KYNU Expression Promotes Cisplatin Resistance in Esophageal Cancer.

Background: Chemotherapy resistance is a barrier to effective cancer prognoses. Cisplatin (CDDP) resistance is a major challenge for esophageal cancer (EC) therapy. A deeper understanding of the fundamental mechanisms of cisplatin resistance and improved targeting strategies are required in clinical settings. This study was performed to identify and characterize a marker of cisplatin resistance in EC cells. Method: KYSE140 and Eca-109 cells were subjected to escalating concentrations of cisplatin, resulting in the development of cisplatin-resistant KYSE140/CDDP and Eca-109/CDDP cell lines, respectively. RNA Sequencing (RNA-seq) was utilized to screen for the genes exhibiting differential expression between cisplatin-resistant and parental cells. Reverse transcription quantitative PCR was conducted to assess gene expression, and western blotting was employed to analyze protein levels. A sphere-formation assay was performed to validate tumor cell stemness. Cell counting kit-8 (CCK-8) experiments were conducted to confirm the sensitivity of cells to cisplatin. We examined the relationship between target genes and the clinicopathological features of patients with EC. Furthermore, the expression of target genes in EC tissues was evaluated via western blotting and fluorescence probe in situ hybridization (FISH). Results: KYNU was upregulated in cisplatin-resistant EC cells (KYSE140/CDDP and Eca-109/CDDP cells) and in EC tissues compared to that in the respective parental cell lines (KYSE140 and Eca-109 cells) and non-carcinoma tissues. Downregulation of KYNU increased cell sensitivity to cisplatin and suppressed tumor stemness, whereas abnormal KYNU expression had the opposite effect. KYNU expression was correlated with the expression of tumor stemness-associated factors (SOX2, Nanog, and OCT4) and the tumor size. Conclusions: KYNU may promote drug resistance in EC by regulating cancer stemness, and could serve as a biomarker and therapeutic target for EC.

Ubiquitin ligase subunit FBXO9 inhibits V-ATPase assembly and impedes lung cancer metastasis.

BACKGROUND: The evolutionarily conserved protein FBXO9 acts as a substrate receptor for the SKP1-cullin-1-RBX1 ubiquitin ligase and is implicated in cancer, exhibiting either tumor-suppressive or oncogenic effects depending on the specific tumor type. However, their role in lung cancer metastasis remains unclear. METHODS: Lentiviral vectors carrying miRNA-based shRNA sequences for gene-specific knockdown were generated, and Lenti-CRISPR-Cas9 vectors containing gene-specific sgRNA sequences were designed. Gene overexpression was achieved using doxycycline-inducible lentiviral constructs, while gene knockdown or knockout cells were generated using shRNA and CRISPR-Cas9, respectively. Functional assays included migration, clonogenic survival assays, tumor sphere assays, and protein interaction studies using mass spectrometry, immunoprecipitation, and immunoblot analysis. RESULTS: This study identified FBXO9 as a crucial regulator that suppresses lung cancer cell migration, tumor sphere growth and restricts metastasis. We showed that FBXO9 facilitates the ubiquitination of the catalytic subunit A (ATP6V1A) of the Vacuolar-type H+-ATPase (V-ATPase), resulting in its interaction with the cytoplasmic chaperone HSPA8 and subsequent sequestration within the cytoplasm. This process hinders the assembly of functional V-ATPase, resulting in reduced vesicular acidification. In contrast, depletion of FBXO9 reduced ATP6V1A ubiquitination, resulting in increased V-ATPase assembly and vesicular acidification, thus promoting pro-metastatic Wnt signaling and metastasis of lung cancer cells. Furthermore, we demonstrated the effectiveness of inhibitors targeting V-ATPase in inhibiting lung cancer metastasis in a mouse model. Finally, we established a correlation between lower FBXO9 levels and poorer survival outcomes in patients with lung cancer. CONCLUSION: These findings collectively elucidate the critical role of FBXO9 in regulating V-ATPase assembly and provide a molecular basis for FBXO9's function in inhibiting lung cancer metastasis. This highlights the potential therapeutic opportunities of FBXO9 supplementation.

