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Deficit of oxygen and nutrients in the tumor microenvironment (TME) triggers abnormal angiogenesis that produces dysfunctional and leaky blood vessels, which fail to adequately perfuse tumor tissues. Resulting hypoxia, exacerbation of metabolic disturbances, and generation of an immunosuppressive TME undermine the efficacy of anticancer therapies. Use of carefully scheduled angiogenesis inhibitors has been suggested to overcome these problems and normalize the TME. Here, we propose an alternative agonist-based normalization approach using a derivative of the C-type natriuretic peptide (dCNP). Multiple gene expression signatures in tumor tissues were affected in mice treated with dCNP. In several mouse orthotopic and subcutaneous solid tumor models including colon and pancreatic adenocarcinomas, this well-tolerated agent stimulated formation of highly functional tumor blood vessels to reduce hypoxia. Administration of dCNP also inhibited stromagenesis and remodeling of the extracellular matrix and decreased tumor interstitial fluid pressure. In addition, treatment with dCNP reinvigorated the antitumor immune responses. Administration of dCNP decelerated growth of primary mouse tumors and suppressed their metastases. Moreover, inclusion of dCNP into the chemo-, radio-, or immune-therapeutic regimens increased their efficacy against solid tumors in immunocompetent mice. These results demonstrate the proof of principle for using vasculature normalizing agonists to improve therapies against solid tumors and characterize dCNP as the first in class among such agents.

Rapidly proliferating cancer cells require a microenvironment where essential metabolic nutrients like glucose, oxygen, and growth factors become scarce as the tumor volume surpasses the established vascular capacity of the tissue. Limits in nutrient availability typically trigger growth arrest and/or apoptosis to prevent cellular expansion. However, tumor cells frequently co-opt cellular survival pathways thereby favoring cell survival under this environmental stress. The unfolded protein response (UPR) pathway is typically engaged by tumor cells to favor adaptation to stress. PERK, an endoplasmic reticulum (ER) protein kinase and UPR effector is activated in tumor cells and contributes tumor cell adaptation by limiting protein translation and balancing redox stress. PERK also induces miRNAs that contribute to tumor adaptation. miR-211 and miR-216b were previously identified as PERK-ATF4-regulated miRNAs that regulate cell survival. We have identified another PERK-responsive miRNA, miR-217, with increased expression under prolonged ER stress. Key targets of miR-217 are identified as TRPM1, the host gene for miR-211 and EZH2. Evidence is provided that miR-217 expression is essential for the rapid loss of miR-211 in prolonged ER stress and provides a functional link for determining whether cells adapt to stress or commit to apoptosis., Implications: PERK-dependent induction of miR-217 limits accumulation and function of the prosurvival miRNA, miR-211, to establish cell fate and promote cell commitment to apoptosis., (©2024 American Association for Cancer Research.)

Since 2019, COVID-19 has triggered a renewed investigation of the urban environment and disease outbreak. While the results have been inconsistent, it has been observed that the quantity of urban green spaces (UGS) is correlated with the risk of COVID-19. However, the spatial pattern has largely been ignored, especially on the community scale. In high-density communities where it is difficult to increase UGS quantity, UGS spatial pattern could be a crucial predictive variable. Thus, this study investigated the relative contribution of quantity and spatial patterns of UGS on COVID-19 risk at the community scale using a random forest (RF) regression model based on (n = 44) communities in Wuhan. Findings suggested that 8 UGS indicators can explain 35% of the risk of COVID-19, and the four spatial pattern metrics that contributed most were core, edge, loop, and branch whereas UGS quantity contributed least. The potential mechanisms between UGS and COVID-19 are discussed, including the influence of UGS on residents' social distance and environmental factors in the community. This study offers a new perspective on optimizing UGS for public health and sustainable city design to combat pandemics and inspire future research on the specific relationship between UGS spatial patterns and pandemics and therefore help establish mechanisms of UGS and pandemics., Competing Interests: The authors declare no conflict of interest.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Cancer-associated fibroblasts (CAF) can promote tumor growth, metastasis, and therapeutic resistance in esophageal squamous cell carcinoma (ESCC), but the mechanisms of action remain elusive. Our objective was to identify secreted factor(s) that mediate the communication between CAFs and ESCC tumor cells with the aim of identifying potential druggable targets. Through unbiased cytokine arrays, we have identified CC motif chemokine ligand 5 (CCL5) as a secreted factor that is increased upon co-culture of ESCC cells and CAFs, which we replicated in esophageal adenocarcinoma (EAC) with CAFs. Loss of tumor-cell-derived CCL5 reduces ESCC cell proliferation in vitro and in vivo and we propose this is mediated, in part, by a reduction in ERK1/2 signaling. Loss of tumor-derived CCL5 reduces the percentage of CAFs recruited to xenograft tumors in vivo. CCL5 is a ligand for the CC motif receptor 5 (CCR5), for which a clinically approved inhibitor exists, namely Maraviroc. Maraviroc treatment reduced tumor volume, CAF recruitment, and ERK1/2 signaling in vivo, thus, mimicking the effects observed with genetic loss of CCL5. High CCL5 or CCR5 expression is associated with worse prognosis in low-grade esophageal carcinomas., Implications: These data highlight the role of CCL5 in tumorigenesis and the therapeutic potential of targeting the CCL5-CCR5 axis in ESCC., (©2023 American Association for Cancer Research.)

Immune suppressive factors of the tumor microenvironment (TME) undermine viability and exhaust the activities of the intratumoral cytotoxic CD8 + T lymphocytes (CTL) thereby evading anti-tumor immunity and decreasing the benefits of immune therapies. To counteract this suppression and improve the efficacy of therapeutic regimens, it is important to identify and understand the critical regulators within CD8 + T cells that respond to TME stress and tumor-derived factors. Here we investigated the regulation and importance of activating transcription factor-4 (ATF4) in CTL using a novel Atf4 ΔCD8 mouse model lacking ATF4 specifically in CD8 + cells. Induction of ATF4 in CD8 + T cells occurred in response to antigenic stimulation and was further increased by exposure to tumor-derived factors and TME conditions. Under these conditions, ATF4 played a critical role in the maintenance of survival and activities of CD8 + T cells. Conversely, selective ablation of ATF4 in CD8 + T cells in mice rendered these Atf4 ΔCD8 hosts prone to accelerated growth of implanted tumors. Intratumoral ATF4-deficient CD8 + T cells were under-represented compared to wild-type counterparts and exhibited impaired activation and increased apoptosis. These findings identify ATF4 as an important regulator of viability and activity of CD8 + T cells in the TME and argue for caution in using agents that could undermine these functions of ATF4 for anti-cancer therapies., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Lung squamous cell carcinoma (LUSC) represents a major subtype of lung cancer with limited treatment options. KMT2D is one of the most frequently mutated genes in LUSC (>20%), and yet its role in LUSC oncogenesis remains unknown. Here, we identify KMT2D as a key regulator of LUSC tumorigenesis wherein Kmt2d deletion transforms lung basal cell organoids to LUSC. Kmt2d loss increases activation of receptor tyrosine kinases (RTKs), EGFR and ERBB2, partly through reprogramming the chromatin landscape to repress the expression of protein tyrosine phosphatases. These events provoke a robust elevation in the oncogenic RTK-RAS signaling. Combining SHP2 inhibitor SHP099 and pan-ERBB inhibitor afatinib inhibits lung tumor growth in Kmt2d-deficient LUSC murine models and in patient-derived xenografts (PDXs) harboring KMT2D mutations. Our study identifies KMT2D as a pivotal epigenetic modulator for LUSC oncogenesis and suggests that KMT2D loss renders LUSC therapeutically vulnerable to RTK-RAS inhibition., Competing Interests: Declaration of interests K.-K.W. is a founder and equity holder of G1 Therapeutics and has sponsored research agreements with Takeda, TargImmune, Bristol-Myers Squibb (BMS), Mirati, Merus, and Alkermes and consulting and sponsored research agreements with AstraZeneca, Janssen, Pfizer, Novartis, Merck, Zentalis, BridgeBio, and Blueprint. A.J.B. has received funding from Bayer, Novartis, Merck, and Repare and is a co-founder with equity in Signet Therapeutics. Y.P., H.Han., H.Z., and K.-K.W. have ownership interest in a patent application., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Overexpression of c-myc via increased transcription or decreased protein degradation is common to many cancer etiologies. c-myc protein degradation is mediated by ubiquitin-dependent degradation, and this ubiquitylation is regulated by several E3 ligases. The primary regulator is Fbxw7, which binds to a phospho-degron within c-myc. Here, we identify a new E3 ligase for c-myc, Fbxl8 (F-box and Leucine Rich Repeat Protein 8), as an adaptor component of the SCF (Skp1-Cullin1-F-box protein) ubiquitin ligase complex, for selective c-myc degradation. SCF Fbxl8 binds and ubiquitylates c-myc, independent of phosphorylation, revealing that it regulates a pool of c-myc distinct from SCF Fbxw7 . Loss of Fbxl8 increases c-myc protein levels, protein stability, and cell division, while overexpression of Fbxl8 reduces c-myc protein levels. Concurrent loss of Fbxl8 and Fbxw7 triggers a robust increase in c-myc protein levels consistent with targeting distinct pools of c-myc. This work highlights new mechanisms regulating c-myc degradation.

