USP13 drives lung squamous cell carcinoma by switching lung club cell lineage plasticity.

Lung squamous cell carcinoma (LUSC) is associated with high mortality and limited targeted therapies. USP13 is one of the most amplified genes in LUSC, yet its role in lung cancer is largely unknown. Here, we established a novel mouse model of LUSC by overexpressing USP13 on KrasG12D/+; Trp53flox/flox background (KPU). KPU-driven lung squamous tumors faithfully recapitulate key pathohistological, molecular features, and cellular pathways of human LUSC. We found that USP13 altered lineage-determining factors such as NKX2-1 and SOX2 in club cells of the airway and reinforced the fate of club cells to squamous carcinoma development. We showed a strong molecular association between USP13 and c-MYC, leading to the upregulation of squamous programs in murine and human lung cancer cells. Collectively, our data demonstrate that USP13 is a molecular driver of lineage plasticity in club cells and provide mechanistic insight that may have potential implications for the treatment of LUSC.

USP13 interacts with cohesin and regulates its ubiquitination in human cells.

Cohesin is a multiprotein ring complex that regulates 3D genome organization, sister chromatid cohesion, gene expression, and DNA repair. Cohesin is known to be ubiquitinated, although the mechanism, regulation, and effects of cohesin ubiquitination remain poorly defined. We previously used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in human HCT116 cells. Here we report that mass spectrometry analysis of dual-affinity purifications identified the USP13 deubiquitinase as a novel cohesin-interacting protein. Subsequent immunoprecipitation/Western blots confirmed the endogenous interaction in HCT116, 293T, HeLa, and RPE-hTERT cells; demonstrated that the interaction occurs specifically in the soluble nuclear fraction (not in the chromatin); requires the ubiquitin-binding domains (UBA1/2) of USP13; and occurs preferentially during DNA replication. Reciprocal dual-affinity purification of endogenous USP13 followed by mass spectrometry demonstrated that cohesin is its primary interactor in the nucleus. Ectopic expression and CRISPR knockout of USP13 showed that USP13 is paradoxically required for both deubiquitination and ubiquitination of cohesin subunits in human cells. USP13 was dispensable for sister chromatid cohesion in HCT116 and HeLa cells, whereas it was required for the dissociation of cohesin from chromatin as cells transit through mitosis. Together these results identify USP13 as a new cohesin-interacting protein that regulates the ubiquitination of cohesin and its cell cycle regulated interaction with chromatin.

TP53 loss creates therapeutic vulnerability in colorectal cancer.

TP53, a well-known tumour suppressor gene that encodes p53, is frequently inactivated by mutation or deletion in most human tumours. A tremendous effort has been made to restore p53 activity in cancer therapies. However, no effective p53-based therapy has been successfully translated into clinical cancer treatment owing to the complexity of p53 signalling. Here we demonstrate that genomic deletion of TP53 frequently encompasses essential neighbouring genes, rendering cancer cells with hemizygous TP53 deletion vulnerable to further suppression of such genes. POLR2A is identified as such a gene that is almost always co-deleted with TP53 in human cancers. It encodes the largest and catalytic subunit of the RNA polymerase II complex, which is specifically inhibited by α-amanitin. Our analysis of The Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE) databases reveals that POLR2A expression levels are tightly correlated with its gene copy numbers in human colorectal cancer. Suppression of POLR2A with α-amanitin or small interfering RNAs selectively inhibits the proliferation, survival and tumorigenic potential of colorectal cancer cells with hemizygous TP53 loss in a p53-independent manner. Previous clinical applications of α-amanitin have been limited owing to its liver toxicity. However, we found that α-amanitin-based antibody-drug conjugates are highly effective therapeutic agents with reduced toxicity. Here we show that low doses of α-amanitin-conjugated anti-epithelial cell adhesion molecule (EpCAM) antibody lead to complete tumour regression in mouse models of human colorectal cancer with hemizygous deletion of POLR2A. We anticipate that inhibiting POLR2A will be a new therapeutic approach for human cancers containing such common genomic alterations.

