ABSTRACT
Triple-negative breast cancer (TNBC) is a highly invasive breast cancer subtype that is challenging to treat due to inherent heterogeneity and absence of estrogen, progesterone, and human epidermal growth factor 2 receptors. Kinase signaling networks drive cancer growth and development, and kinase inhibitors are promising anti-cancer strategies in diverse cancer subtypes. Kinase inhibitor screens are an efficient, valuable means of identifying compounds that suppress cancer cell growth in vitro, facilitating the identification of kinase vulnerabilities to target therapeutically. The Kinase Chemogenomic Set is a well-annotated library of 187 kinase inhibitor compounds that indexes 215 kinases of the 518 in the known human kinome representing various kinase networks and signaling pathways, several of which are understudied. Our screen revealed 14 kinase inhibitor compounds effectively inhibited TNBC cell growth and proliferation. Upon further testing, three compounds, THZ531, THZ1, and PFE-PKIS 29, had the most significant and consistent effects across a range of TNBC cell lines. These cyclin-dependent kinase (CDK)12/CDK13, CDK7, and phosphoinositide 3-kinase inhibitors, respectively, decreased metabolic activity in TNBC cell lines and promote a gene expression profile consistent with the reversal of the epithelial-to-mesenchymal transition, indicating these kinase networks potentially mediate metastatic behavior. These data identified novel kinase targets and kinase signaling pathways that drive metastasis in TNBC.
ABSTRACT
Following metastatic spread, many hormone receptor positive (HR+) patients develop a more aggressive phenotype with an observed loss of the HRs estrogen receptor (ER) and progesterone receptor (PR). During metastasis, breast cancer cells are exposed to high magnitudes of fluid shear stress (FSS). Unfortunately, the role for FSS on the regulation of HR expression and function during metastasis is not fully understood. This study was designed to elucidate the impact of FSS on HR+ breast cancer. Utilizing a microfluidic platform capable of exposing breast cancer cells to FSS that mimics in situ conditions, we demonstrate the impact of FSS exposure on representative HR+ breast cancer cell lines through protein and gene expression analysis. Proteomics results demonstrated that 540 total proteins and 1473 phospho-proteins significantly changed due to FSS exposure and pathways of interest included early and late estrogen response. The impact of FSS on response to 17ß-estradiol (E2) was next evaluated and gene expression analysis revealed repression of ER and E2-mediated genes (PR and SDF1) following exposure to FSS. Western blot demonstrated enhanced phosphorylation of mTOR following exposure to FSS. Taken together, these studies provide initial insight into the effects of FSS on HR signaling in metastatic breast cancer.
Subject(s)
Breast Neoplasms , Gene Expression Regulation, Neoplastic , Receptors, Estrogen , Receptors, Progesterone , Stress, Mechanical , Humans , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/genetics , Female , Receptors, Progesterone/metabolism , Receptors, Progesterone/genetics , Cell Line, Tumor , Receptors, Estrogen/metabolism , Receptors, Estrogen/genetics , Estradiol/pharmacology , Phosphorylation , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Proteomics/methods , MCF-7 Cells , Chemokine CXCL12/metabolism , Chemokine CXCL12/geneticsABSTRACT
Xenohormesis proposes that phytochemicals produced to combat stressors in the host plant exert biochemical effects in animal cells lacking cognate receptors. Xenohormetic phytochemicals such as flavonoids and phytoalexins modulate a range of human cell signaling mechanisms but functional correlations with human pathophysiology are lacking. Here, potent inhibitory effects of grapefruit-derived Naringenin (Nar) and soybean-derived Glyceollins (Gly) in human microphysiological models of bulk tissue vasculogenesis and tumor angiogenesis are reported. Despite this interference of vascular morphogenesis, Nar and Gly are not cytotoxic to endothelial cells and do not prevent cell cycle entry. The anti-vasculogenic effects of Glyceollin are significantly more potent in sex-matched female (XX) models. Nar and Gly do not decrease viability or expression of proangiogenic genes in triple negative breast cancer (TNBC) cell spheroids, suggesting that inhibition of sprouting angiogenesis by Nar and Gly in a MPS model of the (TNBC) microenvironment are mediated via direct effects in endothelial cells. The study supports further research of Naringenin and Glyceollin as health-promoting agents with special attention to mechanisms of action in vascular endothelial cells and the role of biological sex, which can improve the understanding of dietary nutrition and the pharmacology of phytochemical preparations.
Subject(s)
Flavanones , Neovascularization, Pathologic , Phytochemicals , Triple Negative Breast Neoplasms , Humans , Flavanones/pharmacology , Neovascularization, Pathologic/drug therapy , Neovascularization, Pathologic/metabolism , Neovascularization, Pathologic/prevention & control , Phytochemicals/pharmacology , Female , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/pathology , Triple Negative Breast Neoplasms/metabolism , Pterocarpans/pharmacology , Angiogenesis Inhibitors/pharmacology , Cell Line, Tumor , Glycine max/chemistry , Citrus paradisi/chemistry , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Human Umbilical Vein Endothelial Cells/drug effects , AngiogenesisABSTRACT
Both breast cancer and obesity can regulate epigenetic changes or be regulated by epigenetic changes. Due to the well-established link between obesity and an increased risk of developing breast cancer, understanding how obesity-mediated epigenetic changes affect breast cancer pathogenesis is critical. Researchers have described how obesity and breast cancer modulate the epigenome individually and synergistically. In this review, the epigenetic alterations that occur in obesity, including DNA methylation, histone, and chromatin modification, accelerated epigenetic age, carcinogenesis, metastasis, and tumor microenvironment modulation, are discussed. Delineating the relationship between obesity and epigenetic regulation is vital to furthering our understanding of breast cancer pathogenesis.
