KISS1 metastasis suppressor in tumor dormancy: a potential therapeutic target for metastatic cancers?

Present therapeutic approaches do not effectively target metastatic cancers, often limited by their inability to eliminate already-seeded non-proliferative, growth-arrested, or therapy-resistant tumor cells. Devising effective approaches targeting dormant tumor cells has been a focus of cancer clinicians for decades. However, progress has been limited due to limited understanding of the tumor dormancy process. Studies on tumor dormancy have picked up pace and have resulted in the identification of several regulators. This review focuses on KISS1, a metastasis suppressor gene that suppresses metastasis by keeping tumor cells in a state of dormancy at ectopic sites. The review explores mechanistic insights of KISS1 and discusses its potential application as a therapeutic against metastatic cancers by eliminating quiescent cells or inducing long-term dormancy in tumor cells.

A community engagement training program for basic and translational cancer researchers.

PURPOSE: There is an increasing awareness of the importance of patient engagement in cancer research, but many basic and translational researchers have never been trained to do so. To address this unmet need, a 1-year patient engagement training program for researchers was developed. METHODS: Eleven researchers and eleven paired research advocates participated. This program, designed for virtual delivery, included 3 didactic modules focused on (1) Community Outreach and Engagement principles and methods, (2) Communication skills, and (3) Team Science. This was followed by longitudinal projects to be completed by the researcher/advocate pairs, including learning about the research project, and co-authoring abstracts, manuscripts and grant proposals. Monthly group meetings allowed pairs to share their experiences. The program culminated in the pairs creating and presenting oral abstracts for the University of Kansas Cancer Center's Annual Research Symposium. RESULTS: All participants indicated that the modules had a positive impact on their ability to collaborate in research. Both researcher self-evaluations and patient advocate evaluations of their researcher partner showed an improvement in researcher communication competency. Results from the Patient Engagement in Research Scale showed that advocates were highly engaged. Within 1 year after program completion, participating pairs have completed four abstracts and 9 grant proposals. CONCLUSION: The program will be modified based on participant feedback, and can be adapted for future cohorts if an increased number of sessions per month and shortened program duration are desired. The program's virtual format allows scalability across institutions to potentially benefit large cohorts of researchers.

Synergistic anti-proliferative activity of JQ1 and GSK2801 in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) constitutes 10-20% of breast cancers and is challenging to treat due to a lack of effective targeted therapies. Previous studies in TNBC cell lines showed in vitro growth inhibition when JQ1 or GSK2801 were administered alone, and enhanced activity when co-administered. Given their respective mechanisms of actions, we hypothesized the combinatorial effect could be due to the target genes affected. Hence the target genes were characterized for their expression in the TNBC cell lines to prove the combinatorial effect of JQ1 and GSK2801. METHODS: RNASeq data sets of TNBC cell lines (MDA-MB-231, HCC-1806 and SUM-159) were analyzed to identify the differentially expressed genes in single and combined treatments. The topmost downregulated genes were characterized for their downregulated expression in the TNBC cell lines treated with JQ1 and GSK2801 under different dose concentrations and combinations. The optimal lethal doses were determined by cytotoxicity assays. The inhibitory activity of the drugs was further characterized by molecular modelling studies. RESULTS: Global expression profiling of TNBC cell lines using RNASeq revealed different expression patterns when JQ1 and GSK2801 were co-administered. Functional enrichment analyses identified several metabolic pathways (i.e., systemic lupus erythematosus, PI3K-Akt, TNF, JAK-STAT, IL-17, MAPK, Rap1 and signaling pathways) enriched with upregulated and downregulated genes when combined JQ1 and GSK2801 treatment was administered. RNASeq identified downregulation of PTPRC, MUC19, RNA5-8S5, KCNB1, RMRP, KISS1 and TAGLN (validated by RT-qPCR) and upregulation of GPR146, SCARA5, HIST2H4A, CDRT4, AQP3, MSH5-SAPCD1, SENP3-EIF4A1, CTAGE4 and RNASEK-C17orf49 when cells received both drugs. In addition to differential gene regulation, molecular modelling predicted binding of JQ1 and GSK2801 with PTPRC, MUC19, KCNB1, TAGLN and KISS1 proteins, adding another mechanism by which JQ1 and GSK2801 could elicit changes in metabolism and proliferation. CONCLUSION: JQ1-GSK2801 synergistically inhibits proliferation and results in selective gene regulation. Besides suggesting that combinatorial use could be useful therapeutics for the treatment of TNBC, the findings provide a glimpse into potential mechanisms of action for this combination therapy approach.

