Slow-cycling (dormant) cancer cells in therapy resistance, cancer relapse and metastasis.

It is increasingly appreciated that cancer cell heterogeneity and plasticity constitute major barriers to effective clinical treatments and long-term therapeutic efficacy. Research in the past two decades suggest that virtually all treatment-naive human cancers harbor subsets of cancer cells that possess many of the cardinal features of normal stem cells. Such stem-like cancer cells, operationally defined as cancer stem cells (CSCs), are frequently quiescent and dynamically change and evolve during tumor progression and therapeutic interventions. Intrinsic tumor cell heterogeneity is reflected in a different aspect in that tumors also harbor a population of slow-cycling cells (SCCs) that are not in the proliferative cell cycle and thus are intrinsically refractory to anti-mitotic drugs. In this Perspective, we focus our discussions on SCCs in cancer and on various methodologies that can be employed to enrich and purify SCCs, compare the similarities and differences between SCCs, CSCs and cancer cells undergoing EMT, and present evidence for the involvement of SCCs in surviving anti-neoplastic treatments, mediating tumor relapse, maintaining tumor dormancy and mediating metastatic dissemination. Our discussions make it clear that an in-depth understanding of the biological properties of SCCs in cancer will be instrumental to developing new therapeutic strategies to prevent tumor relapse and distant metastasis.

USP22 controls multiple signaling pathways that are essential for vasculature formation in the mouse placenta.

USP22, a component of the SAGA complex, is overexpressed in highly aggressive cancers, but the normal functions of this deubiquitinase are not well defined. We determined that loss of USP22 in mice results in embryonic lethality due to defects in extra-embryonic placental tissues and failure to establish proper vascular interactions with the maternal circulatory system. These phenotypes arise from abnormal gene expression patterns that reflect defective kinase signaling, including TGFß and several receptor tyrosine kinase pathways. USP22 deletion in endothelial cells and pericytes that are induced from embryonic stem cells also hinders these signaling cascades, with detrimental effects on cell survival and differentiation as well as on the ability to form vessels. Our findings provide new insights into the functions of USP22 during development that may offer clues to its role in disease states.

Concise Review: NANOG in Cancer Stem Cells and Tumor Development: An Update and Outstanding Questions.

The homeobox domain transcription factor NANOG, a key regulator of embryonic development and cellular reprogramming, has been reported to be broadly expressed in human cancers. Functional studies have provided strong evidence that NANOG possesses protumorigenic attributes. In addition to promoting self-renewal and long-term proliferative potential of stem-like cancer cells, NANOG-mediated oncogenic reprogramming may underlie clinical manifestations of malignant disease. In this review, we examine the molecular origin, expression, biological activities, and mechanisms of action of NANOG in various malignancies. We also consider clinical implications such as correlations between NANOG expression and cancer prognosis and/or response to therapy. We surmise that NANOG potentiates the molecular circuitry of tumorigenesis, and thus may represent a novel therapeutic target or biomarker for the diagnosis, prognosis, and treatment outcome of cancer. Finally, we present critical pending questions relating NANOG to cancer stem cells and tumor development.

Regulation of NANOG in cancer cells.

As one of the key pluripotency transcription factors, NANOG plays a critical role in maintaining the self-renewal and pluripotency in normal embryonic stem cells. Recent data indicate that NANOG is expressed in a variety of cancers and its expression correlates with poor survival in cancer patients. Of interest, many studies suggest that NANOG enhances the defined characteristics of cancer stem cells and may thus function as an oncogene to promote carcinogenesis. Therefore, NANOG expression determines the cell fate not only in pluripotent cells but also in cancer cells. Although the regulation of NANOG in normal embryonic stem cells is reasonably well understood, the regulation of NANOG in cancer cells has only emerged recently. The current review provides a most updated summary on how NANOG expression is regulated during tumor development and progression.

Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control.

p38 mitogen-activated protein kinases (MAPKs) participate in autophagic signaling; and previous reports suggest that pyridinyl imidazole p38 MAPK inhibitors, including SB203580 and SB202190, induce cell death in some cancer cell-types through unrestrained autophagy. Subsequent studies, however, have suggested that the associated cytoplasmic vacuolation resulted from off-target inhibition of an unidentified enzyme. Herein, we report that SB203580-induced vacuolation is rapid, reversible, and relies on the class III phosphatidylinositol 3-kinase (PIK3C3) complex and the production of phosphatidylinositol 3-phosphate [PI(3)P] but not on autophagy per se. Rather, vacuolation resulted from the accumulation of Rab7 on late endosome and lysosome (LEL) membranes, combined with an osmotic imbalance that triggered severe swelling in these organelles. Inhibition of PIKfyve, the lipid kinase that converts PI(3)P to PI(3,5)P2 on LEL membranes, produced a similar phenotype in cells; therefore, we performed in vitro kinase assays and discovered that both SB203580 and SB202190 directly inhibited recombinant PIKfyve. Cancer cells treated with either drug likewise displayed significant reductions in the endogenous levels of PI(3,5)P2. Despite these results, SB203580-induced vacuolation was not entirely due to off-target inhibition of PIKfyve, as a drug-resistant p38α mutant suppressed vacuolation; and combined genetic deletion of both p38α and p38ß dramatically sensitized cells to established PIKfyve inhibitors, including YM201636 and apilimod. The rate of vacuole dissolution (i.e., LEL fission), following the removal of apilimod, was also significantly reduced in cells treated with BIRB-796, a structurally unrelated p38 MAPK inhibitor. Thus, our studies indicate that pyridinyl imidazole p38 MAPK inhibitors induce cytoplasmic vacuolation through the combined inhibition of both PIKfyve and p38 MAPKs, and more generally, that p38 MAPKs act epistatically to PIKfyve, most likely to promote LEL fission.

NDUFS4 regulates cristae remodeling in diabetic kidney disease.

The mitochondrial electron transport chain (ETC) is a highly adaptive process to meet metabolic demands of the cell, and its dysregulation has been associated with diverse clinical pathologies. However, the role and nature of impaired ETC in kidney diseases remains poorly understood. Here, we generate diabetic mice with podocyte-specific overexpression of Ndufs4, an accessory subunit of mitochondrial complex I, as a model investigate the role of ETC integrity in diabetic kidney disease (DKD). We find that conditional male mice with genetic overexpression of Ndufs4 exhibit significant improvements in cristae morphology, mitochondrial dynamics, and albuminuria. By coupling proximity labeling with super-resolution imaging, we also identify the role of cristae shaping protein STOML2 in linking NDUFS4 with improved cristae morphology. Together, we provide the evidence on the central role of NDUFS4 as a regulator of cristae remodeling and mitochondrial function in kidney podocytes. We propose that targeting NDUFS4 represents a promising approach to slow the progression of DKD.

Mitochondrial defects and metabolic vulnerabilities in Lynch syndrome-associated MSH2-deficient endometrial cancer.

