mass and strength compared to wild‐type mice. In MISR mice, NFκB suppression also led to an increase in proteasome activity and alterations in several genes and pathways involved in muscle growth and atrophy (e.g., myostatin). We conclude that the mechanism behind aging‐induced sarcopenia is NFκB independent and differs from muscle wasting due to pathologic conditions. Our findings also indicate that, while suppressing NFκB improves insulin sensitivity in aged mice, this transcription factor is important for normal muscle mass maintenance and its sustained inhibition is detrimental to muscle function.

Cytometry by time-of-flight (CyTOF) simultaneously measures multiple cellular proteins at the single-cell level and is used to assess intertumor and intratumor heterogeneity. This approach may be used to investigate the variability of individual tumor responses to treatments. Herein, we stratified lung tumor subpopulations based on AXL signaling as a potential targeting strategy. Integrative transcriptome analyses were used to investigate how TP-0903, an AXL kinase inhibitor, influences redundant oncogenic pathways in metastatic lung cancer cells. CyTOF profiling revealed that AXL inhibition suppressed SMAD4/TGFβ signaling and induced JAK1–STAT3 signaling to compensate for the loss of AXL. Interestingly, high JAK1–STAT3 was associated with increased levels of AXL in treatment-naïve tumors. Tumors with high AXL, TGFβ, and JAK1 signaling concomitantly displayed CD133-mediated cancer stemness and hybrid epithelial-to-mesenchymal transition features in advanced-stage patients, suggesting greater potential for distant dissemination. Diffusion pseudotime analysis revealed cell-fate trajectories among four different categories that were linked to clinicopathologic features for each patient. Patient-derived organoids (PDO) obtained from tumors with high AXL and JAK1 were sensitive to TP-0903 and ruxolitinib (JAK inhibitor) treatments, supporting the CyTOF findings. This study shows that single-cell proteomic profiling of treatment-naïve lung tumors, coupled with ex vivo testing of PDOs, identifies continuous AXL, TGFβ, and JAK1–STAT3 signal activation in select tumors that may be targeted by combined AXL–JAK1 inhibition. Significance: Single-cell proteomic profiling of clinical samples may facilitate the optimal selection of novel drug targets, interpretation of early-phase clinical trial data, and development of predictive biomarkers valuable for patient stratification.

SULT2B1b (SULT2B) is a prostate-expressed hydroxysteroid sulfotransferase, which may regulate intracrine androgen homeostasis by mediating 3β-sulfation of dehydroepiandrosterone (DHEA), the precursor for 5α-dihydrotestosterone (DHT) biosynthesis. The aldo-keto reductase (AKR)1C3 regulates androgen receptor (AR) activity in castration-resistant prostate cancer (CRPC) by promoting tumor tissue androgen biosynthesis from adrenal DHEA and also by functioning as an AR-selective coactivator. Herein we report that SULT2B-depleted CRPC cells, arising from stable RNA interference or gene knockout (KO), are markedly upregulated for AKR1C3, activated for ERK1/2 survival signal, and induced for epithelial-to-mesenchymal (EMT)-like changes. EMT was evident from increased mesenchymal proteins and elevated EMT-inducing transcription factors SNAI1 and TWIST1 in immunoblot and single-cell mass cytometry analyses. SULT2B KO cells showed greater motility and invasion in vitro; growth escalation in xenograft study; and enhanced metastatic potential predicted on the basis of decreased cell stiffness and adhesion revealed from atomic force microscopy analysis. While AR and androgen levels were unchanged, AR activity was elevated, since PSA and FKBP5 mRNA induction by DHT-activated AR was several-fold higher in SULT2B-silenced cells. AKR1C3 silencing prevented ERK1/2 activation and SNAI1 induction in SULT2B-depleted cells. SULT2B was undetectable in nearly all CRPC metastases from 50 autopsy cases. Primary tumors showed variable and Gleason score (GS)-independent SULT2B levels. CRPC metastases lacking SULT2B expressed AKR1C3. Since AKR1C3 is frequently elevated in advanced prostate cancer, the inhibitory influence of SULT2B on AKR1C3 upregulation, ERK1/2 activation, EMT-like induction, and on cell motility and invasiveness may be clinically significant. Pathways regulating the inhibitory SULT2B-AKR1C3 axis may inform new avenue(s) for targeting SULT2B-deficient prostate cancer.

The role of cholesterol content on monocyte biomechanics remains understudied despite the well-established link between cholesterol and monocytes/macrophages in atherosclerosis, and the effect on other cell types. In this work, we have investigated the effect of cholesterol on monocyte deformability and the underlying molecular mechanisms. We altered the baseline cholesterol in human monocytic cell line THP-1, and investigated the changes in monocyte deformability using a custom microfluidic platform and atomic force microscopy. We observed that the cholesterol depletion lowered deformability while enrichment increased deformability compared to untreated cells. As a consequence of altered deformability, cholesterol depleted cells spread more on collagen-coated surfaces with elongated morphology, whereas cholesterol enriched cells had a more rounded morphology. We observed that the decreased deformability in cholesterol depleted cells, despite an increase in the fluidity of the membrane, is due to an increase in phosphorylation of Protein Kinase C (PKC), which translates to a higher degree of actin polymerization. Together, our results highlight the importance of biophysical regulation of monocyte response to cholesterol levels.