Subject(s)
Breast Neoplasms , Epigenesis, Genetic , Humans , Female , Breast Neoplasms/genetics , Breast Neoplasms/pathology , DNA Methylation , Histones/metabolism , Obesity/complications , Obesity/genetics , Tumor Microenvironment/geneticsABSTRACT
Physical characteristics of solid tumors such as dense internal microarchitectures and pathological stiffness influence cancer progression and treatment. While it is routine to engineer culture substrates and scaffolds with elastic moduli that approximate tumors, these models often fail to capture characteristic internal microarchitectures such as densely compacted concentric ECM fibers at the stromal interface. Contractile mesenchymal cells can solve this engineering challenge by deforming, contracting, and compacting extracellular matrix (ECM) hydrogels to decrease tissue volume and increase tissue density. Here we demonstrate that allowing human fibroblasts of varying origins to freely contract collagen type I-containing hydrogels co-seeded with carcinoma cell spheroids produces a tissue engineered construct with structural features that mimic dense solid tumors in vivo. Morphometry and mechanical testing were conducted in tandem with biochemical analysis of proliferation and viability to confirm that dense carcinoma constructs engineered using this approach capture relevant physical characteristics of solid carcinomas in a tractable format that preserves viability and is amenable to extended culture. The reported method is adaptable to the use of multiple mesenchymal cell types and the inclusion of fibrin in the ECM combined with seeding of endothelial cells to produce prevascularized constructs. The physical dense carcinoma constructs engineered using this approach may provide more clinically relevant venues for studying cancer pathophysiology and the challenges associated with the delivery of macromolecular drugs and cellular immunotherapies to solid tumors.
Subject(s)
Carcinoma , Collagen , Humans , Collagen/chemistry , Hydrogels/chemistry , Endothelial Cells , Extracellular Matrix/metabolism , Tissue Engineering/methods , Carcinoma/metabolismABSTRACT
Triple-negative breast cancers (TNBCs) are aggressive forms of breast cancer and tend to grow and spread more quickly than most other types of breast cancer. TNBCs can neither be targeted by hormonal therapies nor the antibody trastuzumab that targets the HER2 protein. There are urgent unmet medical needs to develop targeted drugs for TNBCs. We identified a small molecule NSC260594 from the NCI diversity set IV compound library. NSC260594 exhibited dramatic cytotoxicity in multiple TNBCs in a dose-and time-dependent manner. NSC260594 inhibited the Myeloid cell leukemia-1 (Mcl-1) expression through downregulation of Wnt signaling proteins. Consistent with this, NSC260594 treatment increased apoptosis, which was confirmed by using an Annexin-V/PI assay. Interestingly, NSC260594 treatment reduced the cancer stem cell (CSC) population in TNBCs. To make NSC260594 more clinically relevant, we treated NSC260594 with TNBC cell derived xenograft (CDX) mouse model, and with patient-derived xenograft (PDX) organoids. NSC260594 significantly suppressed MDA-MB-231 tumor growth in vivo, and furthermore, the combination treatment of NSC260594 and everolimus acted synergistically to decrease growth of TNBC PDX organoids. Together, we found that NSC260594 might serve as a lead compound for triple-negative breast cancer therapy through targeting Mcl-1.
Subject(s)
Antineoplastic Agents , Triple Negative Breast Neoplasms , Animals , Humans , Mice , Annexin A5 , Antibodies , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Disease Models, Animal , Triple Negative Breast Neoplasms/drug therapyABSTRACT
Metabolism controls cellular phenotype and fate. In this report, we demonstrate that nicotinamide N-methyltransferase (NNMT), a metabolic enzyme that regulates developmental stem cell transitions and tumor progression, is highly expressed in human idiopathic pulmonary fibrosis (IPF) lungs, and is induced by the pro-fibrotic cytokine, transforming growth factor-ß1 (TGF-ß1) in lung fibroblasts. NNMT silencing reduces the expression of extracellular matrix proteins, both constitutively and in response to TGF-ß1. Furthermore, NNMT controls the phenotypic transition from homeostatic, pro-regenerative lipofibroblasts to pro-fibrotic myofibroblasts. This effect of NNMT is mediated, in part, by the downregulation of lipogenic transcription factors, TCF21 and PPARγ, and the induction of a less proliferative but more differentiated myofibroblast phenotype. NNMT confers an apoptosis-resistant phenotype to myofibroblasts that is associated with the downregulation of pro-apoptotic members of the Bcl-2 family, including Bim and PUMA. Together, these studies indicate a critical role for NNMT in the metabolic reprogramming of fibroblasts to a pro-fibrotic and apoptosis-resistant phenotype and support the concept that targeting this enzyme may promote regenerative responses in chronic fibrotic disorders such as IPF.