LINC01572 is a Novel Prognostic Biomarker and Therapeutic Target in Lung Adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is one of the most common and lethal cancer types worldwide. LINC0572 is a long non-coding RNA (lncRNA) that has been associated with the clinical characteristics of several types of malignancy. However, the biological mechanism of LINC0572 in LUAD is still unclear and remains to be elucidated. METHODS: R packages and online bioinformatic tools were used to investigate the biological characteristics of LINC01572, including its abnormal expression, oncogenic role, and clinical prognostic value. In vitro and in vivo experiments were conducted to investigate the biological functions of LINC01572 in tumorigenesis and development. These included colony formation assays, cell migration assays, flow cytometry, cell counting kit-8 (CCK-8) cell proliferation and tumor transplant growth experiments. RESULTS: Bioinformatics results showed that LINC01572 was overexpressed in both LUAD and lung squamous cell carcinoma (LUSC) patients. LINC01572 overexpression was associated with shorter overall survival (OS) in LUAD. Further study of clinical specimens confirmed that LINC01572 was highly expressed in the tumor tissue of non-small cell lung cancer (NSCLC) patients. In vitro experiments also confirmed that LINC01572 was overexpressed in tumor cell lines. Inhibition of LINC01572 expression significantly impaired cell proliferation, cell migration, and clone formation. Experiments in nude mouse revealed that transplanted tumors with low expression of LINC01572 had significantly slower rates of growth in terms of volume and weight compared to the control group (p < 0.05). In addition, gene set enrichment analysis (GSEA) and immune landscape profiling showed that LINC01572 can promote tumor initiation and progression by deregulating the cell cycle and immunocyte infiltration. CONCLUSIONS: LINC01572 is overexpressed in tumor tissue relative to adjacent normal tissue. Moreover, LUAD patients with high expression of LINC01572 showed a worse survival prognosis. LINC01572 is associated with tumor initiation, progression and immune dysregulation. It therefore has potential value as a novel biomarker and therapeutic target in LUAD.

TRIM27 cooperates with STK38L to inhibit ULK1-mediated autophagy and promote tumorigenesis.

Autophagy represents a fundamental mechanism for maintaining cell survival and tissue homeostasis in response to physiological and pathological stress. Autophagy initiation converges on the FIP200-ATG13-ULK1 complex wherein the serine/threonine kinase ULK1 plays a central role. Here, we reveal that the E3 ubiquitin ligase TRIM27 functions as a negative regulatory component of the FIP200-ATG13-ULK1 complex. TRIM27 directly polyubiquitinates ULK1 at K568 and K571 sites with K48-linked ubiquitin chains, with proteasomal turnover maintaining control over basal ULK1 levels. However, during starvation-induced autophagy, TRIM27 catalyzes non-degradative K6- and K11-linked ubiquitination of the serine/threonine kinase 38-like (STK38L) kinase. In turn, STK38L ubiquitination promotes its activation and phosphorylation of ULK1 at Ser495, rendering ULK1 in a permissive state for TRIM27-mediated hyper-ubiquitination of ULK1. This cooperative mechanism serves to restrain the amplitude and duration of autophagy. Further evidence from mouse models shows that basal autophagy levels are increased in Trim27 knockout mice and that Trim27 differentially regulates tumorigenesis and metastasis. Our study identifies a key role of STK38L-TRIM27-ULK1 signaling axis in negatively controlling autophagy with relevance established in human breast cancer.

KDM6A promotes imatinib resistance through YY1-mediated transcriptional upregulation of TRKA independently of its demethylase activity in chronic myelogenous leukemia.