Background: Non-small cell lung cancer (NSCLC) is a molecularly heterogeneous disease with a high propensity for drug resistance and metastasis. AXL, a member of the Tyro3-AXL-Mer (TAM) family of receptor tyrosine kinases, is a central regulator of epithelial-to-mesenchymal transition (EMT) and enables tumor cells to invade and acquire drug resistance. AXL is overexpressed in NSCLC and its expression correlates positively with tumor invasion, drug resistance, and negatively predicts overall survival. We mechanistically interrogated the effects of the AXL inhibitor, TP-0903, on EMT in NSCLC cells using transcriptomic and proteomic profiling. Methods: Atomic force microscopy, Western blot analysis, RNA sequencing, and mass cytometry (CyTOF) were used to evaluate the phenotypic, transcriptomic, and proteomic profiles of A549 cells treated with 40 nM TP-0903 or shAXL knockdown. A549 and H1650 NSCLC xenograft models were used to explore the consequences of AXL inhibition in vivo. Results: As expected, TP-0903 treatment attenuated AXL signaling and downstream phosphorylation in the A549 cells. Interestingly, the treatment also reduced gene expression responses to TGFβ-Hippo signaling by disrupting the transcriptional complexes formed by SMAD2/3, SMAD4, YAP1, and TAZ. Consistent with AXL inhibition, TP-0903 reversed the mesenchymal phenotype in A549 and H2009 cell lines and decreased their migratory potential in culture. The CyTOF analysis on TP-0903-treated cells identified resistant clones overexpressing TGFβ receptor II (TGFBR2) and TAZ proteins and displaying hybrid EMT phenotypes. TP-0903 was also active in suppressing A549 or H1650 tumor growth in vivo. Conclusions: We are the first to report the interplay between AXL and TGFβ-Hippo signaling axis. TP-0903 has excellent therapeutic promise in NSCLC and we speculate that TP-0903 can target mesenchymal transitional states in NSCLC, possibly through the inhibition of the AXL-TGFβ-Hippo signaling axis. Citation Format: Josephine A. Taverna, Chia-Nung Hung, Chun-Lin Lin, Pawel A. Osmulski, Maria E. Gaczynska, Chiou-Miin Wang, Nicholas D. Lucio, Meizhen Chen, Chih-Wei Chou, Alia Nazarullah, Shellye R. Lampkin, Lianquin Qiu, David J. Bearss, Steve Warner, Lars Mouritsen, Mark Wade, Daniel DeArmond, Ruben Mesa, Nameer Kirma, Tim H.-M. Huang. AXL Inhibitor TP-0903 attenuates TGFβ-Hippo signaling in lung adenocarcinoma cells [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr B29.

Proline- and arginine-rich peptide PR11 is an allosteric inhibitor of 20S proteasome. We modified its sequence inter alia by introducing HbYX, RYX, or RHbX C-terminal extensions (Hb, hydrophobic moiety; R, arginine; Y, tyrosine; X, any residue). Consequently, we were able to improve inhibitory potency or to convert inhibitors into strong activators: the former with an aromatic penultimate Hb residue and the latter with the HbYX motif. The PR peptide activator stimulated 20S proteasome in vitro to efficiently degrade protein substrates, such as α-synuclein and enolase, but also activated proteasome in cultured fibroblasts. The positive and negative PR modulators differently influenced the proteasome conformational dynamics and affected opening of the substrate entry pore. The resolved crystal structure showed PR inhibitor bound far from the active sites, at the proteasome outer face, in the pocket used by natural activators. Our studies indicate the opportunity to tune proteasome activity by allosteric regulators based on PR peptide scaffold.

Proteasome, the essential giant protease, is the hub of the ubiquitin-proteasome pathway critical for maintaining intracellular proteostasis. Inhibitors of the proteasome cause overload of cells with non-degraded protein debris and activate proapoptotic signaling. The effects are especially dramatic in fast metabolizing cancer cells; hence the proteasome inhibitors are used as anti-cancer drugs. There are two FDA-approved drugs directly blocking the proteasome activity, bortezomib and carfilzomib, and several others are in trials. So far, the high effectiveness of these drugs is limited to multiple myeloma and other hematological malignancies. Unfortunately, breast and other solid cancers are much more resistant to apoptosis triggered by attenuation of the ubiquitin-proteasome pathway than majority of blood cancers. The relatively low effectiveness of proteasome inhibitors has been noted even for cancers that have been identified in genome-wide screens as addicted to the proteasome activity, such as triple negative breast cancers. The source of the resistance, noted in cell culture, animal studies and clinical trials, remains unknown. Here we propose that, at least in part, the resistance can be tracked down to protein-protein interactions between the drug target, the proteasome, and a cytosolic heat shock protein, Hsp72 (inducible Hsp70). Indeed, high levels of Hsp72 have been observed in many cancers, including breast, and generally correlate with poor prognosis. The intracellular roles of the ubiquitous chaperone are undoubtedly very diverse, however we postulate a novel molecular action: direct protection of the proteasome from competitive inhibitors of its enzymatic activity. The hypothesis has been inspired by our recent work exploring the very effective ubiquitin-proteasome pathway in the naked mole rats. These small African rodents do not develop cancers and maintain unusually long lifespan and health-span. Interestingly, the proteasome in naked mole rats is exceptionally resistant to inhibition. We discovered that the resistance is bestowed by a cytosolic protein factor containing Hsp72 and Hsp40 chaperones (Biochim. Biophys. Acta-MBD 1842; 2060-2072; 2014). We partially purified the factor and tested its in vitro effectiveness with human proteasomes. The molecular mechanism beneficial for naked mole rats may be also protective for human cancer cells. We used pifithrin μ, a specific small molecule inhibitor blocking the interactions of Hsp72 with other proteins, to test the hypothesis. We prepared lysates from cultured triple negative breast cancer cells MDA-MB-231. Such lysates are rich in proteasome activity and preserve interactions of the proteasomes with natural protein ligands. Interestingly, upon the treatment with pifithrin μ proteasomes in lysates maintained their high activity while becoming significantly more sensitive to inhibition with bortezomib. The studies are in progress to assess the identity of a putative chaperone-based proteasome resistance factor in breast cancer cells. Citation Format: Gaczynska M, Osmulski PA. Are specific interactions of proteasome with Hsp72 responsible for resistance of triple negative breast cancer cells to proteasome inhibitors?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-12.