A novel non-coding RNA lncRNA-JADE connects DNA damage signalling to histone H4 acetylation.

A prompt and efficient DNA damage response (DDR) eliminates the detrimental effects of DNA lesions in eukaryotic cells. Basic and preclinical studies suggest that the DDR is one of the primary anti-cancer barriers during tumorigenesis. The DDR involves a complex network of processes that detect and repair DNA damage, in which long non-coding RNAs (lncRNAs), a new class of regulatory RNAs, may play an important role. In the current study, we identified a novel lncRNA, lncRNA-JADE, that is induced after DNA damage in an ataxia-telangiectasia mutated (ATM)-dependent manner. LncRNA-JADE transcriptionally activates Jade1, a key component in the HBO1 (human acetylase binding to ORC1) histone acetylation complex. Consequently, lncRNA-JADE induces histone H4 acetylation in the DDR. Markedly higher levels of lncRNA-JADE were observed in human breast tumours in comparison with normal breast tissues. Knockdown of lncRNA-JADE significantly inhibited breast tumour growth in vivo. On the basis of these results, we propose that lncRNA-JADE is a key functional link that connects the DDR to histone H4 acetylation, and that dysregulation of lncRNA-JADE may contribute to breast tumorigenesis.

Targeting tumor suppressor genes for cancer therapy.

Cancer drugs are broadly classified into two categories: cytotoxic chemotherapies and targeted therapies that specifically modulate the activity of one or more proteins involved in cancer. Major advances have been achieved in targeted cancer therapies in the past few decades, which is ascribed to the increasing understanding of molecular mechanisms for cancer initiation and progression. Consequently, monoclonal antibodies and small molecules have been developed to interfere with a specific molecular oncogenic target. Targeting gain-of-function mutations, in general, has been productive. However, it has been a major challenge to use standard pharmacologic approaches to target loss-of-function mutations of tumor suppressor genes. Novel approaches, including synthetic lethality and collateral vulnerability screens, are now being developed to target gene defects in p53, PTEN, and BRCA1/2. Here, we review and summarize the recent findings in cancer genomics, drug development, and molecular cancer biology, which show promise in targeting tumor suppressors in cancer therapeutics.

Reduced Fertility and Altered Epididymal and Sperm Integrity in Mice Lacking ADAM7.

The mammalian epididymis is a highly convoluted tubule that connects the testis to the vas deferens. Its proper functions in sperm transport, storage, and maturation are essential for male reproduction. One of the genes predominantly expressed in the epididymis is ADAM7 (a disintegrin and metalloprotease 7). Previous studies have shown that ADAM7 synthesized in the epididymis is secreted into the epididymal lumen and is then transferred to sperm membranes, where it forms a chaperone complex that is potentially involved in sperm fertility. In this study, we generated and analyzed mice with a targeted disruption in the Adam7 gene. We found that the fertility of male mice was modestly but significantly reduced by knockout of Adam7. Histological analyses revealed that the cell heights of the epithelium were dramatically decreased in the caput of the epididymis of Adam7-null mice, suggesting a requirement for ADAM7 in maintaining the integrity of the epididymal epithelium. We found that sperm from Adam7-null mice exhibit decreased motility, tail deformation, and altered tyrosine phosphorylation, indicating that the absence of ADAM7 leads to abnormal sperm functions and morphology. Western blot analyses revealed reduced levels of integral membrane protein 2B (ITM2B) and ADAM2 in sperm from Adam7-null mice, suggesting a requirement for ADAM7 in normal expression of sperm membrane proteins involved in sperm functions. Collectively, our study demonstrates for the first time that ADAM7 is required for normal fertility and is important for the maintenance of epididymal integrity and for sperm morphology, motility, and membrane proteins.

Crosstalk between the DNA damage response pathway and microRNAs.

MicroRNAs (miRNAs) are a family of small, non-coding RNAs that control gene expression at the post-transcriptional level by destabilizing and inhibiting translation of their target messenger RNAs. MiRNAs are involved in the regulation of a number of fundamental biological processes, and their dysregulation is thought to contribute to several disease processes. Emerging evidence suggests that miRNAs also play a critical role in protecting the heritable genome by contributing to the regulation of the DNA damage response. Consequently, much recent investigative effort has been directed towards an improved understanding of how miRNAs are regulated in response to DNA damage. In this review, we discuss the most recent findings regarding the regulation of miRNA expression and the functional roles of miRNAs in the DNA damage response.