BRMS1: a multifunctional signaling molecule in metastasis.

Despite high mortality rates, molecular understanding of metastasis remains limited. It can be regulated by both pro- and anti-metastasis genes. The metastasis suppressor, breast cancer metastasis suppressor 1 (BRMS1), has been positively correlated with patient outcomes, but molecular functions are still being characterized. BRMS1 has been implicated in focal adhesion kinase (FAK), epidermal growth factor receptor (EGFR), and NF-κB signaling pathways. We review evidence that BRMS1 regulates these vast signaling pathways through chromatin remodeling as a member of mSin3 histone deacetylase complexes.

KISS1 in metastatic cancer research and treatment: potential and paradoxes.

The significance of KISS1 goes beyond its original discovery as a metastasis suppressor. Its function as a neuropeptide involved in diverse physiologic processes is more well studied. Enthusiasm regarding KISS1 has cumulated in clinical trials in multiple fields related to reproduction and metabolism. But its cancer therapeutic space is unsettled. This review focuses on collating data from cancer and non-cancer fields in order to understand shared and disparate signaling that might inform clinical development in the cancer therapeutic and biomarker space. Research has focused on amino acid residues 68-121 (kisspeptin 54), binding to the KISS1 receptor and cellular responses. Evidence and counterevidence regarding this canonical pathway require closer look at the covariates so that the incredible potential of KISS1 can be realized.

Roles of mitochondria in the hallmarks of metastasis.

Although mitochondrial contributions to cancer have been recognised for approximately a century, given that mitochondrial DNA (mtDNA) is dwarfed by the size of the nuclear genome (nDNA), nuclear genetics has represented a focal point in cancer biology, often at the expense of mtDNA and mitochondria. However, genomic sequencing and advances in in vivo models underscore the importance of mtDNA and mitochondria in cancer and metastasis. In this review, we explore the roles of mitochondria in the four defined 'hallmarks of metastasis': motility and invasion, microenvironment modulation, plasticity and colonisation. Biochemical processes within the mitochondria of both cancer cells and the stromal cells with which they interact are critical for each metastatic hallmark. We unravel complex dynamics in mitochondrial contributions to cancer, which are context-dependent and capable of either promoting metastasis or being leveraged to prevent it at various points of the metastatic cascade. Ultimately, mitochondrial contributions to cancer and metastasis are rooted in the capacity of these organelles to tune metabolic and genetic responses to dynamic microenvironmental cues.

Suppression of pancreatic cancer liver metastasis by secretion-deficient ITIH5.

BACKGROUND: Previously, we identified ITIH5 as a suppressor of pancreatic ductal adenocarcinoma (PDAC) metastasis in experimental models. Expression of ITIH5 correlated with decreased cell motility, invasion and metastasis without significant inhibition of primary tumour growth. Here, we tested whether secretion of ITIH5 is required to suppress liver metastasis and sought to understand the role of ITIH5 in human PDAC. METHODS: We expressed mutant ITIH5 with deletion of the N-terminal secretion sequence (ITIH5Δs) in highly metastatic human PDAC cell lines. We used a human tissue microarray (TMA) to compare ITIH5 levels in uninvolved pancreas, primary and metastatic PDAC. RESULTS: Secretion-deficient ITIH5Δs was sufficient to suppress liver metastasis. Similar to secreted ITIH5, expression of ITIH5Δs was associated with rounded cell morphology, reduced cell motility and reduction of liver metastasis. Expression of ITIH5 is low in both human primary PDAC and matched metastases. CONCLUSIONS: Metastasis suppression by ITIH5 may be mediated by an intracellular mechanism. In human PDAC, loss of ITIH5 may be an early event and ITIH5-low PDAC cells in primary tumours may be selected for liver metastasis. Further defining the ITIH5-mediated pathway in PDAC could establish future therapeutic exploitation of this biology and reduce morbidity and mortality associated with PDAC metastasis.

KISS1 in breast cancer progression and autophagy.