// Izhar Batth 1, 8 , Huiyoung Yun 2 , Suleman Hussain 2 , Peng Meng 1, 7 , Pawel Osmulski 3 , Tim Hui-Ming Huang 3, 5 , Roble Bedolla 1 , Amanda Profit 4 , Robert Reddick 4 , Addanki Kumar 1, 2, 3, 5, 6 1 Department of Urology, The University of Texas Health Science Center, San Antonio, TX, USA 2 Department of Pharmacology, The University of Texas Health Science Center, San Antonio, TX, USA 3 Department of Molecular Medicine, The University of Texas Health Science Center, San Antonio, TX, USA 4 Department of Pathology, The University of Texas Health Science Center, San Antonio, TX, USA 5 Cancer Therapy and Research Center, The University of Texas Health Science Center, San Antonio, TX, USA 6 The University of Texas Health Science Center at San Antonio and South Texas Veterans Health Care System, San Antonio, TX, USA 7 Current address: Life Sciences Division, Lawrence Berkley National Laboratory, Berkley, CA, USA 8 Current address: Department of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA Correspondence to: Addanki Kumar, e-mail: kumara3@uthscsa.edu Keywords: castrate resistant prostate cancer, apoptosis, FLIP, RON, MST1R Received: November 18, 2015 Accepted: January 29, 2016 Published: February 9, 2016 ABSTRACT Castrate-resistant prostate cancer (CRPC) is the fatal form of prostate cancer. Although reactivation of androgen receptor (AR) occurs following androgen deprivation, the precise mechanism involved is unclear. Here we show that the receptor tyrosine kinase, RON alters mechanical properties of cells to influence epithelial to mesenchymal transition and functions as a transcription factor to differentially regulate AR signaling. RON inhibits AR activation and subset of AR-regulated transcripts in androgen responsive LNCaP cells. However in C4-2B, a castrate-resistant sub-line of LNCaP and AR-negative androgen independent DU145 cells, RON activates subset of AR-regulated transcripts. Expression of AR in PC-3 cells leads to activation of RON under androgen deprivation but not under androgen proficient conditions implicating a role for RON in androgen independence. Consistently, RON expression is significantly elevated in castrate resistant prostate tumors. Taken together our results suggest that RON activation could aid in promoting androgen independence and that inhibition of RON in combination with AR antagonist(s) merits serious consideration as a therapeutic option during hormone deprivation therapy.

We applied Quantitative Nanomechanical Measurements with Atomic Force Microscopy (QNM-AFM) to test a response of triple negative breast cancer (TNBC) cells to treatment with inhibitors of proteasome. Genetic screens identified TNBCs as addicted to the activity of ubiquitin-proteasome pathway, the major intracellular venue of regulated protein degradation, served by the essential protease, the proteasome. Here we tested the effects of treatment of two proteasome inhibitors on the canonical triple-negative cell line, MDA-MB-231. The inhibitors represent two very distinct types of mechanism. Bortezomib (Velcade®) is a competitive inhibitor that targets active sites of the enzyme. On the other hand, B1 is a novel noncompetitive allosteric drug lead that interferes with interactions between the subcomplexes of proteasome, the 20S catalytic core and the 19S regulatory particle ("cap"). In a search for test to predict the putative value of inhibitors we turned to a mechanical phenotype. Mechanical properties of cells constitute a sensitive indicator of physiological status of cells. The best-known and most explored mechanical parameters are elasticity ("softness") represented by the Young modulus, and surface adhesiveness ("stickiness"). It has been well established that cancer cell are much softer and less sticky than the healthy counterparts. These changes in physical phenotype of cancer cells are usually linked to remodeling of their cytoskeleton and altered expression of membrane proteins. Such remodeling may be a very early indicator of the cells’ response to a drug, a less explored but potentially very useful feature. Indeed, it has been shown that often treatment of cancer cells with anticancer drugs at least partially reverses their mechanical phenotype resembling healthy cells. Here we found that 24-hrs exposure of the cells to 10 nM bortezomib increased their stiffness and adhesiveness about two times. Even more striking, a treatment of the cells with 10 nM B1 induced almost threefold increase of stiffness with twofold increase in adhesion. Strikingly, treatments with such concentrations of the inhibitors alone did not significantly influence the viability of the cells, whereas their combination reduced the population of live cells to 50% or less of the control values. The strong effects of the inhibitor treatment on the mechanical phenotype are in stark contrast with little or no effects on cell viability. The results point at extraordinary sensitivity of the mechanical phenotype in detection of cell response to anticancer drugs. We are exploring the potential predictive value of AFM-based cell surface studies in a search for effective drugs or drug combinations. Citation Format: Pawel A Osmulski, Maria Gaczynska. Atomic force microscopy of triple negative breast cancer cells: A predictive value of mechanical phenotype [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P3-06-51.