Heterogeneous Nuclear Ribonucleoprotein K (hnRNPK) is a multifunctional RNA binding protein (RBP) localized in the nucleus and the cytoplasm. Abnormal cytoplasmic enrichment observed in solid tumors often correlates with poor clinical outcome. The mechanism of cytoplasmic redistribution and ensuing functional role of cytoplasmic hnRNPK remain unclear. Here we demonstrate that the SCF Fbxo4 E3 ubiquitin ligase restricts the pro-oncogenic activity of hnRNPK via K63 linked polyubiquitylation, thus limiting its ability to bind target mRNA. We identify SCF Fbxo4 -hnRNPK responsive mRNAs whose products regulate cellular processes including proliferation, migration, and invasion. Loss of SCF Fbxo4 leads to enhanced cell invasion, migration, and tumor metastasis. C-Myc was identified as one target of SCF Fbxo4 -hnRNPK. Fbxo4 loss triggers hnRNPK-dependent increase in c-Myc translation, thereby contributing to tumorigenesis. Increased c-Myc positions SCF Fbxo4 -hnRNPK dysregulated cancers for potential therapeutic interventions that target c-Myc-dependence. This work demonstrates an essential role for limiting cytoplasmic hnRNPK function in order to maintain translational and cellular homeostasis., (© 2022. The Author(s).)

Artisficial ecological corridors (AECs) are internationally recognized as a standard method for restoring the regional ecological environment. However, the coupling relationship between AECs and soil quality has rarely been studied. Harbin, a typical mollisols region in the cold area of China, has severe soil problems and remediation is urgently needed, yet AEC research in this region is lacking. Based on the perspective of soil restoration, the construction factors of ecological corridors are quantitatively evaluated. It can predict the long-term impact of AECs already built along Harbin's Ashi River on soil chemical indices. This research studied the ecological restoration of secondary woodland, cultivated land within the ecological corridor, and cultivated land outside the influence range of the corridor under the influence of continuous recovery time and different locations in the corridor (distance from the Ashe River). Soil samples were taken from 5 plots, with a total of 161 samples, and 12 indices of soil ecological characteristics were monitored. The result are as follows: It is believed that the quality restoration of mollisols through ecological corridors has great application potential. Based on the low-cost natural restoration of ecological corridors, the highest values of total phosphorus (TP) and soil organic matter (SOM) in soil indices were detected in corridors (restored for more than 10 years). In addition, after ten years of recovery, pH and electrical conductivity (EC) in the ecological corridor returned to normal from high levels in cultivated land that far exceeded the reference values. The recovery process of mollisols mass begins to decrease, then increases, and finally reaches and exceeds the reference value of standard mollisols. The redundancy analysis of soil samples found the distance to be a key factor affecting soil total nitrogen (TN), SOM, and cation exchange capacity (CEC). Recovery time is a crucial factor affecting soil total organic carbon (SOC), pH and EC. According to the TN, SOM, and CEC mollisols indices, the ecological corridor's unilateral width is 125-150m. According to the SOC, pH, and EC indices of mollisols, the AECs should complete a natural recovery cycle of a minimum of 13 years. This study reveals the change mechanism of soil quality in mollisols area corridors based on recovery time and location. This research offer ideas and a scientific basis for worldwide governments in mollisols to formulate mollisols restoration policies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xu, Wu and Diehl.)

Effector trogocytosis between malignant cells and tumor-specific cytotoxic T lymphocytes (CTLs) contributes to immune evasion through antigen loss on target cells and fratricide of antigen-experienced CTLs by other CTLs. The mechanisms regulating these events in tumors remain poorly understood. Here, we demonstrate that tumor-derived factors (TDFs) stimulated effector trogocytosis and restricted CTLs' tumoricidal activity and viability in vitro. TDFs robustly altered the CTL's lipid profile, including depletion of 25-hydroxycholesterol (25HC). 25HC inhibited trogocytosis and prevented CTL's inactivation and fratricide. Mechanistically, TDFs induced ATF3 transcription factor that suppressed the expression of 25HC-regulating gene-cholesterol 25-hydroxylase (CH25H). Stimulation of trogocytosis in the intratumoral CTL by the ATF3-CH25H axis attenuated anti-tumor immunity, stimulated tumor growth, and impeded the efficacy of chimeric antigen receptor (CAR) T cell adoptive therapy. Through use of armored CAR constructs or pharmacologic agents restoring CH25H expression, we reversed these phenotypes and increased the efficacy of immunotherapies., Competing Interests: Declaration of interests A.B. and G.S. were employees of and stockholders in Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited producing TAK981, while engaged in the research project. Z.L., N.M., and S.Y.F. are listed as inventors on The University of Pennsylvania’s patent application related to the matter described in this manuscript., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Evasion of antitumor immunity and resistance to therapies in solid tumors are aided by an immunosuppressive tumor microenvironment (TME). We found that TME factors, such as regulatory T cells and adenosine, downregulated type I interferon receptor IFNAR1 on CD8 + cytotoxic T lymphocytes (CTLs). These events relied upon poly-ADP ribose polymerase-11 (PARP11), which was induced in intratumoral CTLs and acted as a key regulator of the immunosuppressive TME. Ablation of PARP11 prevented loss of IFNAR1, increased CTL tumoricidal activity and inhibited tumor growth in an IFNAR1-dependent manner. Accordingly, genetic or pharmacologic inactivation of PARP11 augmented the therapeutic benefits of chimeric antigen receptor T cells. Chimeric antigen receptor CTLs engineered to inactivate PARP11 demonstrated a superior efficacy against solid tumors. These findings highlight the role of PARP11 in the immunosuppressive TME and provide a proof of principle for targeting this pathway to optimize immune therapies., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Bidirectional signalling between the tumour and stroma shapes tumour aggressiveness and metastasis. ATF4 is a major effector of the Integrated Stress Response, a homeostatic mechanism that couples cell growth and survival to bioenergetic demands. Using conditional knockout ATF4 mice, we show that global, or fibroblast-specific loss of host ATF4, results in deficient vascularization and a pronounced growth delay of syngeneic melanoma and pancreatic tumours. Single-cell transcriptomics of tumours grown in Atf4 Δ/Δ mice uncovered a reduction in activation markers in perivascular cancer-associated fibroblasts (CAFs). Atf4 Δ/Δ fibroblasts displayed significant defects in collagen biosynthesis and deposition and a reduced ability to support angiogenesis. Mechanistically, ATF4 regulates the expression of the Col1a1 gene and levels of glycine and proline, the major amino acids of collagen. Analyses of human melanoma and pancreatic tumours revealed a strong correlation between ATF4 and collagen levels. Our findings establish stromal ATF4 as a key driver of CAF functionality, malignant progression and metastasis., (© 2022. The Author(s).)