Rationale: Despite landmark therapy of chronic myelogenous leukemia (CML) with tyrosine kinase inhibitors (TKIs), drug resistance remains problematic. Cancer pathogenesis involves epigenetic dysregulation and in particular, histone lysine demethylases (KDMs) have been implicated in TKI resistance. We sought to identify KDMs with altered expression in CML and define their contribution to imatinib resistance. Methods: Bioinformatics screening compared KDM expression in CML versus normal bone marrow with shRNA knockdown and flow cytometry used to measure effects on imatinib-induced apoptosis in K562 cells. Transcriptomic analyses were performed against KDM6A CRISPR knockout/shRNA knockdown K562 cells along with gene rescue experiments using wildtype and mutant demethylase-dead KDM6A constructs. Co-immunoprecipitation, luciferase reporter and ChIP were employed to elucidate mechanisms of KDM6A-dependent resistance. Results: Amongst five KDMs upregulated in CML, only KDM6A depletion sensitized CML cells to imatinib-induced apoptosis. Re-introduction of demethylase-dead KDM6A as well as wild-type KDM6A restored imatinib resistance. RNA-seq identified NTRK1 gene downregulation after depletion of KDM6A. Moreover, NTRK1 expression positively correlated with KDM6A in a subset of clinical CML samples and KDM6A knockdown in fresh CML isolates decreased NTRK1 encoded protein (TRKA) expression. Mechanistically, KDM6A was recruited to the NTRK1 promoter by the transcription factor YY1 with subsequent TRKA upregulation activating down-stream survival pathways to invoke imatinib resistance. Conclusion: Contrary to its reported role as a tumor suppressor and independent of its demethylase function, KDM6A promotes imatinib-resistance in CML cells. The identification of the KDM6A/YY1/TRKA axis as a novel imatinib-resistance mechanism represents an unexplored avenue to overcome TKI resistance in CML.

BRD4 modulates vulnerability of triple-negative breast cancer to targeting of integrin-dependent signaling pathways.

PURPOSE: Stemming from a myriad of genetic and epigenetic alterations, triple-negative breast cancer (TNBC) is tied to poor clinical outcomes and aspires for individualized therapies. Here we investigated the therapeutic potential of co-inhibiting integrin-dependent signaling pathway and BRD4, a transcriptional and epigenetic mediator, for TNBC. METHODS: Two independent patient cohorts were subjected to bioinformatic and IHC examination for clinical association of candidate cancer drivers. The efficacy and biological bases for co-targeting these drivers were interrogated using cancer cell lines, a protein kinase array, chemical inhibitors, RNAi/CRISPR/Cas9 approaches, and a 4 T1-Balb/c xenograft model. RESULTS: We found that amplification of the chromosome 8q24 region occurred in nearly 20% of TNBC tumors, and that it coincided with co-upregulation or amplification of c-Myc and FAK, a key effector of integrin-dependent signaling. This co-upregulation at the mRNA or protein level correlated with a poor patient survival (p < 0.0109 or p < 0.0402, respectively). Furthermore, we found that 14 TNBC cell lines exhibited high vulnerabilities to the combination of JQ1 and VS-6063, potent pharmacological antagonists of the BRD4/c-Myc and integrin/FAK-dependent pathways, respectively. We also observed a cooperative inhibitory effect of JQ1 and VS-6063 on tumor growth and infiltration of Ly6G+ myeloid-derived suppressor cells in vivo. Finally, we found that JQ1 and VS-6063 cooperatively induced apoptotic cell death by altering XIAP, Bcl2/Bcl-xl and Bim levels, impairing c-Src/p130Cas-, PI3K/Akt- and RelA-associated signaling, and were linked to EMT-inducing transcription factor Snail- and Slug-dependent regulation. CONCLUSION: Based on our results, we conclude that the BRD4/c-Myc- and integrin/FAK-dependent pathways act in concert to promote breast cancer cell survival and poor clinical outcomes. As such, they represent promising targets for a synthetic lethal-type of therapy against TNBC.

Cell-cycle-dependent phosphorylation of RRM1 ensures efficient DNA replication and regulates cancer vulnerability to ATR inhibition.

Ribonucleotide reductase (RNR) catalyzes the rate-limiting step of de novo synthesis of deoxyribonucleotide triphosphates (dNTPs) building blocks for DNA synthesis, and is a well-recognized target for cancer therapy. RNR is a heterotetramer consisting of two large RRM1 subunits and two small RRM2 subunits. RNR activity is greatly stimulated by transcriptional activation of RRM2 during S/G2 phase to ensure adequate dNTP supply for DNA replication. However, little is known about the cell-cycle-dependent regulation of RNR activity through RRM1. Here, we report that RRM1 is phosphorylated at Ser 559 by CDK2/cyclin A during S/G2 phase. And this S559 phosphorylation of RRM1enhances RNR enzymatic activity and is required for maintaining sufficient dNTPs during normal DNA replication. Defective RRM1 S559 phosphorylation causes DNA replication stress, double-strand break, and genomic instability. Moreover, combined targeting of RRM1 S559 phosphorylation and ATR triggers lethal replication stress and profound antitumor effects. Thus, this posttranslational phosphorylation of RRM1 provides an alternative mechanism to finely regulating RNR and therapeutic opportunities for cancer treatment.