Lynch syndrome (LS) is defined by inherited mutations in DNA mismatch repair genes, including MSH2, and carries 60% lifetime risk of developing endometrial cancer (EC). Beyond hypermutability, specific mechanisms for LS-associated endometrial carcinogenesis are not well understood. Here, we assessed the effects of MSH2 loss on EC pathogenesis using a novel mouse model (PR-Cre Msh2 flox/flox , abbreviated Msh2KO), primary cell lines established from this model, human tissues, and human EC cell lines with isogenic MSH2 knockdown. Beginning at eight months of age, 30% of Msh2KO mice exhibited endometrial atypical hyperplasia (AH), a precancerous lesion. At 12 to 16 months of age, 47% of Msh2KO mice exhibited either AH or ECs with histologic features similar to human LS-related ECs. Transcriptomic profiling of EC from Msh2KO mice revealed a transcriptomic signature for mitochondrial dysfunction. Studies in vitro and in vivo revealed mitochondrial dysfunction based upon two mechanisms: marked mitochondrial content reduction, along with pronounced disruptions to the integrity of retained mitochondria. Human LS-related ECs also exhibited mitochondrial content reduction compared with non-LS-related ECs. Functional studies revealed metabolic reprogramming of MSH2-deficient EC cells in vitro , including reduced oxidative phosphorylation and increased susceptibility to glycolysis suppression. We are the first to identify mitochondrial dysfunction and metabolic disruption as a consequence of MSH2 deficiency-related EC. Mitochondrial and metabolic aberrations should be evaluated as novel biomarkers for endometrial carcinogenesis or risk stratification and could serve as targets for cancer interception in women with LS. Significance: This is the first study to report mitochondrial dysfunction contributing to MSH2-deficient endometrial cancer development, identifying a noncanonical pathway for MSH2 deficient carcinogenesis, which also imparts vulnerability to metabolic targeting.

STAT3 protects hematopoietic stem cells by preventing activation of a deleterious autocrine type-I interferon response.

Hematopoietic stem and progenitor cells (HSPCs) maintain blood-forming and immune activity, yet intrinsic regulators of HSPCs remain elusive. STAT3 function in HSPCs has been difficult to dissect as Stat3-deficiency in the hematopoietic compartment induces systemic inflammation, which can impact HSPC activity. Here, we developed mixed bone marrow (BM) chimeric mice with inducible Stat3 deletion in 20% of the hematopoietic compartment to avoid systemic inflammation. Stat3-deficient HSPCs were significantly impaired in reconstitution ability following primary or secondary bone marrow transplantation, indicating hematopoietic stem cell (HSC) defects. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells (LSKs) revealed aberrant activation of cell cycle, p53, and interferon (IFN) pathways in Stat3-deficient HSPCs. Stat3-deficient LSKs accumulated γH2AX and showed increased expression of DNA sensors and type-I IFN (IFN-I), while treatment with A151-ODN inhibited expression of IFN-I and IFN-responsive genes. Further, the blockade of IFN-I receptor signaling suppressed aberrant cell cycling, STAT1 activation, and nuclear p53 accumulation. Collectively, our results show that STAT3 inhibits a deleterious autocrine IFN response in HSCs to maintain long-term HSC function. These data signify the importance of ensuring therapeutic STAT3 inhibitors are targeted specifically to diseased cells to avoid off-target loss of healthy HSPCs.

The cancer gene WWOX behaves as an inhibitor of SMAD3 transcriptional activity via direct binding.

BACKGROUND: The WW domain containing protein WWOX has been postulated to behave as a tumor suppressor in breast and other cancers. Expression of this protein is lost in over 70% of ER negative tumors. This prompted us to investigate the phenotypic and gene expression effects of loss of WWOX expression in breast cells. METHODS: Gene expression microarrays and standard in vitro assays were performed on stably silenced WWOX (shRNA) normal breast cells. Bioinformatic analyses were used to identify gene networks and transcriptional regulators affected by WWOX silencing. Co-immunoprecipitations and GST-pulldowns were used to demonstrate a direct interaction between WWOX and SMAD3. Reporter assays, ChIP, confocal microscopy and in silico analyses were employed to determine the effect of WWOX silencing on TGFß-signaling. RESULTS: WWOX silencing affected cell proliferation, motility, attachment and deregulated expression of genes involved in cell cycle, motility and DNA damage. Interestingly, we detected an enrichment of targets activated by the SMAD3 transcription factor, including significant upregulation of ANGPTL4, FST, PTHLH and SERPINE1 transcripts. Importantly, we demonstrate that the WWOX protein physically interacts with SMAD3 via WW domain 1. Furthermore, WWOX expression dramatically decreases SMAD3 occupancy at the ANGPTL4 and SERPINE1 promoters and significantly quenches activation of a TGFß responsive reporter. Additionally, WWOX expression leads to redistribution of SMAD3 from the nuclear to the cytoplasmic compartment. Since the TGFß target ANGPTL4 plays a key role in lung metastasis development, we performed a meta-analysis of ANGPTL4 expression relative to WWOX in microarray datasets from breast carcinomas. We observed a significant inverse correlation between WWOX and ANGPTL4. Furthermore, the WWOX(lo)/ANGPTL4(hi) cluster of breast tumors is enriched in triple-negative and basal-like sub-types. Tumors with this gene expression signature could represent candidates for anti-TGFß targeted therapies. CONCLUSIONS: We show for the first time that WWOX modulates SMAD3 signaling in breast cells via direct WW-domain mediated binding and potential cytoplasmic sequestration of SMAD3 protein. Since loss of WWOX expression increases with breast cancer progression and it behaves as an inhibitor of SMAD3 transcriptional activity these observations may help explain, at least in part, the paradoxical pro-tumorigenic effects of TGFß signaling in advanced breast cancer.