Mitochondria and endoplasmic reticulum (ER) share structural and functional networks and activate well-orchestrated signaling processes to shape cells' fate and function. While persistent ER stress (ERS) response leads to mitochondrial collapse, moderate ERS promotes mitochondrial function. Strategies to boost antitumor T-cell function by targeting ER-mitochondria cross-talk have not yet been exploited. Here, we used carbon monoxide (CO), a short-lived gaseous molecule, to test whether engaging moderate ERS conditions can improve mitochondrial and antitumor functions in T cells. In melanoma antigen-specific T cells, CO-induced transient activation of ERS sensor protein kinase R-like endoplasmic reticulum kinase (PERK) significantly increased antitumor T-cell function. Furthermore, CO-induced PERK activation temporarily halted protein translation and induced protective autophagy, including mitophagy. The use of LC3-GFP enabled differentiation between the cells that prepare themselves to undergo active autophagy (LC3-GFPpos) and those that fail to enter the process (LC3-GFPneg). LC3-GFPpos T cells showed strong antitumor potential, whereas LC3-GFPneg cells exhibited a T regulatory-like phenotype, harbored dysfunctional mitochondria, and accumulated abnormal metabolite content. These anomalous ratios of metabolites rendered the cells with a hypermethylated state and distinct epigenetic profile, limiting their antitumor activity. Overall, this study shows that ERS-activated autophagy pathways modify the mitochondrial function and epigenetically reprogram T cells toward a superior antitumor phenotype to achieve robust tumor control., Significance: Transient activation of ER stress with carbon monoxide drives mitochondrial biogenesis and protective autophagy that elicits superior antitumor T-cell function, revealing an approach to improving adoptive cell efficacy therapy., (©2022 American Association for Cancer Research.)

Cyclin D1 is a regulatory subunit of -Cyclin Dependent Kinases 4 and 6 (CDK4/6) and regulates progression from G1 to S phase of the cell cycle. Dysregulated cyclin D1-CDK4/6 contributes to abnormal cell proliferation and tumor development. Phosphorylation of threonine 286 of cyclin D1 is necessary for ubiquitin-dependent degradation. Non-phosphorylatable cyclin D1 mutants are stabilized and concentrated in the nucleus, contributing to genomic instability and tumor development. Studies investigating the tumor-promoting functions of cyclin D1 mutants have focused on the use of artificial promoters to drive the expression which unfortunately may not accurately reflect tumorigenic functions of mutant cyclin D1 in cancer development. We have generated a conditional knock-in mouse model where cyclin D1T286A is expressed under the control of its endogenous promoter following Cre-dependent excision of a lox-stop-lox sequence. Acute expression of cyclin D1T286A following tamoxifen-inducible Cre recombinase triggers inflammation, lymphocyte abnormality and ultimately mesenteric tumors in the intestine. Tissue-specific expression of cyclin D1T286A in the uterus and endometrium cooperates with Pten loss to drive endometrial hyperplasia and cancer. Mechanistically, cyclin D1T286A mutant activates NF-κB signaling, augments inflammation, and contributes to tumor development. These results indicate that mutation of cyclin D1 at threonine 286 has a critical role in regulating inflammation and tumor development., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Objective: Oesophageal squamous cell carcinoma (OSCC), like other squamous carcinomas, harbour highly recurrent cell cycle pathway alterations, especially hyperactivation of the CCND1/CDK4/6 axis, raising the potential for use of existing CDK4/6 inhibitors in these cancers. Although CDK4/6 inhibition has shown striking success when combined with endocrine therapy in oestrogen receptor positive breast cancer, CDK4/6 inhibitor palbociclib monotherapy has not revealed evidence of efficacy to date in OSCC clinical studies. Herein, we sought to elucidate the identification of key dependencies in OSCC as a foundation for the selection of targets whose blockade could be combined with CDK4/6 inhibition., Design: We combined large-scale genomic dependency and pharmaceutical screening datasets with preclinical cell line models, to identified potential combination therapies in squamous cell cancer., Results: We identified sensitivity to inhibitors to the ERBB family of receptor kinases, results clearly extending beyond the previously described minority of tumours with EGFR amplification/dependence, specifically finding a subset of OSCCs with dual dependence on ERBB3 and ERBB2. Subsequently. we demonstrated marked efficacy of combined pan-ERBB and CDK4/6 inhibition in vitro and in vivo. Furthermore, we demonstrated that squamous lineage transcription factor KLF5 facilitated activation of ERBBs in OSCC., Conclusion: These results provide clear rationale for development of combined ERBB and CDK4/6 inhibition in these cancers and raises the potential for KLF5 expression as a candidate biomarker to guide the use of these agents. These data suggested that by combining existing Food and Drug Administration (FDA)-approved agents, we have the capacity to improve therapy for OSCC and other squamous cancer., Competing Interests: Competing interests: AB receives research funding from Bayer, Merck and Novartis and is a consultant to Earli, and HelixNano and a cofounder of Signet Therapeutics. K-KW is a founder and equity holder of G1 Therapeutics, and he has consulting/sponsored research agreements with MedImmune, Takeda, TargImmune, BMS, AstraZeneca, Janssen, Pfizer, Novartis, Merck, Ono and Array., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Alcohol (ethanol) consumption is a major risk factor for head and neck and esophageal squamous cell carcinomas (SCCs). However, how ethanol (EtOH) affects SCC homeostasis is incompletely understood., Methods: We utilized three-dimensional (3D) organoids and xenograft tumor transplantation models to investigate how EtOH exposure influences intratumoral SCC cell populations including putative cancer stem cells defined by high CD44 expression (CD44H cells)., Results: Using 3D organoids generated from SCC cell lines, patient-derived xenograft tumors, and patient biopsies, we found that EtOH is metabolized via alcohol dehydrogenases to induce oxidative stress associated with mitochondrial superoxide generation and mitochondrial depolarization, resulting in apoptosis of the majority of SCC cells within organoids. However, CD44H cells underwent autophagy to negate EtOH-induced mitochondrial dysfunction and apoptosis and were subsequently enriched in organoids and xenograft tumors when exposed to EtOH. Importantly, inhibition of autophagy increased EtOH-mediated apoptosis and reduced CD44H cell enrichment, xenograft tumor growth, and organoid formation rate., Conclusions: This study provides mechanistic insights into how EtOH may influence SCC cells and establishes autophagy as a potential therapeutic target for the treatment of EtOH-associated SCC.