The ubiquitin-proteasome pathway (UPP) is a major venue for controlled intracellular protein degradation in Eukaryota. The machinery of several hundred proteins is involved in recognizing, tagging, transporting, and cleaving proteins, all in a highly regulated manner. Short-lived transcription factors, misfolded translation products, stress-damaged polypeptides, or worn-out long-lived proteins, all can be found among the substrates of UPP. Carefully choreographed protein-protein interactions (PPI) are involved in each step of the pathway. For many of the steps small-molecule inhibitors have been identified and often they directly or indirectly target PPI. The inhibitors may destabilize intracellular proteostasis and trigger apoptosis. So far this is the most explored option used as an anticancer strategy. Alternatively, substrate-specific polyubiquitination may be regulated for a precise intervention aimed at a particular metabolic pathway. This very attractive opportunity is moving close to clinical application. The best known drug target in UPP is the proteasome: the end point of the journey of a protein destined for degradation. The proteasome alone is a perfect object to study the mechanisms and roles of PPI on many levels. This giant protease is built from multisubunit modules and additionally utilizes a service from transient protein ligands, for example, delivering substrates. An elaborate set of PPI within the highest-order proteasome assembly is involved in substrate recognition and processing. Below we will outline PPI involved in the UPP and discuss the growing prospects for their utilization in pharmacological interventions.

The ubiquitin-proteasome pathway (UPP) is a major venue for controlled intracellular protein degradation in Eukaryota. The machinery of several hundred proteins is involved in recognizing, tagging, transporting, and cleaving proteins, all in a highly regulated manner. Short-lived transcription factors, misfolded translation products, stress-damaged polypeptides, or worn-out long-lived proteins, all can be found among the substrates of UPP. Carefully choreographed protein-protein interactions (PPI) are involved in each step of the pathway. For many of the steps small-molecule inhibitors have been identified and often they directly or indirectly target PPI. The inhibitors may destabilize intracellular proteostasis and trigger apoptosis. So far this is the most explored option used as an anticancer strategy. Alternatively, substrate-specific polyubiquitination may be regulated for a precise intervention aimed at a particular metabolic pathway. This very attractive opportunity is moving close to clinical application. The best known drug target in UPP is the proteasome: the end point of the journey of a protein destined for degradation. The proteasome alone is a perfect object to study the mechanisms and roles of PPI on many levels. This giant protease is built from multisubunit modules and additionally utilizes a service from transient protein ligands, for example, delivering substrates. An elaborate set of PPI within the highest-order proteasome assembly is involved in substrate recognition and processing. Below we will outline PPI involved in the UPP and discuss the growing prospects for their utilization in pharmacological interventions.

Cholesterol is an important risk factor of atherosclerosis, due to its active uptake by monocytes/macrophages. Monocyte recruitment from flowing blood to atherosclerotic foci is the key first step in the development of atherosclerosis. Cholesterol content alters cell membrane stiffness, and lateral lipid and protein diffusion. We hypothesized that cholesterol content will modulate the recruitment of monocytes to inflamed endothelial surface by altering the dynamics of adhesion receptors. We depleted or enriched the cellular cholesterol levels using methyl-β-cyclodextran in freshly isolated human monocytes. We investigated the effect of these changes on the mechanics of monocyte rolling on E-selectin surfaces at 1 dyn/cm2 in microchannels. Using imaging flow cytometry and atomic force microscopy, we characterized the distribution of lipid rafts and the E-selectin counterreceptor CD44 on the monocyte surface. We observed that lower levels of cholesterol resulted in the uniform, CD44-mediated rolling of monocytes on the E-selectin-coated surfaces. We also observed that cells depleted of cholesterol had higher membrane fluidity, and more uniform distribution of CD44 counterreceptor, which resulted in smooth motion of the cells compared to cells enriched with cholesterol. This work demonstrates that cholesterol can modulate monocyte adhesion by regulating the receptor mobility, and our results provide insights into the biophysical regulation of inflammation for the better understanding of diseases like atherosclerosis and hypercholesterolemia.

Significance: The proteasome is the essential protease that is responsible for regulated cleavage of the bulk of intracellular proteins. Its central role in cellular physiology has been exploited in therapies against aggressive cancers where proteasome-specific competitive inhibitors that block proteasome active centers are very effectively used. However, drugs regulating this essential protease are likely to have broader clinical usefulness. The non-catalytic sites of the proteasome emerge as an attractive alternative target in search of highly specific and diverse proteasome regulators. Recent Advances: Crystallographic models of the proteasome leave the false impression of fixed structures with minimal molecular dynamics lacking long-distance allosteric signaling. However, accumulating biochemical and structural observations strongly support the notion that the proteasome is regulated by precise allosteric interactions arising from protein dynamics, encouraging the active search for allosteric regulators. Here, we discuss properties of several promising compounds that affect substrate gating and processing in antechambers, and interactions of the catalytic core with regulatory proteins. Critical Issues: Given the structural complexity of proteasome assemblies, it is a painstaking process to better understand their allosteric regulation and molecular dynamics. Here, we discuss the challenges and achievements in this field. We place special emphasis on the role of atomic force microscopy imaging in probing the allostery and dynamics of the proteasome, and in dissecting the mechanisms involving small-molecule allosteric regulators. Future Directions: New small-molecule allosteric regulators may become a next generation of drugs targeting the proteasome, which is critical to the development of new therapies in cancers and other diseases. Antioxid. Redox Signal. 21, 2286–2301.