Tumor suppressors are cellular proteins typically expressed in normal (non-cancer) cells that not only regulate such cellular functions as proliferation, migration and adhesion, but can also be secreted into extracellular space and serve as biomarkers for pathological conditions or tumor progression. KISS1, a precursor for several shorter peptides, known as metastin (Kisspeptin-54), Kisspeptin-14, Kisspeptin-13 and Kisspeptin-10, is one of those metastasis suppressor proteins, whose expression is commonly downregulated in the metastatic tumors of various origins. The commonly accepted role of KISS1 in metastatic tumor progression mechanism is the ability of this protein to suppress colonization of disseminated cancer cells in distant organs critical for the formation of the secondary tumor foci. Besides, recent evidence suggests involvement of KISS1 in the mechanisms of tumor angiogenesis, autophagy and apoptosis regulation, suggesting a possible role in both restricting and promoting cancer cell invasion. Here, we discuss the role of KISS1 in regulating metastases, the link between KISS1 expression and the autophagy-related biology of cancer cells and the perspectives of using KISS1 as a potential diagnostic marker for cancer progression as well as a new anti-cancer therapeutics.

The Histone Demethylase KDM3A, Increased in Human Pancreatic Tumors, Regulates Expression of DCLK1 and Promotes Tumorigenesis in Mice.

BACKGROUND & AIMS: The histone lysine demethylase 3A (KDM3A) demethylates H3K9me1 and H3K9Me2 to increase gene transcription and is upregulated in tumors, including pancreatic tumors. We investigated its activities in pancreatic cancer cell lines and its regulation of the gene encoding doublecortin calmodulin-like kinase 1 (DCLK1), a marker of cancer stem cells. METHODS: We knocked down KDM3A in MiaPaCa-2 and S2-007 pancreatic cancer cell lines and overexpressed KDM3A in HPNE cells (human noncancerous pancreatic ductal cell line); we evaluated cell migration, invasion, and spheroid formation under hypoxic and normoxic conditions. Nude mice were given orthotopic injections of S2-007 cells, with or without (control) knockdown of KDM3A, and HPNE cells, with or without (control) overexpression of KDM3A; tumor growth was assessed. We analyzed pancreatic tumor tissues from mice and pancreatic cancer cell lines by immunohistochemistry and immunoblotting. We performed RNA-sequencing analysis of MiaPaCa-2 and S2-007 cells with knockdown of KDM3A and evaluated localization of DCLK1 and KDM3A by immunofluorescence. We analyzed the cancer genome atlas for levels of KDM3A and DCLK1 messenger RNA in human pancreatic ductal adenocarcinoma (PDAC) tissues and association with patient survival time. RESULTS: Levels of KDM3A were increased in human pancreatic tumor tissues and cell lines, compared with adjacent nontumor pancreatic tissues, such as islet and acinar cells. Knockdown of KDM3A in S2-007 cells significantly reduced colony formation, invasion, migration, and spheroid formation, compared with control cells, and slowed growth of orthotopic tumors in mice. We identified KDM3A-binding sites in the DCLK1 promoter; S2-007 cells with knockdown of KDM3A had reduced levels of DCLK1. HPNE cells that overexpressed KDM3A formed foci and spheres in culture and formed tumors and metastases in mice, whereas control HPNE cells did not. Hypoxia induced sphere formation and increased levels of KDM3A in S2-007 cells and in HPNE cells that overexpressed DCLK1, but not control HPNE cells. Levels of KDM3A and DCLK1 messenger RNA were higher in human PDAC than nontumor pancreatic tissues and correlated with shorter survival times of patients. CONCLUSIONS: We found human PDAC samples and pancreatic cancer cell lines to overexpress KDM3A. KDM3A increases expression of DCLK1, and levels of both proteins are increased in human PDAC samples. Knockdown of KDM3A in pancreatic cancer cell lines reduced their invasive and sphere-forming activities in culture and formation of orthotopic tumors in mice. Hypoxia increased expression of KDM3A in pancreatic cancer cells. Strategies to disrupt this pathway might be developed for treatment of pancreatic cancer.

Roles of the mitochondrial genetics in cancer metastasis: not to be ignored any longer.

Mitochondrial DNA (mtDNA) encodes for only a fraction of the proteins that are encoded within the nucleus, and therefore has typically been regarded as a lesser player in cancer biology and metastasis. Accumulating evidence, however, supports an increased role for mtDNA impacting tumor progression and metastatic susceptibility. Unfortunately, due to this delay, there is a dearth of data defining the relative contributions of specific mtDNA polymorphisms (SNP), which leads to an inability to effectively use these polymorphisms to guide and enhance therapeutic strategies and diagnosis. In addition, evidence also suggests that differences in mtDNA impact not only the cancer cells but also the cells within the surrounding tumor microenvironment, suggesting a broad encompassing role for mtDNA polymorphisms in regulating the disease progression. mtDNA may have profound implications in the regulation of cancer biology and metastasis. However, there are still great lengths to go to understand fully its contributions. Thus, herein, we discuss the recent advances in our understanding of mtDNA in cancer and metastasis, providing a framework for future functional validation and discovery.