Intercellular biomolecule transfer (ICBT) between malignant and benign cells is a major driver of tumor growth, resistance to anticancer therapies, and therapy-triggered metastatic disease. Here we characterized cholesterol 25-hydroxylase (CH25H) as a key genetic suppressor of ICBT between malignant and endothelial cells (ECs) and of ICBT-driven angiopoietin-2-dependent activation of ECs, stimulation of intratumoral angiogenesis, and tumor growth. Human CH25H was downregulated in the ECs from patients with colorectal cancer and the low levels of stromal CH25H were associated with a poor disease outcome. Knockout of endothelial CH25H stimulated angiogenesis and tumor growth in mice. Pharmacologic inhibition of ICBT by reserpine compensated for CH25H loss, elicited angiostatic effects (alone or combined with sunitinib), augmented the therapeutic effect of radio-/chemotherapy, and prevented metastatic disease induced by these regimens. We propose inhibiting ICBT to improve the overall efficacy of anticancer therapies and limit their prometastatic side effects.

Primary open-angle glaucoma (POAG) constitutes the most common type of glaucoma. Emerging evidence suggests that Endoplasmic Reticulum (ER) stress and the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-mediated Unfolded Protein Response (UPR) signaling pathway play a key role in POAG pathogenesis. Thus, the main aim of the study was to evaluate the effectiveness of the PERK inhibitor LDN-0060609 in cellular model of glaucoma using primary human trabecular meshwork (HTM) cells. To evaluate the level of the ER stress marker proteins, Western blotting and TaqMan gene expression assay were used. The cytotoxicity was measured by XTT, LDH assays and Giemsa staining, whereas genotoxicity via comet assay. Changes in cell morphology were assessed by phase-contrast microscopy. Analysis of apoptosis was performed by caspase-3 assay and flow cytometry (FC), whereas cell cycle progression by FC. The results obtained have demonstrated that LDN-0060609 triggered a significant decrease of ER stress marker proteins within HTM cells with induced ER stress conditions. Moreover, LDN-0060609 effectively increased viability, reduced DNA damage, increased proliferation, restored normal morphology, reduced apoptosis and restored normal cell cycle distribution of HTM cells with induced ER stress conditions. Thereby, PERK inhibitors, such as LDN-0060609, may provide an innovative, ground-breaking treatment strategy against POAG.

Esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers worldwide and evolves often to lung metastasis. P53 R175H (homologous to Trp53 R172H in mice) is a common hot spot mutation. How metastasis is regulated by p53 R175H in ESCC remains to be investigated. To investigate p53 R175H -mediated molecular mechanisms, we used a carcinogen-induced approach in Trp53 R172H/- mice to model ESCC. In the primary Trp53 R172H/- tumor cell lines, we depleted Trp53 R172H (shTrp53) and observed a marked reduction in cell invasion in vitro and lung metastasis burden in a tail-vein injection model in comparing isogenic cells (shCtrl). Furthermore, we performed bulk RNA-seq to compare gene expression profiles of metastatic and primary shCtrl and shTrp53 cells. We identified the YAP- BIRC5 axis as a potential mediator of Trp53 R172H -mediated metastasis. We demonstrate that expression of Survivin, an antiapoptotic protein encoded by BIRC5 , increases in the presence of Trp53 R172H Furthermore, depletion of Survivin specifically decreases Trp53 R172H -driven lung metastasis. Mechanistically, Trp53 R172H but not wild-type Trp53, binds with YAP in ESCC cells, suggesting their cooperation to induce Survivin expression. Furthermore, Survivin high expression level is associated with increased metastasis in several GI cancers. Taken together, this study unravels new insights into how mutant p53 mediates metastasis., (© 2021 Tang et al.; Published by Cold Spring Harbor Laboratory Press.)

The initiation of cell division integrates a large number of intra- and extracellular inputs. D-type cyclins (hereafter, cyclin D) couple these inputs to the initiation of DNA replication 1 . Increased levels of cyclin D promote cell division by activating cyclin-dependent kinases 4 and 6 (hereafter, CDK4/6), which in turn phosphorylate and inactivate the retinoblastoma tumour suppressor. Accordingly, increased levels and activity of cyclin D-CDK4/6 complexes are strongly linked to unchecked cell proliferation and cancer 2,3 . However, the mechanisms that regulate levels of cyclin D are incompletely understood 4,5 . Here we show that autophagy and beclin 1 regulator 1 (AMBRA1) is the main regulator of the degradation of cyclin D. We identified AMBRA1 in a genome-wide screen to investigate the genetic basis of  the response to CDK4/6 inhibition. Loss of AMBRA1 results in high levels of cyclin D in cells and in mice, which promotes proliferation and decreases sensitivity to CDK4/6 inhibition. Mechanistically, AMBRA1 mediates ubiquitylation and proteasomal degradation of cyclin D as a substrate receptor for the cullin 4 E3 ligase complex. Loss of AMBRA1 enhances the growth of lung adenocarcinoma in a mouse model, and low levels of AMBRA1 correlate with worse survival in patients with lung adenocarcinoma. Thus, AMBRA1 regulates cellular levels of cyclin D, and contributes to cancer development and the response of cancer cells to CDK4/6 inhibitors.

Inositol-requiring enzyme type 1 (IRE1) is a serine/threonine kinase acting as one of three branches of the Unfolded Protein Response (UPR) signaling pathway, which is activated upon endoplasmic reticulum (ER) stress conditions. It is known to be capable of inducing both pro-survival and pro-apoptotic cellular responses, which are strictly related to numerous human pathologies. Among others, IRE1 activity has been confirmed to be increased in cancer, neurodegeneration, inflammatory and metabolic disorders, which are associated with an accumulation of misfolded proteins within ER lumen and the resulting ER stress conditions. Emerging evidence suggests that genetic or pharmacological modulation of IRE1 may have a significant impact on cell viability, and thus may be a promising step forward towards development of novel therapeutic strategies. In this review, we extensively describe the structural analysis of IRE1 molecule, the molecular dynamics associated with IRE1 activation, and interconnection between it and the other branches of the UPR with regard to its potential use as a therapeutic target. Detailed knowledge of the molecular characteristics of the IRE1 protein and its activation may allow the design of specific kinase or RNase modulators that may act as drug candidates.

Overexpression of D-type cyclins in human cancer frequently occurs as a result of protein stabilization, emphasizing the importance of identification of the machinery that regulates their ubiqutin-dependent degradation. Cyclin D3 is overexpressed in ~50% of Burkitt's lymphoma correlating with a mutation of Thr-283. However, the E3 ligase that regulates phosphorylated cyclin D3 and whether a stabilized, phosphorylation deficient mutant of cyclin D3, has oncogenic activity are undefined. We describe the identification of SCF-Fbxl8 as the E3 ligase for Thr-283 phosphorylated cyclin D3. SCF-Fbxl8 poly-ubiquitylates p-Thr-283 cyclin D3 targeting it to the proteasome. Functional investigation demonstrates that Fbxl8 antagonizes cell cycle progression, hematopoietic cell proliferation, and oncogene-induced transformation through degradation of cyclin D3, which is abolished by expression of cyclin D3T283A, a non-phosphorylatable mutant. Clinically, the expression of cyclin D3 is inversely correlated with the expression of Fbxl8 in lymphomas from human patients implicating Fbxl8 functions as a tumor suppressor.