WWOX P47T partial loss-of-function mutation induces epilepsy, progressive neuroinflammation, and cerebellar degeneration in mice phenocopying human SCAR12.

WWOX gene loss-of-function (LoF) has been associated with neuropathologies resulting in developmental, epileptic, and ataxic phenotypes of varying severity based on the level of WWOX dysfunction. WWOX gene biallelic germline variant p.Pro47Thr (P47T) has been causally associated with a new form of autosomal recessive cerebellar ataxia with epilepsy and intellectual disability (SCAR12, MIM:614322). This mutation affecting the WW1 protein binding domain of WWOX, impairs its interaction with canonical proline-proline-X-tyrosine motifs in partner proteins. We generated a mutant knock-in mouse model of Wwox P47T mutation that phenocopies human SCAR12. WwoxP47T/P47T mice displayed epilepsy, profound social behavior and cognition deficits, and poor motor coordination, and unlike KO models that survive only for 1 month, live beyond 1 year of age. These deficits progressed with age and mice became practically immobile, suggesting severe cerebellar dysfunction. WwoxP47T/P47T mice brains revealed signs of progressive neuroinflammation with elevated astro-microgliosis that increased with age. Cerebellar cortex displayed significantly reduced molecular and granular layer thickness and a strikingly reduced number of Purkinje cells with degenerated dendrites. Transcriptome profiling from various brain regions of WW domain LoF mice highlighted widespread changes in neuronal and glial pathways, enrichment of bioprocesses related to neuroinflammation, and severe cerebellar dysfunction. Our results show significant pathobiological effects and potential mechanisms through which WWOX partial LoF leads to epilepsy, cerebellar neurodegeneration, neuroinflammation, and ataxia. Additionally, the mouse model described here will be a useful tool to understand the role of WWOX in common neurodegenerative conditions in which this gene has been identified as a novel risk factor.

PRMT blockade induces defective DNA replication stress response and synergizes with PARP inhibition.

Multiple cancers exhibit aberrant protein arginine methylation by both type I arginine methyltransferases, predominately protein arginine methyltransferase 1 (PRMT1) and to a lesser extent PRMT4, and by type II PRMTs, predominately PRMT5. Here, we perform targeted proteomics following inhibition of PRMT1, PRMT4, and PRMT5 across 12 cancer cell lines. We find that inhibition of type I and II PRMTs suppresses phosphorylated and total ATR in cancer cells. Loss of ATR from PRMT inhibition results in defective DNA replication stress response activation, including from PARP inhibitors. Inhibition of type I and II PRMTs is synergistic with PARP inhibition regardless of homologous recombination function, but type I PRMT inhibition is more toxic to non-malignant cells. Finally, we demonstrate that the combination of PARP and PRMT5 inhibition improves survival in both BRCA-mutant and wild-type patient-derived xenografts without toxicity. Taken together, these results demonstrate that PRMT5 inhibition may be a well-tolerated approach to sensitize tumors to PARP inhibition.