Discovery of novel 2-aminonicotinonitrile derivatives with new potential autophagy activity.

Background: To clarify the molecular mechanism of novel 2-aminonicotinonitrile autophagy enhancers, two series of novel 2-aminonicotinonitrile derivatives are synthesized and their structure-activity relationship and biological activity were analyzed. Results & methodology: Structure-activity relationship analysis revealed that substituents at C-4 and C-6 position of 7a contribute to enhance their autophagy-inducing activity, while C-5 position substituents have the opposite effect. The most promising compound 7g showed the strongest autophagy-inducing activity and better antiproliferative activity by inducing cell apoptosis and blocking cell cycle G1 arrest in SGC-7901 cells. Conclusion: The novel 2-aminonicotinonitrile autophagy enhancers were for the first time discovered and 7g might be a promising new autophagy enhancer with potential anticancer activity.

Transgenic expression of Sag/Rbx2 E3 causes early stage tumor promotion, late stage cytogenesis and acinar loss in the Kras-PDAC model.

SAG (Sensitive to Apoptosis Gene), also known as RBX2 or ROC2, is a RING component of CRL (Cullin-RING ligase), required for its activity. Our previous studies showed that Sag/Rbx2 co-operated with Kras or Pten loss to promote tumorigenesis in the lung and prostate, respectively, but antagonized Kras to inhibit skin tumorigenesis, suggesting a tissue/context dependent function of Sag. The role of SAG in KRAS-induced pancreatic tumorigenesis is unknown. In this study, we mined a cancer database and found that SAG is overexpressed in pancreatic cancer tissues and correlates with decreased patient survival. Whether Sag overexpression plays a causal role in pancreatic tumorigenesis is unknown. Here, we reported the generation of Sag transgenic mouse model alone (CS), or in combination with KrasG12D, driven by p48-Cre (KCS mice) for pancreatic specific Sag expression. Sag transgenic expression alone has no phenotypical abnormality, but in combination with KrasG12D promotes ADM (acinar-to-ductal metaplasia) conversion in vitro and mPanIN1 formation in vivo at the early stage, and impairs pancreatic functions at the late stage, as evidenced by poor glucose tolerance and significantly reduced α-Amylase activity, and induction of cytogenesis and acinar cell loss, eventually leading to atrophic pancreata and shortened mouse life-span. Mechanistically, Sag transgenic expression altered several key signaling pathways, particularly inactivation of mTORC1 signaling due to Deptor accumulation, and activation of the antioxidant Nrf2-Nqo1 axis. Thus, Sag plays a stage dependent promotion (early) and fate-changing (late) role during Kras-pancreatic tumorigenesis, likely via regulating its key substrates, which control growth-related signal transduction pathways.

The cross talk of two family members of ß-TrCP in the regulation of cell autophagy and growth.

ß-transducin repeat-containing protein (ß-TrCP), one of the best-characterized substrate recognition components of the SKP1-CUL1-F-box (SCF) E3 ligase, has two distinct paralogs, ß-TrCP1 and ß-TrCP2, expressed in mammals. Through governing the ubiquitination and degradation of numerous key regulators, ß-TrCP1/2 regulates various cellular physiological and pathological processes. However, whether and how these two proteins cross talk and whether they regulate cell autophagy and proliferation in different manners is completely unknown. Herein, we report that ß-TrCP1 and ß-TrCP2 are the physiological substrates of SCF E3 ligase and target each other for degradation that is dependent on their ß-TrCP degron sequences. Furthermore, glucose deprivation activates AMPK kinase to phosphorylate ß-TrCP1 and promotes the subsequent ubiquitination and degradation of ß-TrCP1 by ß-TrCP2, but does not promote ß-TrCP2 degradation by ß-TrCP1. Finally, we found that ß-TrCP2, not ß-TrCP1, preferentially degrades DEPTOR and REDD1, the inhibitors of mTORC1, to activate mTORC1, leading to autophagy inhibition and cell growth. Thus, our study demonstrates that ß-TrCP1 and ß-TrCP2 mutually target each other for degradation and that ß-TrCP2 acts as a dominant paralog in the regulation of cell autophagy and growth, which might be a promising anticancer target.