D cyclins include three isoforms: D1, D2, and D3. D cyclins heterodimerize with cyclin-dependent kinase 4/6 (CDK4/6) to form kinase complexes that can phosphorylate and inactivate Rb. Inactivation of Rb triggers the activation of E2F transcription factors, which in turn regulate the expression of genes whose products drive cell cycle progression. Because D-type cyclins function as mitogenic sensors that link growth factor signaling directly with G 1 phase progression, it is not surprising that D cyclin accumulation is dysregulated in a variety of human tumors. Elevated expression of D cyclins results from gene amplification, increased gene transcription and protein translation, decreased microRNA levels, and inefficiency or loss of ubiquitylation-mediated protein degradation. This review focuses on the clinicopathological importance of D cyclins, how dysregulation of Ubiquitin-Proteasome System (UPS) contributes to the overexpression of D cyclins, and the therapeutic potential through targeting D cyclin-related machinery in human tumors., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Esophageal cancers comprise adenocarcinoma and squamous cell carcinoma, two distinct histologic subtypes. Both are difficult to treat and among the deadliest human malignancies. We describe protocols to initiate, grow, passage, and characterize patient-derived organoids (PDO) of esophageal cancers, as well as squamous cell carcinomas of oral/head-and-neck and anal origin. Formed rapidly (<14 days) from a single-cell suspension embedded in basement membrane matrix, esophageal cancer PDO recapitulate the histology of the original tumors. Additionally, we provide guidelines for morphological analyses and drug testing coupled with functional assessment of cell response to conventional chemotherapeutics and other pharmacological agents in concert with emerging automated imaging platforms. Predicting drug sensitivity and potential therapy resistance mechanisms in a moderate-to-high throughput manner, esophageal cancer PDO are highly translatable in personalized medicine for customized esophageal cancer treatments. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Generation of esophageal cancer PDO Basic Protocol 2: Propagation and cryopreservation of esophageal cancer PDO Basic Protocol 3: Imaged-based monitoring of organoid size and growth kinetics Basic Protocol 4: Harvesting esophageal cancer PDO for histological analyses Basic Protocol 5: PDO content analysis by flow cytometry Basic Protocol 6: Evaluation of drug response with determination of the half-inhibitory concentration (IC 50 ) Support Protocol: Production of RN in HEK293T cell conditioned medium., (© 2020 John Wiley & Sons, Inc.)

Primary tumor-derived factors (TDFs) act upon normal cells to generate a pre-metastatic niche, which promotes colonization of target organs by disseminated malignant cells. Here we report that TDFs-induced activation of the p38α kinase in lung fibroblasts plays a critical role in the formation of a pre-metastatic niche in the lungs and subsequent pulmonary metastases. Activation of p38α led to inactivation of type I interferon signaling and stimulation of expression of fibroblast activation protein (FAP). FAP played a key role in remodeling of the extracellular matrix as well as inducing the expression of chemokines that enable lung infiltration by neutrophils. Increased activity of p38 in normal cells was associated with metastatic disease and poor prognosis in human melanoma patients whereas inactivation of p38 suppressed lung metastases. We discuss the p38α-driven mechanisms stimulating the metastatic processes and potential use of p38 inhibitors in adjuvant therapy of metastatic cancers., Competing Interests: Competing Interests Statement The authors have declared that no conflict of interest exists

Higher prevalence of neurodegenerative diseases is strictly connected with progressive aging of the world population. Interestingly, a broad range of age-related, neurodegenerative diseases is characterized by a common pathological mechanism-accumulation of misfolded and unfolded proteins within the cells. Under certain circumstances, such protein aggregates may evoke endoplasmic reticulum (ER) stress conditions and subsequent activation of the unfolded protein response (UPR) signaling pathways via the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-dependent manner. Under mild to moderate ER stress, UPR has a pro-adaptive role. However, severe or long-termed ER stress conditions directly evoke shift of the UPR toward its pro-apoptotic branch, which is considered to be a possible cause of neurodegeneration. To this day, there is no effective cure for Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), or prion disease. Currently available treatment approaches for these diseases are only symptomatic and cannot affect the disease progression. Treatment strategies, currently under detailed research, include inhibition of the PERK-dependent UPR signaling branches. The newest data have reported that the use of small-molecule inhibitors of the PERK-mediated signaling branches may contribute to the development of a novel, ground-breaking therapeutic approach for neurodegeneration. In this review, we critically describe all the aspects associated with such targeted therapy against neurodegenerative proteopathies.

Background: Colorectal cancer constitutes one of the most common cancer with a high mortality rate. The newest data has reported that activation of the pro-apoptotic PERK-dependent unfolded protein response signaling pathway by small-molecule inhibitors may constitute an innovative anti-cancer treatment strategy., Objective: In the presented study, we evaluated the effectiveness of the PERK-dependent unfolded protein response signaling pathway small-molecule inhibitor 42215 both on HT-29 human colon adenocarcinoma and CCD 841 CoN normal human colon epithelial cell lines., Methods: Cytotoxicity of the PERK inhibitor was evaluated by the resazurin-based and lactate dehydrogenase (LDH) tests. Apoptotic cell death was measured by flow cytometry using the FITCconjugated Annexin V to indicate apoptosis and propidium iodide to indicate necrosis as well as by colorimetric caspase-3 assay. The effect of tested PERK inhibitor on cell cycle progression was measured by flow cytometry using the propidium iodide staining. The level of the phosphorylated form of the eukaryotic initiation factor 2 alpha was detected by the Western blot technique., Results: Obtained results showed that investigated PERK inhibitor is selective only toward cancer cells, since inhibited their viability in a dose- and time-dependent manner and induced their apoptosis and G2/M cell cycle arrest. Furthermore, 42215 PERK inhibitor evoked significant inhibition of eIF2α phosphorylation within HT-29 cancer cells., Conclusion: Highly-selective PERK inhibitors may provide a ground-breaking, anti-cancer treatment strategy via activation of the pro-apoptotic branch of the PERK-dependent unfolded protein response signaling pathway., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The cyclin-dependent kinase 4/6 (CDK4/6) kinase is dysregulated in melanoma, highlighting it as a potential therapeutic target. CDK4/6 inhibitors are being evaluated in trials for melanoma and additional cancers. While beneficial, resistance to therapy is a concern, and the molecular mechanisms of such resistance remain undefined. We demonstrate that reactivation of mammalian target of rapamycin 1 (mTORC1) signaling through increased expression of the amino acid transporter, solute carrier family 36 member 1 (SLC36A1), drives resistance to CDK4/6 inhibitors. Increased expression of SLC36A1 reflects two distinct mechanisms: (i) Rb loss, which drives SLC36A1 via reduced suppression of E2f; (ii) fragile X mental retardation syndrome-associated protein 1 overexpression, which promotes SLC36A1 translation and subsequently mTORC1. Last, we demonstrate that a combination of a CDK4/6 inhibitor with an mTORC1 inhibitor has increased therapeutic efficacy in vivo, providing an important avenue for improved therapeutic intervention in aggressive melanoma.