The naked mole-rat maintains robust proteostasis and high levels of proteasome-mediated proteolysis for most of its exceptional (~31years) life span. Here, we report that the highly active proteasome from the naked mole-rat liver resists attenuation by a diverse suite of proteasome-specific small molecule inhibitors. Moreover, mouse, human, and yeast proteasomes exposed to the proteasome-depleted, naked mole-rat cytosolic fractions, recapitulate the observed inhibition resistance, and mammalian proteasomes also show increased activity. Gel filtration coupled with mass spectrometry and atomic force microscopy indicates that these traits are supported by a protein factor that resides in the cytosol. This factor interacts with the proteasome and modulates its activity. Although Heat shock protein 72 kDa (HSP72) and Heat shock protein 40 kDa (Homolog of bacterial DNAJ1) (HSP40(Hdj1)) are among the constituents of this factor, the observed phenomenon, such as increasing peptidase activity and protecting against inhibition cannot be reconciled with any known chaperone functions. This novel function may contribute to the exceptional protein homeostasis in the naked mole-rat and allow it to successfully defy aging.

BACKGROUND. Emerging evidence shows that nanomechanical phenotypes of circulating tumor cells (CTC) could become potential biomarkers for metastatic castration resistant prostate cancer (mCRPC). METHODS. To determine the nanomechanical phenotypes of CTCs we applied atomic force microscopy (AFM) employing the PeakForce quantitative nanomechanical (QNM) imaging. We assessed biophysical parameters (elasticity, deformation, and adhesion) of 130 CTCs isolated from blood samples from five castration sensitive (CS) and 12 castration resistant prostate cancer (CRPCa) patients. RESULTS. We found that CTCs from CRPCa patients are three times softer, three times more deformable, and seven times more adhesive than counterparts from CSPCa patients. Both nonsupervised hierarchical clustering and principle component analysis show that three combined nanomechanical parameters could constitute a valuable set to distinguish between CSPCa and CRPCa. CONLUSIONS. Our study indicates that nanomechanical phenotypes of CTCs may serve as novel and effective biomarkers for mCRPC. Prostate

Proteasome is a 'proteolytic factory' that constitutes an essential part of the ubiquitin-proteasome pathway. The involvement of proteasome in regulation of all major aspects of cellular physiology makes it an attractive drug target. So far, only inhibitors of the proteasome entered the clinic as anti-cancer drugs. However, proteasome regulators may also be useful for treatment of inflammatory and neurodegenerative diseases. We established in our previous studies that the peptide Tat2, comprising the basic domain of HIV-1 Tat protein: R(49) KKRRQRR(56) , supplemented with Q(66) DPI(69) fragment, inhibits the 20S proteasome in a noncompetitive manner. Mechanism of Tat2 likely involves allosteric regulation because it competes with the proteasome natural 11S activator for binding to the enzyme noncatalytic subunits. In this study, we performed alanine walking coupled with biological activity measurements and FTIR and CD spectroscopy to dissect contribution of a charge and conformation of Tat2 to its capability to influence peptidase activity of the proteasome. In solution, Tat2 and most of its analogs with a single Ala substitution preferentially adopted a conformation containing PPII/turn structural motifs. Replacing either Asp10 or two or more adjacent Arg/Lys residues induced a random coil conformation, probably by disrupting ionic interactions responsible for stabilization of the peptides ordered structure. The random coil Tat2 analogs lost their capability to activate the latent 20S proteasome. In contrast, inhibitory properties of the peptides more significantly depended on their positive charge. The data provide valuable clues for the future optimization of the Tat2-based proteasome regulators.

Purpose: Little is known about adaptive selection of tumor cells transiting from in situ proliferation to distant colonization through blood circulation. This study used genomic, transcriptomic, and biophysical analyses at single-cell levels to explore biological and physical properties of circulating tumor cells (CTCs) undergoing hemodynamic stress. The aims are trying to understand how CTCs strive to survive in the bloodstream and to develop strategies for therapeutic intervention. Experimental Design: We compared genomic profiles of CTCs in blood and primary tumor cells shed in urine of prostate cancer patients to identify amplified regions preferentially retained in CTCs. Single-cell RNA-seq was used to confirm amplification-associated overexpression of genes in CTCs. Results: Among 261 recurrently amplified genomic regions in the analysis, 70 were found predominantly shown in CTCs relative to primary tumor cells. In line with the results at the genomic level, the transcriptomic data of CTCs demonstrate a great amount of cells showing high expression levels in the oxidative phosphorylation (OXPHOS) pathway compared to other hallmark gene sets. The finding suggests that tumor cells with these pre-existing genomic alterations were adaptively selected for transcription reprogramming during blood circulation. Specifically, amplified genes associated with OXPHOS were exploited by CTCs for alternative fuels. As to the upstream of this transcription event, we found the expression of MEN1, encoding menin known to form a transcription factor complex with the mixed-lineage leukemia protein (MLL) in prostate cancer cells, was positively correlated with 11 of the 14 OXPHOS loci in the Cancer Genomic Atlas prostate cancer cohort. In vitro assay showed that MEN1 knockdown by shRNA resulted in attenuation of both mRNA and protein expression of OXPHOS loci in PC-3 cells. Taken together pre-existing amplification of OXPHOS loci can be used as a transcription apparatus by menin for metabolic reprogramming of tumor cells in response to harsh microenvironments in the bloodstream. Conclusions: Single-cell profiling identified signaling pathways that are crucial for CTCs to survive in the bloodstream. The finding suggests that a metabolic shift from Warburg to OXPHOS metabolism can be associated with a hybrid mesenchymal-epithelial phenotype of CTCs. Moreover, the study demonstrates the feasibility of routinely conducting single-cell analysis of exfoliated tumor cells for minimally invasive monitoring of disease progression and treatment response in prostate cancer patients Citation Format: Chun-Lin Lin, Tan Xi, Chia-Nung Hung, Pawel A. Osmulski, Chih-Wei Chou, Meizhen Chen, Chiou-Miin Wang, Kohzoh Mitsuya, Nameer Kirma, Maria E. Gaczynska, Chun-Liang Chen, Tim H.-M. Huang. Amplification-associated upregulation of genes involved in oxidative phosphorylation for disseminated prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2766.