Mitochondrial polymorphisms contribute to aging phenotypes in MNX mouse models.

Many inbred strains of mice develop spontaneous tumors as they age. Recent awareness of the impacts of mitochondrial DNA (mtDNA) on cancer and aging has inspired developing a mitochondrial-nuclear exchange (MNX) mouse model in which nuclear DNA is paired with mitochondrial genomes from other strains of mouse. MNX mice exhibit mtDNA influences on tumorigenicity and metastasis upon mating with transgenic mice. However, we also wanted to investigate spontaneous tumor phenotypes as MNX mice age. Utilizing FVB/NJ, C57BL/6J, C3H/HeN, and BALB/cJ wild-type inbred strains, previously documented phenotypes were observed as expected in MNX mice with the same nuclear background. However, aging nuclear matched MNX mice exhibited decreased occurrence of mammary tumors in C3H/HeN mice containing C57BL/6J mitochondria compared to wild-type C3H/HeN mice. Although aging tumor phenotypes appear to be driven by nuclear genes, evidence suggesting that some differences are modified by the mitochondrial genome is presented.

The isolated C-terminal nuclear localization sequence of the breast cancer metastasis suppressor 1 is disordered.

BRMS1 is a 246-residue-long protein belonging to the family of metastasis suppressors. It is a predominantly nuclear protein, although it can also function in the cytoplasm. At its C terminus, it has a region that is predicted to be a nuclear localization sequence (NLS); this region, NLS2, is necessary for metastasis suppression. We have studied in vitro and in silico the conformational preferences in aqueous solution of a peptide (NLS2-pep) that comprises the NLS2 of BRMS1, to test whether it has a preferred conformation that could be responsible for its function. Our spectroscopic (far-UV circular dichroism, DOSY-NMR and 2D-NMR) and computational (all-atom molecular dynamics) results indicate that NLS2-pep was disordered in aqueous solution. Furthermore, it did not acquire a structure even when experiments were performed in a more hydrophobic environment, such as the one provided by 2,2,2-trifluoroethanol (TFE). The hydrodynamic radius of the peptide in water was identical to that of a random-coil sequence, in agreement with both our molecular simulations and other theoretical predictions. Thus, we suggest that NLS2 is a disordered region, with non pre-formed structure, that participates in metastasis suppression.

Suppression of pancreatic cancer growth and metastasis by HMP19 identified through genome-wide shRNA screen.

Therapeutic effectiveness against metastatic or even locally advanced pancreatic ductal adenocarcinoma (PDAC) is dismal, with 5-year survival less than 5%. Even in patients who undergo potentially curative resection, most patients' tumors recur in the liver. Improving therapies targeting or preventing liver metastases is crucial for improving prognosis. To identify genes suppressing metastasis, a genome-wide shRNA screen was done using the human non-metastatic PDAC cell line, S2-028. After identification of candidates, functional validation was done using intrasplenic and orthotopic injections in athymic mice. HMP19 strongly inhibited metastasis but also partially attenuated tumor growth in the pancreas. Knockdown of HMP19 increased localization of activated ERK1/2 in the nucleus, corresponding to facilitated cell proliferation, decreased p27(Kip1) and increased cyclin E1. Over-expression of HMP19 exerted the opposite effects. Using a tissue microarray of 84 human PDAC, patients with low expression of HMP19 showed significantly higher incidence of liver metastasis (p = 0.0175) and worse prognosis (p = 0.018) after surgery. HMP19, a new metastasis/tumor suppressor in PDAC, appears to alter signaling that leads to cell proliferation and appears to offer prognostic value in human PDAC.

Allelic variation and differential expression of the mSIN3A histone deacetylase complex gene Arid4b promote mammary tumor growth and metastasis.

Accumulating evidence suggests that breast cancer metastatic progression is modified by germline polymorphism, although specific modifier genes have remained largely undefined. In the current study, we employ the MMTV-PyMT transgenic mouse model and the AKXD panel of recombinant inbred mice to identify AT-rich interactive domain 4B (Arid4b; NM_194262) as a breast cancer progression modifier gene. Ectopic expression of Arid4b promoted primary tumor growth in vivo as well as increased migration and invasion in vitro, and the phenotype was associated with polymorphisms identified between the AKR/J and DBA/2J alleles as predicted by our genetic analyses. Stable shRNA-mediated knockdown of Arid4b caused a significant reduction in pulmonary metastases, validating a role for Arid4b as a metastasis modifier gene. ARID4B physically interacts with the breast cancer metastasis suppressor BRMS1, and we detected differential binding of the Arid4b alleles to histone deacetylase complex members mSIN3A and mSDS3, suggesting that the mechanism of Arid4b action likely involves interactions with chromatin modifying complexes. Downregulation of the conserved Tpx2 gene network, which is comprised of many factors regulating cell cycle and mitotic spindle biology, was observed concomitant with loss of metastatic efficiency in Arid4b knockdown cells. Consistent with our genetic analysis and in vivo experiments in our mouse model system, ARID4B expression was also an independent predictor of distant metastasis-free survival in breast cancer patients with ER+ tumors. These studies support a causative role of ARID4B in metastatic progression of breast cancer.