Cancer constitutes a grave problem nowadays in view of the fact that it has become one of the main causes of death worldwide. Poor clinical prognosis is presumably due to cancer cells metabolism as tumor microenvironment is affected by oxidative stress. This event triggers adequate cellular response and thereby creates appropriate conditions for further cancer progression. Endoplasmic reticulum (ER) stress occurs when the balance between an ability of the ER to fold and transfer proteins and the degradation of the misfolded ones become distorted. Since ER is an organelle relatively sensitive to oxidative damage, aforementioned conditions swiftly cause the activation of the unfolded protein response (UPR) signaling pathway. The output of the UPR, depending on numerous factors, may vary and switch between the pro-survival and the pro-apoptotic branch, and hence it displays opposing effects in deciding the fate of the cancer cell. The role of UPR-related proteins in tumorigenesis, such as binding the immunoglobulin protein (BiP) and inositol-requiring enzyme-1α (IRE1α), activating transcription factor 6 (ATF6) or the protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), has already been specifically described so far. Nevertheless, due to the paradoxical outcomes of the UPR activation as well as gaps in current knowledge, it still needs to be further investigated. Herein we would like to elicit the actual link between neoplastic diseases and the UPR signaling pathway, considering its major branches and discussing its potential use in the development of a novel, anti-cancer, targeted therapy.

The c-Myc oncogene drives malignant progression and induces robust anabolic and proliferative programmes leading to intrinsic stress. The mechanisms enabling adaptation to MYC-induced stress are not fully understood. Here we reveal an essential role for activating transcription factor 4 (ATF4) in survival following MYC activation. MYC upregulates ATF4 by activating general control nonderepressible 2 (GCN2) kinase through uncharged transfer RNAs. Subsequently, ATF4 co-occupies promoter regions of over 30 MYC-target genes, primarily those regulating amino acid and protein synthesis, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), a negative regulator of translation. 4E-BP1 relieves MYC-induced proteotoxic stress and is essential to balance protein synthesis. 4E-BP1 activity is negatively regulated by mammalian target of rapamycin complex 1 (mTORC1)-dependent phosphorylation and inhibition of mTORC1 signalling rescues ATF4-deficient cells from MYC-induced endoplasmic reticulum stress. Acute deletion of ATF4 significantly delays MYC-driven tumour progression and increases survival in mouse models. Our results establish ATF4 as a cellular rheostat of MYC activity, which ensures that enhanced translation rates are compatible with survival and tumour progression.

Hyperactive oncogenic Myc stimulates protein synthesis that induces the unfolded protein response, which requires the function of the eukaryotic translation initiation factor 2-alpha kinase 3, also known as protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK). Activated PERK acts to limit mRNA translation, enable proper protein folding, and restore the homeostasis in the endoplasmic reticulum. Given that Myc activation contributes to many types of lymphoid and myeloid human leukemias, we used a mouse model to examine the importance of PERK in development and progression of Myc-induced leukemias. We found that genetic ablation of Perk does not suppress the generation of the leukemic cells in the bone marrow. However, the cell-autonomous Perk deficiency restricts the dissemination of leukemic cells into peripheral blood, lymph nodes, and vital peripheral organs. Whereas the loss of the IFNAR1 chain of type I IFN receptor stimulated leukemia, Perk ablation did not stabilize IFNAR1, suggesting that PERK stimulates the leukemic cells' dissemination in an IFNAR1-independent manner. We discuss the rationale for using PERK inhibitors against Myc-driven leukemias. IMPLICATIONS: The role of PERK in dissemination of Myc-induced leukemic cells demonstrated in this study argues for the use of PERK inhibitors against leukemia progression., (©2019 American Association for Cancer Research.)

Understanding and overcoming resistance to cyclin-dependent kinase 4/6 (CDK4/6) inhibitors will be challenging. Recent work reveals that dysregulation of F-Box Protein 4 (FBXO4)-Cyclin D1 axis leads to mitochondrial dysfunction and drives glutamine-addiction in esophageal squamous cell carcinoma. This metabolism feature makes these tumors susceptible to combined treatment with glutaminase (GLS) inhibitor and metformin even when resisting to CDK4/6 inhibitors.

The dysregulation of Fbxo4-cyclin D1 axis occurs at high frequency in esophageal squamous cell carcinoma (ESCC), where it promotes ESCC development and progression. However, defining a therapeutic vulnerability that results from this dysregulation has remained elusive. Here we demonstrate that Rb and mTORC1 contribute to Gln-addiction upon the dysregulation of the Fbxo4-cyclin D1 axis, which leads to the reprogramming of cellular metabolism. This reprogramming is characterized by reduced energy production and increased sensitivity of ESCC cells to combined treatment with CB-839 (glutaminase 1 inhibitor) plus metformin/phenformin. Of additional importance, this combined treatment has potent efficacy in ESCC cells with acquired resistance to CDK4/6 inhibitors in vitro and in xenograft tumors. Our findings reveal a molecular basis for cancer therapy through targeting glutaminolysis and mitochondrial respiration in ESCC with dysregulated Fbxo4-cyclin D1 axis as well as cancers resistant to CDK4/6 inhibitors.

The characteristic hallmark of Alzheimer's disease (AD) are progressive changes in the brain structure and function, caused by aggregation of senile plagues, composed of improperly folded amyloid β(Aβ) protein, in the brain tissue. Recent research has suggested that causes of AD are closely associated with perturbation on the molecular level caused by the activation of the pro-apoptotic, PERKdependent Unfolded Protein Response (UPR) signaling pathway activated under Endoplasmic Reticulum (ER) stress conditions., Aim: The aims of the study were evaluation of the activity of the smallmolecule inhibitors of PERK kinase, GSK2606414 and LDN-0060609, via the analysis of the level of the phosphorylation of eIF2α as one of the main markers of the UPR signaling pathway activation as well as evaluation of the cytotoxicity of the inhibitor LDN-0060609., Materials and Methods: The study was conducted on commercially available cell lines of wild type mouse embryotic fibroblasts 3T3 MEFs WT and with deletion of PERK gene 3T3 MEFs KO, mouse neurons CATH.a and human neuroblastoma SH-SY5Y with overexpression of amyloid precursor protein (APP). Cells were treated with commercially available inhibitor GSK2606414 or LDN-0060609, selected from the small-molecule compounds library Laboratory for Drug Discovery in Neurodegeneration, on appropriate cell culture medium with thapsigargin as an activator of Endoplasmic Reticulum (ER) stress conditions. To evaluate the level of eIF2α phosphorylation we used the Western blot technique. Detection of immune complexes was performed using the chemiluminescence. Evaluation of the LDN-0060609 compound cytotoxicity was carried out on SH-SY5Y cells using the XTT assay., Results: The results of the study showed that the commercially available GSK2606414 inhibitor at a concentration of 1 μM causes >85% inhibition of the phosphorylation of eIF2α in all tested cell lines. The newly tested LDN-0060609 inhibitor showed the highest inhibitory activity at 25 μM resulting in 52% inhibition of eIF2α phosphorylation. In addition, the LDN-0060609 inhibitor did not induce a cytotoxic effect at any used concentrations and incubation times. Conclusions. It is believed that the LDN-0060609., Conclusions: It is believed that the LDN-0060609 inhibitor, that in comparison with commercially available GSK2606414 inhibitor does not evoke a cytotoxic effect, may constitute a potential factor inhibiting activation of the PERK-dependent UPR signaling pathway responsible for neurodegenerative processes in AD. Small-molecule PERK inhibitors may constitute an innovative therapeutic strategy for AD treatment., (© 2019 MEDPRESS.)