Background: Non-small cell lung cancer (NSCLC) is a molecularly heterogeneous disease with a high propensity for drug resistance and metastasis. AXL, a member of the Tyro3-AXL-Mer family of receptor tyrosine kinases, is a central regulator of epithelial-to-mesenchymal transition (EMT) and enables tumor cells to invade and acquire drug resistance. AXL is overexpressed in lung tumors, correlates positively with tumor invasion, drug resistance, and negatively predicts overall survival. We mechanistically interrogate the effects of AXL inhibitor TP-0903 on EMT in lung adenocarcinoma cells using transcriptomic and proteomic profiling. Methods: Atomic force microscopy, Western blot analysis, RNA sequencing and mass cytometry (CyTOF) were all used to scrutinize the biomechanical properties, phenotypic, transcriptomic and proteomic profiles of A549 cells treated with 40nM TP0903 or shAXL knockdown. Results: TP-0903 attenuates total AXL/AXL phosphorylation and blunts transcriptional responses to TGFβ-Hippo signaling by disrupting the transcriptional complexes formed by SMAD2/3, SMAD4, YAP1 and TAZ. AXL knockdown or TP-0903 reverses EMT phenotype and reduces migration potential in A549 and H2009 adenocarcinoma cell lines. CyTOF data also identified resistant clones that overexpress TGFβ receptor II, TAZ protein and display hybrid EMT phenotypes. Conclusions: We are the first to report the interplay between AXL and TGFβ-Hippo signaling axis. TP-0903 study agent has excellent therapeutic promise in NSCLC and we speculate that TP-0903 drug can target epithelial to mesenchymal transitional states in lung cancer cells possibly through the inhibition of the AXL-TGFβ-Hippo signaling axis. Citation Format: Josephine Amalia Taverna, Chia-Nung Hung, Chun-Lin Lin, Pawel Osmulski, Meizhen Chen, Chiou-Miin Wang, Nicholas L. L. Lucio, Nameer Kirma, Chih-Wei Chou, Maria E. Gaczynska, Alia Nazarullah, Mark Wade, Lars Mouritsen, Tim Huang. AXL inhibitor TP-0903 attenuates AXL-TGFbeta Hippo signaling axis in lung adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2193.

Endometrial cancer is the most common gynecologic cancer. It is divided into two major types, a less aggressive endometrioid type and a more aggressive serous type. Recent clinical observations underscore obesity as a major risk factor for endometrial cancer. In this study, we hypothesized that obesity can result in long term epigenetic modifications in the endometrial compartment, leading to the promotion of endometrial cancer. We examined the role of adipose-derived stem cells (ASCs), which are adipocyte progenitors, in the endometrial tumor microenvironment. By staining for ASCs in a tumor tissue microarray, we observed ASC infiltration that was specific to samples from obese patients, correlating with body mass index (R2=0.77, p Citation Format: Li-Ling Lin, Srikanth Polusani, Guangcun Huang, Chun-Lin Lin, Chiou-Miin Wang, Nicholas Lucio, Mikhail Kolonin, Alexes Daquinag, Pawel Osmulski, Bruce Nicholson, Edward Kost, Tim Huang, Nameer B. Kirma. Adipose-derived stem cell disruption of gap junction intercellular communication in obesity-associated endometrial cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5150.

Tumors express intracellular PD-L1 that we showed increases ovarian cancer (OC) and melanoma virulence by increasing mTORC1 and tumor stem cells (TSC). We made PD-L1KO(CRISPR) in ES2 human OC and B16 melanoma cells and re-expressed PD-L1 only on cell surface (PD-L1surf) or in cytoplasm (PD-L1cyto) with specific expression vectors. Subcellular PD-L1 location in all clones was validated by immunoblots, confocal microscopy, and co-IP/mass spec. We selected clones with PD-L1 surf or cyto expression similar to parental cells. PD-L1KO reduced cell diameter 30% which was restored partially in PD-L1surf and fully in PD-L1cyto in ES2 and B16. PD-L1KO significantly decreased viability in all cell lines tested which was fully rescued in PD-L1surf and PD-L1cyto. PD-L1surf ES2 and B16 grew slower in vitro and in vivo than PD-L1cyto and only PD-L1cyto rescued F-actin polymerization phenotype seen in PD-L1KO ES2 and B16. RNA-seq showed that surf and cyto PD-L1 differentially altered canonical signaling pathways, notably mTOR, stemness and gene regulation. PD-L1cyto grew much slower than PD-L1surf B16 in NSG mice but generated more TSC in vitro, and PD-L1surf but not PD-L1cyto B16 responded to αPD-L1 in WT. PD-L1cyto but not PD-L1surf induced mTORC1 activation (p-rpS6, p-P70S6K) through mTORC1 lysosomal docking through LAMTOR1/2 seen by confocal and confirmed by co-IP in ES2 and B16. Imaging CyTOF of human melanomas showed PD-L1cyto association with activated mTOR. Thus, surf versus cyto PD-L1 differentially affects tumor biology, signaling, pathology, TIL and treatment response in OC and melanoma. We define a mechanism for PD-L1-driven mTORC1 activation. Surface vs. cytoplasmic PD-L1 and/or mTORC1 could be response biomarkers for αPD-L1 or αPD-1.