Myosin regulatory light chains are required to maintain the stability of myosin II and cellular integrity.

Myosin II is an actin-binding protein composed of MHC (myosin heavy chain) IIs, RLCs (regulatory light chains) and ELCs (essential light chains). Myosin II expressed in non-muscle tissues plays a central role in cell adhesion, migration and division. The regulation of myosin II activity is known to involve the phosphorylation of RLCs, which increases the Mg2+-ATPase activity of MHC IIs. However, less is known about the details of RLC-MHC II interaction or the loss-of-function phenotypes of non-muscle RLCs in mammalian cells. In the present paper, we investigate three highly conserved non-muscle RLCs of the mouse: MYL (myosin light chain) 12A (referred to as MYL12A), MYL12B and MYL9 (MYL12A/12B/9). Proteomic analysis showed that all three are associated with the MHCs MYH9 (NMHC IIA) and MYH10 (NMHC IIB), as well as the ELC MYL6, in NIH 3T3 fibroblasts. We found that knockdown of MYL12A/12B in NIH 3T3 cells results in striking changes in cell morphology and dynamics. Remarkably, the levels of MYH9, MYH10 and MYL6 were reduced significantly in knockdown fibroblasts. Comprehensive interaction analysis disclosed that MYL12A, MYL12B and MYL9 can all interact with a variety of MHC IIs in diverse cell and tissue types, but do so optimally with non-muscle types of MHC II. Taken together, our study provides direct evidence that normal levels of non-muscle RLCs are essential for maintaining the integrity of myosin II, and indicates that the RLCs are critical for cell structure and dynamics.

A Dual Role of DDX3X in dsRNA-Derived Innate Immune Signaling.

DEAD-Box Helicase 3 X-Linked (DDX3X) is essential for RNA metabolism and participates in various cellular processes involving RNA. DDX3X has been implicated in cancer growth and metastasis. DDX3X is involved in antiviral responses for viral RNAs and contributes to pro- or anti-microbial responses. A better understanding of how human cells regulate innate immune response against the viral "non-self" double-stranded RNAs (dsRNAs) and endogenous viral-like "self" dsRNAs is critical to understanding innate immune sensing, anti-microbial immunity, inflammation, immune cell homeostasis, and developing novel therapeutics for infectious, immune-mediated diseases, and cancer. DDX3X has known for activating the viral dsRNA-sensing pathway and innate immunity. However, accumulating research reveals a more complex role of DDX3X in regulating dsRNA-mediated signaling in cells. Here, we discuss the role of DDX3X in viral dsRNA- or endogenous dsRNA-mediated immune signaling pathways.

CK2-Mediated Phosphorylation Upregulates the Stability of USP13 and Promotes Ovarian Cancer Cell Proliferation.

Ubiquitin-specific Peptidase 13 (USP13) is a deubiquitinating enzyme that regulates the stability or function of its substrate. USP13 is highly amplified in human ovarian cancer, and elevated expression of USP13 promotes tumorigenesis and metastasis of ovarian cancer. However, there is little known about USP13 post-translational modifications and their role in ovarian cancer. Here, we found that USP13 is phosphorylated at Thr122 in ovarian cancer cells. Phosphorylated Thr122 (pT122) on endogenous USP13 was observed in most human ovarian cancer cells, and the abundance of this phosphorylation was correlated to the total level of USP13. We further demonstrated that Casein kinase 2 (CK2) directly interacts with and phosphorylates USP13 at Thr122, which promotes the stability of USP13 protein. Finally, we showed that Threonine 122 is important for cell proliferation of ovarian cancer cells. Our findings may reveal a novel regulatory mechanism for USP13, which may lead to novel therapeutic targeting of USP13 in ovarian cancer.

USP13 promotes development and metastasis of high-grade serous ovarian carcinoma in a novel mouse model.