Tumor-derived extracellular vesicles (TEV) "educate" healthy cells to promote metastases. We found that melanoma TEV downregulated type I interferon (IFN) receptor and expression of IFN-inducible cholesterol 25-hydroxylase (CH25H). CH25H produces 25-hydroxycholesterol, which inhibited TEV uptake. Low CH25H levels in leukocytes from melanoma patients correlated with poor prognosis. Mice incapable of downregulating the IFN receptor and Ch25h were resistant to TEV uptake, TEV-induced pre-metastatic niche, and melanoma lung metastases; however, ablation of Ch25h reversed these phenotypes. An anti-hypertensive drug, reserpine, suppressed TEV uptake and disrupted TEV-induced formation of the pre-metastatic niche and melanoma lung metastases. These results suggest the importance of CH25H in defense against education of normal cells by TEV and argue for the use of reserpine in adjuvant melanoma therapy., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Multiple, both endogenous and exogenous, sources may induce DNA damage and DNA replication stress. Cells have developed DNA damage response (DDR) signaling pathways to maintain genomic stability and effectively detect and repair DNA lesions. Serine/ threonine kinases such as Ataxia-telangiectasia mutated (ATM) and Ataxia-telangiectasia and Rad3-Related (ATR) are the major regulators of DDR, since after sensing stalled DNA replication forks, DNA double- or single-strand breaks, may directly phosphorylate and activate their downstream targets, that play a key role in DNA repair, cell cycle arrest and apoptotic cell death. Interestingly, key components of DDR signaling networks may constitute an attractive target for anti-cancer therapy through two distinct potential approaches: as chemoand radiosensitizers to enhance the effectiveness of currently used genotoxic treatment or as single agents to exploit defects in DDR in cancer cells via synthetic lethal approach. Moreover, the newest data reported that serine/threonine protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) is also closely associated with cancer development and progression. Thereby, utilization of small-molecule, serine/threonine kinase inhibitors may provide a novel, groundbreaking, anti-cancer treatment strategy. Currently, a range of potent, highlyselective toward ATM, ATR and PERK inhibitors has been discovered, but after foregoing study, additional investigations are necessary for their future clinical use., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Objectives: There is a body of evidence that neurodegenerative disease entities are directly correlated with the perturbations on the molecular level. Hence, the ER stress-mediated Unfolded Protein Response (UPR) is activated resulting in PERK-dependent phosphorylation of the Eukaryotic initiation factor 2 (eIF2α). Thus, the levels of ATF4 and CHOP proteins are significantly increased, which subsequently switches the pro-adaptive branch of the UPR into the pro-apoptotic directly leading to neuronal loss and initiation of the neurodegenerative process. The aim of the presented study was the evaluation of the biological activity of highly specific, small-molecule inhibitors of the PERKdependent UPR signaling pathway., Methods: The study was conducted on rat astrocytic DI TNC1 cell line. The level of p-eIF2α was measured by Western blot technique, the cytotoxicity of the investigated compound was assessed by the MTT assay and using the FITC-conjugated Annexin V (Annexin V-FITC) to indicate apoptosis and propidium iodide (PI) to indicate necrosis. The effect of tested compound on cell cycle progression was measured by flow cytometry, where the PI-labelled nuclei were analysed for DNA content., Results: As a result one of the investigated compound LDN-0060609 triggers a significant inhibition of the eIF2α phosphorylation in DI TNC1 cell line. Moreover, we showed that compound LDN-0060609 is non-cytotoxic and has no effect on cell cycle progression., Conclusion: In conclusion, LDN-0060609 may constitute a novel, targeted treatment approach against neurodegenerative diseases, including Alzheimer's disease (AD), where pathogenesis and progression are closely associated with the overactivation of the PERK-dependent UPR signaling pathway., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Overexpression of the deubiquitylase ubiquitin-specific peptidase 22 (USP22) is a marker of aggressive cancer phenotypes like metastasis, therapy resistance, and poor survival. Functionally, this overexpression of USP22 actively contributes to tumorigenesis, as USP22 depletion blocks cancer cell cycle progression in vitro, and inhibits tumor progression in animal models of lung, breast, bladder, ovarian, and liver cancer, among others. Current models suggest that USP22 mediates these biological effects via its role in epigenetic regulation as a subunit of the Spt-Ada-Gcn5-acetyltransferase (SAGA) transcriptional cofactor complex. Challenging the dogma, we report here a nontranscriptional role for USP22 via a direct effect on the core cell cycle machinery: that is, the deubiquitylation of the G1 cyclin D1 (CCND1). Deubiquitylation by USP22 protects CCND1 from proteasome-mediated degradation and occurs separately from the canonical phosphorylation/ubiquitylation mechanism previously shown to regulate CCND1 stability. We demonstrate that control of CCND1 is a key mechanism by which USP22 mediates its known role in cell cycle progression. Finally, USP22 and CCND1 levels correlate in patient lung and colorectal cancer samples and our preclinical studies indicate that targeting USP22 in combination with CDK inhibitors may offer an approach for treating cancer patients whose tumors exhibit elevated CCND1., Competing Interests: Conflict of interest statement: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

KRAS mutation is present in approximately 30% of human lung adenocarcinomas. Although recent advances in targeted therapy have shown great promise, effective targeting of KRAS remains elusive, and concurrent alterations in tumor suppressors render KRAS- mutant tumors even more resistant to existing therapies. Contributing to the refractoriness of KRAS -mutant tumors are immunosuppressive mechanisms, such as increased presence of suppressive regulatory T cells (Treg) in tumors and elevated expression of the inhibitory receptor PD-1 on tumor-infiltrating T cells. Treatment with BET bromodomain inhibitors is beneficial for hematologic malignancies, and they have Treg-disruptive effects in a non-small cell lung cancer (NSCLC) model. Targeting PD-1-inhibitory signals through PD-1 antibody blockade also has substantial therapeutic impact in lung cancer, although these outcomes are limited to a minority of patients. We hypothesized that the BET bromodomain inhibitor JQ1 would synergize with PD-1 blockade to promote a robust antitumor response in lung cancer. In the present study, using Kras +/LSL-G12D ; Trp53 L/L (KP) mouse models of NSCLC, we identified cooperative effects between JQ1 and PD-1 antibody. The numbers of tumor-infiltrating Tregs were reduced and activation of tumor-infiltrating T cells, which had a T-helper type 1 (Th1) cytokine profile, was enhanced, underlying their improved effector function. Furthermore, lung tumor-bearing mice treated with this combination showed robust and long-lasting antitumor responses compared with either agent alone, culminating in substantial improvement in the overall survival of treated mice. Thus, combining BET bromodomain inhibition with immune checkpoint blockade offers a promising therapeutic approach for solid malignancies such as lung adenocarcinoma. Cancer Immunol Res; 6(10); 1234-45. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Background & Aims: Oropharyngeal and esophageal squamous cell carcinomas, especially the latter, are a lethal disease, featuring intratumoral cancer cell heterogeneity and therapy resistance. To facilitate cancer therapy in personalized medicine, three-dimensional (3D) organoids may be useful for functional characterization of cancer cells ex vivo . We investigated the feasibility and the utility of patient-derived 3D organoids of esophageal and oropharyngeal squamous cell carcinomas., Methods: We generated 3D organoids from paired biopsies representing tumors and adjacent normal mucosa from therapy-naïve patients and cell lines. We evaluated growth and structures of 3D organoids treated with 5-fluorouracil ex vivo ., Results: Tumor-derived 3D organoids were grown successfully from 15 out of 21 patients (71.4%) and passaged with recapitulation of the histopathology of the original tumors. Successful formation of tumor-derived 3D organoids was associated significantly with poor response to presurgical neoadjuvant chemotherapy or chemoradiation therapy in informative patients ( P = 0.0357, progressive and stable diseases, n = 10 vs. partial response, n = 6). The 3D organoid formation capability and 5-fluorouracil resistance were accounted for by cancer cells with high CD44 expression and autophagy, respectively. Such cancer cells were found to be enriched in patient-derived 3D organoids surviving 5-fluorouracil treatment., Conclusions: The single cell-based 3D organoid system may serve as a highly efficient platform to explore cancer therapeutics and therapy resistance mechanisms in conjunction with morphological and functional assays with implications for translation in personalized medicine.