SPINDOC binds PARP1 to facilitate PARylation.

SPINDOC is tightly associated with the histone H3K4me3 effector protein SPIN1. To gain a better understanding of the biological roles of SPINDOC, we identified its interacting proteins. Unexpectedly, SPINDOC forms two mutually exclusive protein complexes, one with SPIN1 and the other with PARP1. Consistent with its ability to directly interact with PARP1, SPINDOC expression is induced by DNA damage, likely by KLF4, and recruited to DNA lesions with dynamics that follows PARP1. In SPINDOC knockout cells, the levels of PARylation are reduced, in both the absence and presence of DNA damage. The SPINDOC/PARP1 interaction promotes the clearance of PARP1 from damaged DNA, and also impacts the expression of known transcriptional targets of PARP1. To address the in vivo roles of SPINDOC in PARP1 regulation, we generate SPINDOC knockout mice, which are viable, but slightly smaller than their wildtype counterparts. The KO mice display reduced levels of PARylation and, like PARP1 KO mice, are hypersensitive to IR-induced DNA damage. The findings identify a SPIN1-independent role for SPINDOC in the regulation of PARP1-mediated PARylation and the DNA damage response.

Functional evidence that the self-renewal gene NANOG regulates human tumor development.

Tumor development has long been known to resemble abnormal embryogenesis. The embryonic stem cell (ESC) self-renewal gene NANOG is purportedly expressed by some epithelial cancer cells but a causal role in tumor development has remained unclear. Here, we provide compelling evidence that cultured cancer cells, as well as xenograft- and human primary prostate cancer cells express a functional variant of NANOG. NANOG mRNA in cancer cells is derived predominantly from a retrogene locus termed NANOGP8. NANOG protein is detectable in the nucleus of cancer cells and is expressed higher in patient prostate tumors than matched benign tissues. NANOGP8 mRNA and/or NANOG protein levels are enriched in putative cancer stem/progenitor cell populations. Importantly, extensive loss-of-function analysis reveals that RNA interference-mediated NANOG knockdown inhibits tumor development, establishing a functional significance for NANOG expression in cancer cells. Nanog short hairpin RNA transduced cancer cells exhibit decreased long-term clonal and clonogenic growth, reduced proliferation and, in some cases, altered differentiation. Thus, our results demonstrate that NANOG, a cell-fate regulatory molecule known to be important for ESC self-renewal, also plays a novel role in tumor development.

Abnormal differentiation, hyperplasia and embryonic/perinatal lethality in BK5-T/t transgenic mice.

The cell-of-origin has a great impact on the types of tumors that develop and the stem/progenitor cells have long been considered main targets of malignant transformation. The SV40 (SV40-Simian Virus 40) large T and small t antigens (T/t), have been targeted to multiple-differentiated cellular compartments in transgenic mice. In most of these studies, transgenic animals develop tumors without apparent defects in animal development. In this study, we used the bovine keratin 5 (BK5) promoter to target the T/t antigens to stem/progenitor cell-containing cytokeratin 5 (CK5) cellular compartment. A transgene construct, BK5-T/t, was made and microinjected into the male pronucleus of FVB/N mouse oocytes. After implanting approximately 1700 embryos, only 7 transgenics were obtained, including 4 embryos (E9.5, E13, E15, and E20) and 3 postnatal animals, which died at P1, P2, and P18, respectively. Immunohistological analysis revealed aberrant differentiation and prominent hyperplasia in several transgenic CK5 tissues, especially the upper digestive organs (tongue, oral mucosa, esophagus, and forestomach) and epidermis, the latter of which also showed focal dysplasia. Altogether, these results indicate that constitutive expression of the T/t antigens in CK5 cellular compartment results in abnormal epithelial differentiation and leads to embryonic/perinatal animal lethality.

Author Correction: LRIG1 is a pleiotropic androgen receptor-regulated feedback tumor suppressor in prostate cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Methodologies in assaying prostate cancer stem cells.