MCF-7 cells expressing nuclear associated lysyl oxidase-like 2 (LOXL2) exhibit an epithelial-to-mesenchymal transition (EMT) phenotype and are highly invasive in vitro.

LOXL2 is a copper- and lysine tyrosylquinone-dependent amine oxidase that has been proposed to function both extracellularly and intracellularly to activate oncogenic signaling pathways leading to EMT and invasion of breast cancer cells. In this study, we selected MCF-7 cells that stably express forms of recombinant LOXL2 differing in their subcellular localizations and catalytic competencies. This enabled us to dissect the molecular functions of intracellular and extracellular LOXL2s and examine their contributions to breast cancer metastasis/invasion. We discovered that secreted LOXL2 (~100-kDa) is N-glycosylated at Asn-455 and Asn-644, whereas intracellular LOXL2 (~75-kDa) is nonglycosylated and N-terminally processed, and is primarily associated with the nucleus. Both forms of LOXL2 can oxidize lysine in solution. However, we found that expression of intracellular LOXL2 is more strongly associated with EMT and invasiveness than secreted LOXL2 in vitro. The results indicate that nuclear associated LOXL2 contributes to the stabilization of Snail1 transcription factor at the protein level to induce EMT and promote invasion in vitro, through repression of E-cadherin, occludin, and estrogen receptor-α, and up-regulation of vimentin, fibronectin, and MT1-MMP.

Expression of metastasis suppressor BRMS1 in breast cancer cells results in a marked delay in cellular adhesion to matrix.

Metastatic dissemination is a multi-step process that depends on cancer cells' ability to respond to microenvironmental cues by adapting adhesion abilities and undergoing cytoskeletal rearrangement. Breast Cancer Metastasis Suppressor 1 (BRMS1) affects several steps of the metastatic cascade: it decreases survival in circulation, increases susceptibility to anoikis, and reduces capacity to colonize secondary organs. In this report, BRMS1 expression is shown to not significantly alter expression levels of integrin monomers, while time-lapse and confocal microscopy revealed that BRMS1-expressing cells exhibited reduced activation of both ß1 integrin and focal adhesion kinase, and decreased localization of these molecules to sites of focal adhesions. Short-term plating of BRMS1-expressing cells onto collagen or fibronectin markedly decreased cytoskeletal reorganization and formation of cellular adhesion projections. Under 3D culture conditions, BRMS1-expressing cells remained rounded and failed to reorganize their cytoskeleton and form invasive colonies. Taken together, BRMS1-expressing breast cancer cells are greatly attenuated in their ability to respond to microenvironment changes. © 2013 Wiley Periodicals, Inc.

Mitochondrial genetic background modulates bioenergetics and susceptibility to acute cardiac volume overload.

Dysfunctional bioenergetics has emerged as a key feature in many chronic pathologies such as diabetes and cardiovascular disease. This has led to the mitochondrial paradigm in which it has been proposed that mtDNA sequence variation contributes to disease susceptibility. In the present study we show a novel animal model of mtDNA polymorphisms, the MNX (mitochondrial-nuclear exchange) mouse, in which the mtDNA from the C3H/HeN mouse has been inserted on to the C57/BL6 nuclear background and vice versa to test this concept. Our data show a major contribution of the C57/BL6 mtDNA to the susceptibility to the pathological stress of cardiac volume overload which is independent of the nuclear background. Mitochondria harbouring the C57/BL6J mtDNA generate more ROS (reactive oxygen species) and have a higher mitochondrial membrane potential relative to those with C3H/HeN mtDNA, independent of nuclear background. We propose this is the primary mechanism associated with increased bioenergetic dysfunction in response to volume overload. In summary, these studies support the 'mitochondrial paradigm' for the development of disease susceptibility, and show that the mtDNA modulates cellular bioenergetics, mitochondrial ROS generation and susceptibility to cardiac stress.