DEPTOR is an in vivo tumor suppressor that inhibits prostate tumorigenesis via the inactivation of mTORC1/2 signals.

The DEPTOR-mTORC1/2 axis has been shown to play an important, but a context dependent role in the regulation of proliferation and the survival of various cancer cells in cell culture settings. The in vivo role of DEPTOR in tumorigenesis remains elusive. Here we showed that the levels of both DEPTOR protein and mRNA were substantially decreased in human prostate cancer tissues, which positively correlated with disease progression. DEPTOR depletion accelerated proliferation and survival, migration, and invasion in human prostate cancer cells. Mechanistically, DEPTOR depletion not only activated both mTORC1 and mTORC2 signals to promote cell proliferation and survival, but also induced an AKT-dependent epithelial-mesenchymal transition (EMT) and ß-catenin nuclear translocation to promote cell migration and invasion. Abrogation of mTOR or AKT activation rescued the biological consequences of DEPTOR depletion. Importantly, in a Deptor-KO mouse model, Deptor knockout accelerated prostate tumorigenesis triggered by Pten loss via the activation of mTOR signaling. Collectively, our study demonstrates that DEPTOR is a tumor suppressor in the prostate, and its depletion promotes tumorigenesis via the activation of mTORC1 and mTORC2 signals. Thus, DEPTOR reactivation via a variety of means would have therapeutic potential for the treatment of prostate cancer.

Deletion of tetraspanin CD151 alters the Wnt oncogene-induced mammary tumorigenesis: A cell type-linked function and signaling.

Tetraspanin CD151 is increasingly implicated as a multifaceted mediator of cancer development and progression. Here we investigated the role of CD151 in breast cancer in the context of the Wnt oncogenic activation. Our data showed that removal of one or both of CD151 alleles in the MMTV-Wnt1 model significantly decreased the tumor-free survival of mice from 34 weeks on average to 22 weeks and 18 weeks, respectively. This effect coincided with an accelerated tumor growth and an increased number of Ki-67+ proliferative cells. Mechanistically, the CD151-deficient tumors were largely ER+, and exhibited hyperactivation of the Wnt pathway as reflected by a marked upregulation in ß-catenin and Cyclin D1, and their target genes. In addition, E-cadherin displayed a cytosolic distribution and transcription factor Snail was markedly upregulated. Collectively, this data implies that CD151 suppresses the Wnt1-driven tumorigenesis, at least in part, via counteracting the epithelial-mesenchymal transition (EMT)-like program in luminal epithelial cells. Meanwhile, the proportion of tumor cells expressing CK5 or p63, the biomarkers of myoepithelial/basal cells, markedly decreased in the absence of CD151. This change was accompanied by a decreased invasiveness of tumors and their incompetence to form a long-term cell culture. Consistent with this basal cell-linked role, the CD151 downregulation impairs mammosphere formation in MCF-10A cells and the defect was rescued by re-expression of intact CD151 ORF, but not its integrin binding-defective mutant. Overall, our study suggests that CD151 is a key player in the Wnt oncogene-driven tumorigenesis and impacts breast cancer malignancy in a cell type-dependent manner.

Cardiac function modulation depends on the A-kinase anchoring protein complex.