The cancer stem cell (CSC) theory posits that only a small population of tumor cells within the tumor has the ability to reinitiate tumor development and is responsible for tumor homeostasis and progression. Tumor initiation is a defining property of putative CSCs, which have been reported in both blood malignancies and solid tumors. In order to test whether any given human tumor cell population has CSC properties, the relatively enriched single cells have to be put into a foreign microenvironment in a recipient animal to test their tumorigenic potential. Furthermore, various in vitro assays need be performed to demonstrate that the presumed CSCs have certain biological properties normally associated with the stem cells (SCs). Herein, we present a comprehensive review of the experimental methodologies that our lab has been using in assaying putative prostate cancer (PCa) SCs in culture, xenograft tumors, and primary tumor samples.

LRIG1 is a pleiotropic androgen receptor-regulated feedback tumor suppressor in prostate cancer.

LRIG1 has been reported to be a tumor suppressor in gastrointestinal tract and epidermis. However, little is known about the expression, regulation and biological functions of LRIG1 in prostate cancer (PCa). We find that LRIG1 is overexpressed in PCa, but its expression correlates with better patient survival. Functional studies reveal strong tumor-suppressive functions of LRIG1 in both AR+ and AR- xenograft models, and transgenic expression of LRIG1 inhibits tumor development in Hi-Myc and TRAMP models. LRIG1 also inhibits castration-resistant PCa and exhibits therapeutic efficacy in pre-established tumors. We further show that 1) AR directly transactivates LRIG1 through binding to several AR-binding sites in LRIG1 locus, and 2) LRIG1 dampens ERBB expression in a cell type-dependent manner and inhibits ERBB2-driven tumor growth. Collectively, our study indicates that LRIG1 represents a pleiotropic AR-regulated feedback tumor suppressor that functions to restrict oncogenic signaling from AR, Myc, ERBBs, and, likely, other oncogenic drivers.

Histone 2B-GFP Label-Retaining Prostate Luminal Cells Possess Progenitor Cell Properties and Are Intrinsically Resistant to Castration.

The existence of slow-cycling luminal cells in the prostate has been suggested, but their identity and functional properties remain unknown. Using a bigenic mouse model to earmark, isolate, and characterize the quiescent stem-like cells, we identify a label-retaining cell (LRC) population in the luminal cell layer as luminal progenitors. Molecular and biological characterizations show that these luminal LRCs are significantly enriched in the mouse proximal prostate, exhibit relative dormancy, display bipotency in both in vitro and in vivo assays, and express a stem/progenitor gene signature with resemblance to aggressive prostate cancer. Importantly, these LRCs, compared with bulk luminal cells, maintain a lower level of androgen receptor (AR) expression and are less androgen dependent and also castration resistant in vivo. Finally, analysis of phenotypic markers reveals heterogeneity within the luminal progenitor cell pool. Our study establishes luminal LRCs as progenitors that may serve as a cellular origin for castration-resistant prostate cancer.

GCN5 Regulates FGF Signaling and Activates Selective MYC Target Genes during Early Embryoid Body Differentiation.

Precise control of gene expression during development is orchestrated by transcription factors and co-regulators including chromatin modifiers. How particular chromatin-modifying enzymes affect specific developmental processes is not well defined. Here, we report that GCN5, a histone acetyltransferase essential for embryonic development, is required for proper expression of multiple genes encoding components of the fibroblast growth factor (FGF) signaling pathway in early embryoid bodies (EBs). Gcn5-/- EBs display deficient activation of ERK and p38, mislocalization of cytoskeletal components, and compromised capacity to differentiate toward mesodermal lineage. Genomic analyses identified seven genes as putative direct targets of GCN5 during early differentiation, four of which are cMYC targets. These findings established a link between GCN5 and the FGF signaling pathway and highlighted specific GCN5-MYC partnerships in gene regulation during early differentiation.