Epithelial ovarian cancer is the most lethal gynecologic malignancy and one of the most common causes of cancer mortality among women worldwide. Ubiquitin-Specific Peptidase 13 (USP13) gene copy is strongly amplified in human epithelial ovarian cancer, and high USP13 expression is correlated with poor survival outcomes. Yet, its pathological contribution to ovarian tumorigenesis remains unknown. We crossed a conditional Usp13 overexpressing knock-in mouse with a conditional knockout of Trp53 and Pten mouse and generated a novel ovarian cancer genetically engineered mouse model (GEMM), which closely recapitulates the genetic changes driving ovarian cancer in humans. Overexpression of USP13 with deletion of Trp53 and Pten in murine ovarian surface epithelium accelerated ovarian tumorigenesis and led to decreased survival in mice. Notably, USP13 greatly enhanced peritoneal metastasis of ovarian tumors with frequent development of hemorrhagic ascites. The primary and metastatic tumors exhibited morphology and clinical behavior similar to human high-grade serous ovarian cancer. Co-inhibition of USP13 and AKT significantly decreased the viability of the primary murine ovarian cancer cells isolated from the GEMM. USP13 also increased the tumorigenic and metastatic abilities of primary murine ovarian cancer cells in a syngeneic mouse study. These findings suggest a critical role of USP13 in ovarian cancer development and reveal USP13 as a potential therapeutic target for ovarian cancer.

Identification of heat shock protein 5, calnexin and integral membrane protein 2B as Adam7-interacting membrane proteins in mouse sperm.

In mammals, sperm acquire their motility and ability to fertilize eggs in the epididymis. This maturation process involves the acquisition of particular proteins from the epididymis. One such secretory protein specifically expressed in the epididymis is Adam7 (a disintegrin and metalloprotease 7). Previous studies have shown that Adam7 that resides in an intracellular compartment of epididymal cells is transferred to sperm membranes, where its levels are dependent on the expression of Adam2 and Adam3, which have critical roles in fertilization. Here, using a proteomics approach based on mass spectrometry, we identified proteins that interact with Adam7 in sperm membranes. This analysis revealed that Adam7 forms complexes with calnexin (Canx), heat shock protein 5 (Hspa5), and integral membrane protein 2B (Itm2b). Canx and Hspa5 are molecular chaperones, and Itm2b is a type II integral membrane protein implicated in neurodegeneration. The interaction of Adam7 with these proteins was confirmed by immunoprecipitation-Western blot analysis. We found that Adam7 and Itm2b are located in detergent-resistant regions known to be highly correlated with membrane lipid rafts. We further found that the association of Adam7 with Itm2b is remarkably promoted during sperm capacitation owing to a conformational change of Adam7 that occurs in concert with the capacitation process. Thus, our results suggest that Adam7 functions in fertilization through the formation of a chaperone complex and enhanced association with Itm2b during capacitation in sperm.

Targeting DDX3X Triggers Antitumor Immunity via a dsRNA-Mediated Tumor-Intrinsic Type I Interferon Response.

Induction of nucleic acid sensing-mediated type I interferon (IFN) has emerged as a novel approach to activate the immune system against cancer. Here we show that the depletion of DEAD-box RNA helicase 3X (DDX3X) triggers a tumor-intrinsic type I IFN response in breast cancer cells. Depletion or inhibition of DDX3X activity led to aberrant cytoplasmic accumulation of cellular endogenous double-stranded RNAs (dsRNA), which triggered type I IFN production through the melanoma differentiation-associated gene 5 (MDA5)-mediated dsRNA-sensing pathway. Furthermore, DDX3X interacted with dsRNA-editing ADAR1 and dual depletion of DDX3X and ADAR1 synergistically activated the cytosolic dsRNA pathway in breast cancer cells. Loss of DDX3X in mouse mammary tumors enhanced antitumor activity by increasing the tumor-intrinsic type I IFN response, antigen presentation, and tumor infiltration of cytotoxic T and dendritic cells. These findings may lead to the development of a novel therapeutic approach for breast cancer by targeting DDX3X in combination with immune-checkpoint blockade. SIGNIFICANCE: This study elucidates the novel role of DDX3X in regulating endogenous cellular dsRNA homeostasis and type I IFN signaling in breast cancer. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/13/3607/F1.large.jpg.