The A-kinase anchoring proteins (AKAPs) are a group of structurally diverse proteins identified in various species and tissues. These proteins are able to anchor protein kinase and other signalling proteins to regulate cardiac function. Acting as a scaffold protein, AKAPs ensure specificity in signal transduction by enzymes close to their appropriate effectors and substrates. Over the decades, more than 70 different AKAPs have been discovered. Accumulative evidence indicates that AKAPs play crucial roles in the functional regulation of cardiac diseases, including cardiac hypertrophy, myofibre contractility dysfunction and arrhythmias. By anchoring different partner proteins (PKA, PKC, PKD and LTCCs), AKAPs take part in different regulatory pathways to function as regulators in the heart, and a damaged structure can influence the activities of these complexes. In this review, we highlight recent advances in AKAP-associated protein complexes, focusing on local signalling events that are perturbed in cardiac diseases and their roles in interacting with ion channels and their regulatory molecules. These new findings suggest that AKAPs might have potential therapeutic value in patients with cardiac diseases, particularly malignant rhythm.

Targeting deubiquitinase USP28 for cancer therapy.

As one of the most important post-translational modifications, ubiquitination plays versatile roles in cancer-related pathways, and is involved in protein metabolism, cell-cycle progression, apoptosis, and transcription. Counteracting the activities of the E3 ligases, the deubiquitylating enzymes have been suggested as another important mechanism to modulate the ubiquitination process, and are implicated in cancer as well. In this article, we review the emerging roles of USP28 in cancer pathways as revealed by recent studies. We discuss the major mechanisms by which USP28 is involved in the cancer-related pathways, whereby USP28 regulates physiological homeostasis of ubiquitination process, DNA-damage response, and cell cycle during genotoxic stress. We further review the studies where USP28 was targeted for treating multiples cancers including non-small cell lung cancer, breast cancer, intestinal cancers, gliomas, and bladder cancer. As a result, the clinical significance of targeting USP28 for cancer therapy merits further exploration and demonstration.

Oral Delivery of Tumor-Targeting Salmonella to Treat Cancer in Mice.

Tumor-targeting bacteria have been developed as novel anticancer agents recently. To achieve their therapeutic effects, bacteria have conventionally been injected intravenously or intraperitoneally into animals or humans. Here, we describe the oral delivery of tumor-targeting Salmonella for cancer therapy in a mouse tumor model. We detail the experimental procedures for establishing a mouse tumor model, preparing bacterial culture, mouse gavage, and detection of the tumor-targeting capability of bacteria administered orally. We also discuss technical notes and provide practical advice that will help the users of this oral delivery model.

Radiosensitization of metformin in pancreatic cancer cells via abrogating the G2 checkpoint and inhibiting DNA damage repair.

Recent evidences have demonstrated the potential of metformin as a novel agent for cancer prevention and treatment. Here, we investigated its ability of radiosensitization and the underlying mechanisms in human pancreatic cancer cells. In this study, we found that metformin at 5 mM concentration enhanced the radiosensitivity of MIA PaCa-2 and PANC-1 cells, with sensitization enhancement ratios of 1.39 and 1.27, respectively. Mechanistically, metformin caused abrogation of the G2 checkpoint and increase of mitotic catastrophe, associated with suppression of Wee1 kinase and in turn CDK1 Tyr15 phosphorylation. Furthermore, metformin inhibited both expression and irradiation-induced foci formation of Rad51, a key player in homologous recombination repair, ultimately leading to persistent DNA damage, as reflected by γ-H2AX and 53BP1 signaling. Finally, metformin-mediated AMPK/mTOR/p70S6K was identified as a possible upstream pathway controlling translational regulation of Wee1 and Rad51. Our data suggest that metformin radiosensitizes pancreatic cancer cells in vitro via abrogation of the G2 checkpoint and inhibition of DNA damage repair. However, the in vivo study is needed to further confirm the findings from the in vitro study.

Erbin is a novel substrate of the Sag-ßTrCP E3 ligase that regulates KrasG12D-induced skin tumorigenesis.