Proteasome is an essential protease of the proteasome-ubiquitin pathway, responsible for maintaining intracellular proteostasis. It is a multifunctional, multisubunit enzyme built from interchangeable modules and intricately regulated on the molecular and cellular levels. Proteasome inhibitors display proapoptotic properties, preferentially aimed at cancerous cells. Consequently, targeted therapy with bortezomib, the competitive inhibitor of the proteasome, has been very successful with refractory/relapsed multiple myeloma and with lymphomas. Unfortunately, breast cancers performed disappointingly in clinical trials with bortezomib. We propose that a new generation of small-molecule proteasome inhibitors distinct from bortezomib may prove successful with breast cancers. In contrast to bortezomib which directly blocks proteasome active sites, the new inhibitors are allosteric ligands binding to noncatalytic sites and disrupting the enzyme's function by targeting long-distance transfer of structural signals within the enzyme's molecule. The advantages of allosteric ligands, now rapidly gaining popularity in targeted drug design, include versatility of molecular actions, defying drug resistance mechanisms, and usually high specificity with low toxicity. For example, one of the small-molecule allosteric ligands we created compromises interactions between the catalytic core proteasome and its regulatory sub-assembly 19S (“cap”, regulatory particle) responsible for recognizing and processing the majority of intracellular protein substrates tagged for degradation by polyubiquitin. We found that treatment with nanomolar concentration of the compound induced cell death in Mcf7 human breast cancer cultured cells. Interestingly, this cell line is known for its resistance to bortezomib treatment. Even more, treatment with the allosteric ligand together with bortezomib produced a synergistic effect and sensitized the Mcf7 cells to the actions of competitive inhibitor. Remarkable, the cytotoxic effects of allosteric ligand or its combination with bortezomib were not observed with the noncancerous human breast cell line, Mcf10A. Summarizing, allosteric regulation of the proteasome by small-molecule ligands, alone or in combination with established drugs competitively inhibiting proteasome, seem to be a promising strategy to utilize proteasome as a drug target in breast cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-17.

// Guangcun Huang 1 , Pawel A. Osmulski 1 , Hakim Bouamar 2 , Devalingam Mahalingam 3 , Chun-Lin Lin 1 , Michael A. Liss 4 , Addanki Pratap Kumar 4, 5 , Chun-Liang Chen 1 , Ian M. Thompson 4 , Lu-Zhe Sun 2 , Maria E. Gaczynska 1 , Tim H.-M. Huang 1 1 Departments of Molecular Medicine Cancer Research and Therapy Center and School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA 2 Departments of Cellular and Structural Biology Cancer Research and Therapy Center and School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA 3 Departments of Medicine Cancer Research and Therapy Center and School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA 4 Departments of Urology Cancer Research and Therapy Center and School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA 5 Departments of Radiation Oncology Cancer Research and Therapy Center and School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA Correspondence to: Tim H.-M. Huang, email: huangt3@uthscsa.edu Maria E. Gaczynska, email: gaczynska@uthscsa.edu Keywords: tumor metastasis, circulating tumor cells, epithelial-mesenchymal transition (EMT), transforming growth factor-β (TGF-β), positive feedback signaling Received: August 08, 2016 Accepted: October 12, 2016 Published: October 21, 2016 ABSTRACT Activation of TGF-β signaling is known to promote epithelial-mesenchymal transition (EMT) for the development of metastatic castration-resistant prostate cancer (mCRPC). To determine whether targeting TGF-β signaling alone is sufficient to mitigate mCRPC, we used the CRISPR/Cas9 genome-editing approach to generate a dominant-negative mutation of the cognate receptor TGFBRII that attenuated TGF-β signaling in mCRPC cells. As a result, the delicate balance of oncogenic homeostasis is perturbed, profoundly uncoupling proliferative and metastatic potential of TGFBRII -edited tumor xenografts. This signaling disturbance triggered feedback rewiring by enhancing ERK signaling known to promote EMT-driven metastasis. Circulating tumor cells displaying upregulated EMT genes had elevated biophysical deformity and an increase in interactions with chaperone macrophages for facilitating metastatic extravasation. Treatment with an ERK inhibitor resulted in decreased aggressive features of CRPC cells in vitro . Therefore, combined targeting of TGF-β and its backup partner ERK represents an attractive strategy for treating mCRPC patients.

Overexpression of epithelial cell adhesion molecule (EpCAM) has been implicated in advanced endometrial cancer, but its roles in this progression remain to be elucidated. In addition to its structural role in modulating cell-surface adhesion, here we demonstrate that EpCAM is a regulatory molecule in which its internalization into the nucleus turns on a transcription program. Activation of EGF/EGFR signal transduction triggered cell-surface cleavage of EpCAM, leading to nuclear internalization of its cytoplasmic domain EpICD. ChIP-seq analysis identified target genes that are coregulated by EpICD and its transcription partner, LEF-1. Network enrichment analysis further uncovered a group of 105 genes encoding functions for tight junction, adherent, and cell migration. Furthermore, nanomechanical analysis by atomic force microscopy revealed increased softness and decreased adhesiveness of EGF-stimulated cancer cells, implicating acquisition of an epithelial–mesenchymal transition (EMT) phenotype. Thus, genome editing of EpCAM could be associated with altering these nanomechanical properties towards a less aggressive phenotype. Using this integrative genomic–biophysical approach, we demonstrate for the first time an intricate relationship between EpCAM-regulated transcription and altered biophysical properties of cells that promote EMT in advanced endometrial cancer. Cancer Res; 76(21); 6171–82. ©2016 AACR.