Characterization of eight novel proteins with male germ cell-specific expression in mouse.

BACKGROUND: Spermatogenesis and fertilization are highly unique processes. Discovery and characterization of germ cell-specific genes are important for the understanding of these reproductive processes. We investigated eight proteins encoded by novel spermatogenic cell-specific genes previously identified from the mouse round spermatid UniGene library. METHODS: Polyclonal antibodies were generated against the novel proteins and western blot analysis was performed with various protein samples. Germ cell specificity was investigated using testes from germ cell-less mutant mice. Developmental expression pattern was examined in testicular germ cells, testicular sperm and mature sperm. Subcellular localization was assessed by cell surface biotin labeling and trypsinization. Protein localization and properties in sperm were investigated by separation of head and tail fractions, and extractabilities by a non-ionic detergent and urea. RESULTS: The authenticity of the eight novel proteins and their specificity to spermatogenic cells were confirmed. In examining the developmental expression patterns, we found the presence of four proteins only in testicular germ cells, a single protein in testicular germ cells and testicular sperm, and three proteins in the testicular stages and mature sperm from the epididymis. Further analysis of the three proteins present in sperm disclosed that one is located at the surface of the acrosomal region and the other two are associated with cytoskeletal structures in the sperm flagellum. We name the genes for these sperm proteins Shsp1 (Sperm head surface protein 1), Sfap1 (Sperm flagellum associated protein 1) and Sfap2 (Sperm flagellum associated protein 2). CONCLUSION: We analyzed eight novel germ cell-specific proteins, providing new and inclusive information about their developmental and cellular characteristics. Our findings will facilitate future investigation into the biological roles of these novel proteins in spermatogenesis and sperm functions.

Regulation of protein metabolism in cancer.

Our recent studies determined molecular interactions between genes in the ubiquitin-proteasome pathways and cancer cell metabolism. Ubiquitin-specific peptidase 13 (USP13) specifically deubiquitinates and thus upregulates ATP citrate lyase and oxoglutarate dehydrogenase that drive ovarian cancer metabolism. These findings may lead to the development of USP13 inhibitors and new-targeted therapies in ovarian cancers.

Targeting 17q23 amplicon to overcome the resistance to anti-HER2 therapy in HER2+ breast cancer.

Chromosome 17q23 amplification occurs in ~11% of human breast cancers. Enriched in HER2+ breast cancers, the 17q23 amplification is significantly correlated with poor clinical outcomes. In addition to the previously identified oncogene WIP1, we uncover an oncogenic microRNA gene, MIR21, in a majority of the WIP1-containing 17q23 amplicons. The 17q23 amplification results in aberrant expression of WIP1 and miR-21, which not only promotes breast tumorigenesis, but also leads to resistance to anti-HER2 therapies. Inhibiting WIP1 and miR-21 selectively inhibits the proliferation, survival and tumorigenic potential of the HER2+ breast cancer cells harboring 17q23 amplification. To overcome the resistance of trastuzumab-based therapies in vivo, we develop pH-sensitive nanoparticles for specific co-delivery of the WIP1 and miR-21 inhibitors into HER2+ breast tumors, leading to a profound reduction of tumor growth. These results demonstrate the great potential of the combined treatment of WIP1 and miR-21 inhibitors for the trastuzumab-resistant HER2+ breast cancers.

Differentially expressed genes implicated in unexplained recurrent spontaneous abortion.