Purpose: Loss of cell-cycle control is a hallmark of cancer, which can be targeted with agents, including cyclin-dependent kinase-4/6 (CDK4/6) kinase inhibitors that impinge upon the G 1 -S cell-cycle checkpoint via maintaining activity of the retinoblastoma tumor suppressor (RB). This class of drugs is under clinical investigation for various solid tumor types and has recently been FDA-approved for treatment of breast cancer. However, development of therapeutic resistance is not uncommon. Experimental Design: In this study, palbociclib (a CDK4/6 inhibitor) resistance was established in models of early stage, RB-positive cancer. Results: This study demonstrates that acquired palbociclib resistance renders cancer cells broadly resistant to CDK4/6 inhibitors. Acquired resistance was associated with aggressive in vitro and in vivo phenotypes, including proliferation, migration, and invasion. Integration of RNA sequencing analysis and phosphoproteomics profiling revealed rewiring of the kinome, with a strong enrichment for enhanced MAPK signaling across all resistance models, which resulted in aggressive in vitro and in vivo phenotypes and prometastatic signaling. However, CDK4/6 inhibitor-resistant models were sensitized to MEK inhibitors, revealing reliance on active MAPK signaling to promote tumor cell growth and invasion. Conclusions: In sum, these studies identify MAPK reliance in acquired CDK4/6 inhibitor resistance that promotes aggressive disease, while nominating MEK inhibition as putative novel therapeutic strategy to treat or prevent CDK4/6 inhibitor resistance in cancer. Clin Cancer Res; 24(17); 4201-14. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The tumor microenvironment (TME) plays a major role in the pathogenesis of multiple cancer types, including upper-gastrointestinal (GI) cancers that currently lack effective therapeutic options. Cancer-associated fibroblasts (CAF) are an essential component of the TME, contributing to tumorigenesis by secreting growth factors, modifying the extracellular matrix, supporting angiogenesis, and suppressing antitumor immune responses. Through an unbiased approach, we have established that IL-6 mediates cross-talk between tumor cells and CAF not only by supporting tumor cell growth, but also by promoting fibroblast activation. As a result, IL-6 receptor (IL6Rα) and downstream effectors offer opportunities for targeted therapy in upper-GI cancers. IL-6 loss suppressed tumorigenesis in physiologically relevant three-dimensional (3D) organotypic and 3D tumoroid models and murine models of esophageal cancer. Tocilizumab, an anti-IL6Rα antibody, suppressed tumor growth in vivo in part via inhibition of STAT3 and MEK/ERK signaling. Analysis of a pan-cancer TCGA dataset revealed an inverse correlation between IL-6 and IL6Rα overexpression and patient survival. Therefore, we expanded evaluation of tocilizumab to head and neck squamous cell carcinoma patient-derived xenografts and gastric adenocarcinoma xenografts, demonstrating suppression of tumor growth and altered STAT3 and ERK1/2 gene signatures. We used small-molecule inhibitors of STAT3 and MEK1/2 signaling to suppress tumorigenesis in the 3D organotypic model of esophageal cancer. We demonstrate that IL6 is a major contributor to the dynamic cross-talk between tumor cells and CAF in the TME. Our findings provide a translational rationale for inhibition of IL6Rα and downstream signaling pathways as a novel targeted therapy in oral-upper-GI cancers. Significance: These findings demonstrate the interaction of esophageal cancer and cancer-associated fibroblasts through IL-6 signaling, providing rationale for a novel therapeutic approach to target these cancers. Cancer Res; 78(17); 4957-70. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The role of KRAS, when activated through canonical mutations, has been well established in cancer 1 . Here we explore a secondary means of KRAS activation in cancer: focal high-level amplification of the KRAS gene in the absence of coding mutations. These amplifications occur most commonly in esophageal, gastric and ovarian adenocarcinomas 2-4 . KRAS-amplified gastric cancer models show marked overexpression of the KRAS protein and are insensitive to MAPK blockade owing to their capacity to adaptively respond by rapidly increasing KRAS-GTP levels. Here we demonstrate that inhibition of the guanine-exchange factors SOS1 and SOS2 or the protein tyrosine phosphatase SHP2 can attenuate this adaptive process and that targeting these factors, both genetically and pharmacologically, can enhance the sensitivity of KRAS-amplified models to MEK inhibition in both in vitro and in vivo settings. These data demonstrate the relevance of copy-number amplification as a mechanism of KRAS activation, and uncover the therapeutic potential for targeting of these tumors through combined SHP2 and MEK inhibition.

RNA-binding proteins (RBP) and noncoding RNAs (ncRNA), such as long noncoding RNAs (lncRNA) and microRNAs (miRNA), control co- and posttranscriptional gene regulation (PTR). At the PTR level, RBPs and ncRNAs contribute to pre-mRNA processing, mRNA maturation, transport, localization, turnover, and translation. Deregulation of RBPs and ncRNAs promotes the onset of cancer progression and metastasis. Both RBPs and ncRNAs are altered by signaling cascades to cooperate or compete with each other to bind their nucleic acid targets. Most importantly, transforming growth factor-beta (TGFβ) signaling plays a significant role in controlling gene expression patterns by targeting RBPs and ncRNAs. Because of TGFβ signaling in cancer, RBP-RNA or RNA-RNA interactions are altered and cause enhanced cell growth and tumor cell dissemination. This review focuses on the emerging concepts of TGFβ signaling on posttranscriptional gene regulation and highlights the implications of RBPs and ncRNAs in cancer progression and metastasis. Mol Cancer Res; 16(4); 567-79. ©2018 AACR ., (©2018 American Association for Cancer Research.)

The unfolded protein response (UPR) is a stress-activated signalling pathway that regulates cell proliferation, metabolism and survival. The circadian clock coordinates metabolism and signal transduction with light/dark cycles. We explore how UPR signalling interfaces with the circadian clock. UPR activation induces a 10 h phase shift in circadian oscillations through induction of miR-211, a PERK-inducible microRNA that transiently suppresses both Bmal1 and Clock, core circadian regulators. Molecular investigation reveals that miR-211 directly regulates Bmal1 and Clock via distinct mechanisms. Suppression of Bmal1 and Clock has the anticipated impact on expression of select circadian genes, but we also find that repression of Bmal1 is essential for UPR-dependent inhibition of protein synthesis and cell adaptation to stresses that disrupt endoplasmic reticulum homeostasis. Our data demonstrate that c-Myc-dependent activation of the UPR inhibits Bmal1 in Burkitt's lymphoma, thereby suppressing both circadian oscillation and ongoing protein synthesis to facilitate tumour progression.

This corrects the article DOI: 10.1038/ncb3595.