The ubiquitin-proteasome pathway plays a crucial role in regulation of intracellular protein turnover. Proteasome, the central protease of the pathway, encompasses multi-subunit assemblies sharing a common catalytic core supplemented by regulatory modules and localizing to different subcellular compartments. To better comprehend age-related functions of the proteasome we surveyed content, composition and catalytic properties of the enzyme in cytosolic, microsomal and nuclear fractions obtained from mouse livers subjected to organismal aging. We found that during aging subunit composition and subcellular distribution of proteasomes changed without substantial alterations in the total level of core complexes. We observed that the general decline in proteasomes functions was limited to nuclear and cytosolic compartments. Surprisingly, the observed changes in activity and specificity were linked to the amount of the activator module and distinct composition of the catalytic subunits. In contrast, activity, specificity and composition of the microsomal-associated proteasomes remained mostly unaffected by aging; however their relative contribution to the total activity was substantially elevated. Unexpectedly, the nuclear proteasomes were affected most profoundly by aging possibly triggering significant changes in cellular signaling and transcription. Collectively, the data indicate an age-related refocusing of proteasome from the compartment-specific functions towards general protein maintenance.

The 26S proteasome is a multisubunit enzyme composed of a cylindrical catalytic core (20S) and a regulatory particle (19S) that together perform the essential degradation of cellular proteins tagged by ubiquitin. To date, however, substrate trajectory within the complex remains elusive. Here we describe a previously unknown functional unit within the 19S, comprising two subunits, Rpn1 and Rpn2. These toroids physically link the site of substrate recruitment with the site of proteolysis. Rpn2 interfaces with the 20S, whereas Rpn1 sits atop Rpn2, serving as a docking site for a substrate-recruitment factor. The 19S ATPases encircle the Rpn1-Rpn2 stack, covering the remainder of the 20S surface. Both Rpn1-Rpn2 and the ATPases are required for substrate translocation and gating of the proteolytic channel. Similar pairing of units is found in unfoldases and nuclear transporters, exposing common features of these protein nanomachines.

Proteasome is the essential multicatalytic protease from the ubiquitin-proteasome pathway, responsible for the regulated degradation of the bulk of intracellular proteins. Cancer cells are more susceptible than healthy cells to blocking of the proteasome catalytic activity. Consequently, proteasome inhibitors are used as successful drugs against blood cancers. Still, many patients do not respond to the treatment or develop resistance. Since all the currently used proteasome-targeting drugs are competitive inhibitors blocking the active sites, we turned our attention to novel noncompetitive regulators affecting protein-protein interactions (PPI), especially attractive for the multisubunit and modular enzyme like the proteasome. Testing effects of competitive inhibitors requires only monitoring of peptidase activity of the proteasome catalytic core. The PPI-targeting compounds often perform disappointingly weak in such tests. Here we show that biophysical methods provide straightforward tools to follow the impact of PPI disruption on proteasome. We assessed small-molecule compounds that distinctly affect assembly and activity of the proteasome: (i) analogues of the rapamycin binding domain, (ii) Tat peptide mimetics modeled after the proteasome-binding domain of HIV-1Tat protein, (iii) derivatives of Pro and Arg-rich fragment of cathelicidin PR39, and (iv) TCH imidazoline derivatives. Probing topography of the catalytic core with AFM identified the gate status and alpha face dynamics as an excellent sensor of a type of the compounds proteasome interactions. Intrinsic fluorescence of tryptophans revealed divergent communication pathways affected by the compounds. AFM based analysis of proteasomal super-assemblies demonstrated distinct modes of compounds-induced disruption of PPIs. Finally, Peak Force QNM AFM demonstrated profound changes in cell elasticity, adhesiveness and roughness of cultured cells treated with the compounds. Based on these findings we attempted to build a model that can help predict the effects of small molecules on proteasome and cell physiology.

The proteasome is a giant protease responsible for degradation of the majority of cytosolic proteins. Competitive inhibitors of the proteasome are used against aggressive blood cancers. However, broadening the use of proteasome-targeting drugs requires new mechanistic approaches to the enzyme’s inhibition. In our previous studies we described Tat1 peptide, an allosteric inhibitor of the proteasome derived from a fragment of the basic domain of HIV-Tat1 protein. Here, we attempted to dissect the structural determinants of the proteasome inhibition by Tat1. Single- and multiple- alanine walking scans were performed. Tat1 analogs with stabilized beta-turn conformation at positions 4–5 and 8–9, pointed out by the molecular dynamics modeling and the alanine scan, were synthesized. Structure of Tat1 analogs were analyzed by circular dichroism, Fourier transform infrared and nuclear magnetic resonance spectroscopy studies, supplemented by molecular dynamics simulations. Biological activity tests and structural studies revealed that high flexibility and exposed positive charge are hallmarks of Tat1 peptide. Interestingly, stabilization of a beta-turn at the 8–9 position was necessary to significantly improve the inhibitory potency.

Protein-protein interactions (PPI) are at the center of molecular mechanisms of life. The protein ligands convene for regulation of biological function: adding, enhancing or inhibiting activity, for assistance in structural integrity or to enable subsequent PPI. All these general roles of PPI are represented in the proteasome, the giant proteolytic factory universally present in human cells. The proteasome is a renowned target for anti-cancer drugs and a considered target for drugs curbing inflammation. The essential function of the proteasome, the degradation of a majority of intracellular proteins via the ubiquitin-proteasome pathway, relies on proper interactions between multiple subunits of the enzyme and between multiple modules forming distinct super-assemblies covered by the "proteasome" name. The interface regions between constitutive, alternative or transient protein components of the proteasome provide a rich platform for design of drugs with potentially very diverse actions. Still, the resource remains largely untapped since all proteasome-targeting drugs used so far in humans are classical competitive inhibitors blocking catalytic centers. In this review, we will discuss the opportunities and challenges of targeting PPI in the hub enzyme for intracellular protein catabolism, the proteasome.