Recurrent spontaneous abortion occurs in approximately 3% of women with diagnosed pregnancies. The etiology in approximately 40% of recurrent spontaneous abortion is unexplained. To elucidate unexplained recurrent spontaneous abortion at the molecular level, we systemically identified differentially expressed genes during implantation window period in unexplained recurrent spontaneous abortion and characterized their functions in a human endometrial cell line. Expression levels of implantation-related genes selected from previously reported, various microarray data were determined to identify differentially expressed genes between normal fertile and unexplained recurrent spontaneous abortion subjects by real-time quantitative RT-PCR. Of 29 implantation-related genes, the transcript levels of cellular retinoic acid binding protein 2 and olfactomedin 1 were higher, whereas that of complement component 4 binding protein alpha was lower in subjects with unexplained recurrent spontaneous abortion, compared to normal fertile subjects. A correlation was evident between the transcript and protein levels of complement component 4 binding protein alpha and cellular retinoic acid binding protein 2. Expression of cellular retinoic acid binding protein 2 was positively correlated with retinoic acid-related genes in normal fertile subjects, but no significant association was observed in unexplained recurrent spontaneous abortion subjects. In relation to complement component 4 binding protein alpha, C5a receptor protein level was significantly higher in subjects with unexplained recurrent spontaneous abortion. Stable expression of cellular retinoic acid binding protein 2 and olfactomedin 1 in a human endometrial cell line inhibited cell growth and induced cell accumulation in the S and G(2)-M phase fractions, but did not trigger apoptosis. This study represents the first systematic identification of differentially expressed genes in unexplained recurrent spontaneous abortion. Defective cell growth by the differentially expressed genes suggests their implication in implantation failure in women with unexplained recurrent spontaneous abortion.

Integrative characterization of germ cell-specific genes from mouse spermatocyte UniGene library.

BACKGROUND: The primary regulator of spermatogenesis, a highly ordered and tightly regulated developmental process, is an intrinsic genetic program involving male germ cell-specific genes. RESULTS: We analyzed the mouse spermatocyte UniGene library containing 2155 gene-oriented transcript clusters. We predict that 11% of these genes are testis-specific and systematically identified 24 authentic genes specifically and abundantly expressed in the testis via in silico and in vitro approaches. Northern blot analysis disclosed various transcript characteristics, such as expression level, size and the presence of isoform. Expression analysis revealed developmentally regulated and stage-specific expression patterns in all of the genes. We further analyzed the genes at the protein and cellular levels. Transfection assays performed using GC-2 cells provided information on the cellular characteristics of the gene products. In addition, antibodies were generated against proteins encoded by some of the genes to facilitate their identification and characterization in spermatogenic cells and sperm. Our data suggest that a number of the gene products are implicated in transcriptional regulation, nuclear integrity, sperm structure and motility, and fertilization. In particular, we found for the first time that Mm.333010, predicted to contain a trypsin-like serine protease domain, is a sperm acrosomal protein. CONCLUSION: We identify 24 authentic genes with spermatogenic cell-specific expression, and provide comprehensive information about the genes. Our findings establish a new basis for future investigation into molecular mechanisms underlying male reproduction.

Systematic identification and integrative analysis of novel genes expressed specifically or predominantly in mouse epididymis.

BACKGROUND: Maturation of spermatozoa, including development of motility and the ability to fertilize the oocyte, occurs during transit through the microenvironment of the epididymis. Comprehensive understanding of sperm maturation requires identification and characterization of unique genes expressed in the epididymis. RESULTS: We systematically identified 32 novel genes with epididymis-specific or -predominant expression in the mouse epididymis UniGene library, containing 1505 gene-oriented transcript clusters, by in silico and in vitro analyses. The Northern blot analysis revealed various characteristics of the genes at the transcript level, such as expression level, size and the presence of isoform. We found that expression of the half of the genes is regulated by androgens. Further expression analyses demonstrated that the novel genes are region-specific and developmentally regulated. Computational analysis showed that 15 of the genes lack human orthologues, suggesting their implication in male reproduction unique to the mouse. A number of the novel genes are putative epididymal protease inhibitors or beta-defensins. We also found that six of the genes have secretory activity, indicating that they may interact with sperm and have functional roles in sperm maturation. CONCLUSION: We identified and characterized 32 novel epididymis-specific or -predominant genes by an integrative approach. Our study is unique in the aspect of systematic identification of novel epididymal genes and should be a firm basis for future investigation into molecular mechanisms underlying sperm maturation in the epididymis.