SAG/RBX2 is the RING (really interesting new gene) component of Cullin-RING ligase, which is required for its activity. An organ-specific role of SAG in tumorigenesis is unknown. We recently showed that Sag/Rbx2, upon lung-targeted deletion, suppressed Kras(G12D)-induced tumorigenesis via inactivating NF-κB and mammalian target of rapamycin pathways. In contrast, we report here that, upon skin-targeted deletion, Sag significantly accelerated Kras(G12D)-induced papillomagenesis. In Kras(G12D)-expressing primary keratinocytes, Sag deletion promotes proliferation by inhibiting autophagy and senescence, by inactivating the Ras-Erk pathway, and by blocking reactive oxygen species (ROS) generation. This is achieved by accumulation of Erbin to block Ras activation of Raf and Nrf2 to scavenge ROS and can be rescued by knockdown of Nrf2 or Erbin. Simultaneous one-allele deletion of the Erbin-encoding gene Erbb2ip partially rescued the phenotypes. Finally, we characterized Erbin as a novel substrate of SAG-ßTrCP E3 ligase. By degrading Erbin and Nrf2, Sag activates the Ras-Raf pathway and causes ROS accumulation to trigger autophagy and senescence, eventually delaying Kras(G12D)-induced papillomagenesis and thus acting as a skin-specific tumor suppressor.

Synergistic inhibition of autophagy and neddylation pathways as a novel therapeutic approach for targeting liver cancer.

Liver cancer is the second-most frequent cause of cancer death in the world and is highly treatment resistant. We reported previously that inhibition of neddylation pathway with specific NAE inhibitor MLN4924, suppressed the malignant phenotypes of liver cancer. However, during the process, MLN4924 induces pro-survival autophagy as a mechanism of drug resistance. Here, we report that blockage of autophagy with clinically-available autophagy inhibitors (e.g. chloroquine) significantly enhanced the efficacy of MLN4924 on liver cancer cells by triggering apoptosis. Mechanistically, chloroquine enhanced MLN4924-induced up-regulation of pro-apoptotic proteins (e.g. NOXA) and down-regulation of anti-apoptotic proteins. Importantly, the down-regulation of NOXA expression via siRNA silencing substantially attenuated apoptosis of liver cancer cells. Further mechanistic studies revealed that blockage of autophagy augmented MLN4924-induced DNA damage and reactive oxygen species (ROS) generation. The elimination of DNA damage or blockage of ROS production significantly reduced the expression of NOXA, and thereby attenuated apoptosis and reduced growth inhibition of liver cancer cells. Moreover, blockage of autophagy enhanced the efficacy of MLN4924 in an orthotopic model of human liver cancer, with induction of NOXA and apoptosis in tumor tissues. These findings provide important preclinical evidence for clinical investigation of synergistic inhibition of neddylation and autophagy in liver cancer.

Targeting mcl-1 for radiosensitization of pancreatic cancers.

In order to identify targets whose inhibition may enhance the efficacy of chemoradiation in pancreatic cancer, we previously conducted an RNAi library screen of 8,800 genes. We identified Mcl-1 (myeloid cell leukemia-1), an anti-apoptotic member of the Bcl-2 family, as a target for sensitizing pancreatic cancer cells to chemoradiation. In the present study we investigated Mcl-1 inhibition by either genetic or pharmacological approaches as a radiosensitizing strategy in pancreatic cancer cells. Mcl-1 depletion by siRNA produced significant radiosensitization in BxPC-3 and Panc-1 cells in association with Caspase-3 activation and PARP cleavage, but only minimal radiosensitization in MiaPaCa-2 cells. We next tested the ability of the recently identified, selective, small molecule inhibitor of Mcl-1, UMI77, to radiosensitize in pancreatic cancer cells. UMI77 caused dissociation of Mcl-1 from the pro-apoptotic protein Bak and produced significant radiosensitization in BxPC-3 and Panc-1 cells, but minimal radiosensitization in MiaPaCa-2 cells. Radiosensitization by UMI77 was associated with Caspase-3 activation and PARP cleavage. Importantly, UMI77 did not radiosensitize normal small intestinal cells. In contrast, ABT-737, an established inhibitor of Bcl-2, Bcl-XL, and Bcl-w, failed to radiosensitize pancreatic cancer cells suggesting the unique importance of Mcl-1 relative to other Bcl-2 family members to radiation survival in pancreatic cancer cells. Taken together, these results validate Mcl-1 as a target for radiosensitization of pancreatic cancer cells and demonstrate the ability of small molecules which bind the canonical BH3 groove of Mcl-1, causing displacement of Mcl-1 from Bak, to selectively radiosensitize pancreatic cancer cells.