Subject(s)
Myofibroblasts , Nicotinamide N-Methyltransferase , Humans , Apoptosis , Basic Helix-Loop-Helix Transcription Factors/metabolism , Fibroblasts/metabolism , Fibrosis , Idiopathic Pulmonary Fibrosis/metabolism , Lung/metabolism , Myofibroblasts/metabolism , Nicotinamide N-Methyltransferase/metabolism , Transforming Growth Factor beta1/metabolismABSTRACT
Triple-negative breast cancers (TNBC) tend to become invasive and metastatic at early stages in their development. Despite some treatment successes in early-stage localized TNBC, the rate of distant recurrence remains high, and long-term survival outcomes remain poor. In a search for new therapeutic targets for this disease, we observed that elevated expression of the serine/threonine kinase calcium/calmodulin (CaM)-dependent protein kinase kinase 2 (CaMKK2) is highly correlated with tumor invasiveness. In validation studies, genetic disruption of CaMKK2 expression or inhibition of its activity with small molecule inhibitors disrupted spontaneous metastatic outgrowth from primary tumors in murine xenograft models of TNBC. High-grade serous ovarian cancer (HGSOC), a high-risk, poor prognosis ovarian cancer subtype, shares many features with TNBC, and CaMKK2 inhibition effectively blocked metastatic progression in a validated xenograft model of this disease. Mechanistically, CaMKK2 increased the expression of the phosphodiesterase PDE1A, which hydrolyzed cyclic guanosine monophosphate (cGMP) to decrease the cGMP-dependent activity of protein kinase G1 (PKG1). Inhibition of PKG1 resulted in decreased phosphorylation of vasodilator-stimulated phosphoprotein (VASP), which in its hypophosphorylated state binds to and regulates F-actin assembly to facilitate cell movement. Together, these findings establish a targetable CaMKK2-PDE1A-PKG1-VASP signaling pathway that controls cancer cell motility and metastasis by impacting the actin cytoskeleton. Furthermore, it identifies CaMKK2 as a potential therapeutic target that can be exploited to restrict tumor invasiveness in patients diagnosed with early-stage TNBC or localized HGSOC. SIGNIFICANCE: CaMKK2 regulates actin cytoskeletal dynamics to promote tumor invasiveness and can be inhibited to suppress metastasis of breast and ovarian cancer, indicating CaMKK2 inhibition as a therapeutic strategy to arrest disease progression.
Subject(s)
Ovarian Neoplasms , Triple Negative Breast Neoplasms , Animals , Female , Humans , Mice , Actins/metabolism , Cell Movement , Ovarian Neoplasms/drug therapy , Protein KinasesABSTRACT
The liver is a major organ that is involved in essential biological functions such as digestion, nutrient storage, and detoxification. Furthermore, it is one of the most metabolically active organs with active roles in regulating carbohydrate, protein, and lipid metabolism. Hepatocellular carcinoma is a cancer of the liver that is associated in settings of chronic inflammation such as viral hepatitis, repeated toxin exposure, and fatty liver disease. Furthermore, liver cancer is the most common cause of death associated with cirrhosis and is the 3rd leading cause of global cancer deaths. LKB1 signaling has been demonstrated to play a role in regulating cellular metabolism under normal and nutrient deficient conditions. Furthermore, LKB1 signaling has been found to be involved in many cancers with most reports identifying LKB1 to have a tumor suppressive role. In this review, we use the KMPlotter database to correlate RNA levels of LKB1 signaling genes and hepatocellular carcinoma patient survival outcomes with the hopes of identifying potential biomarkers clinical usage. Based on our results STRADß, CAB39L, AMPKα, MARK2, SIK1, SIK2, BRSK1, BRSK2, and SNRK expression has a statistically significant impact on patient survival.
Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , AMP-Activated Protein Kinase Kinases , AMP-Activated Protein Kinases/metabolism , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolismABSTRACT
As breast cancer cells transition from letrozole-sensitive to letrozole-resistant, they over-express epidermal growth factor receptor (EGFR), mitogen-activated protein kinase (MAPK), and human epidermal growth factor receptor 2 (HER2) while acquiring enhanced motility and epithelial-to-mesenchymal transition (EMT)-like characteristics that are attenuated and reversed by glyceollin treatment, respectively. Interestingly, glyceollin inhibits the proliferation and tumor progression of triple-negative breast cancer (TNBC) and estrogen-independent breast cancer cells; however, it is unlikely that a single phytochemical would effectively target aromatase-inhibitor (AI)-resistant metastatic breast cancer in the clinical setting. Since our previous report indicated that the combination of lapatinib and glyceollin induced apoptosis in hormone-dependent AI-resistant breast cancer cells, we hypothesized that combination therapy would also be beneficial for hormone independent letrozole-resistant breast cancer cells (LTLT-Ca) compared to AI-sensitive breast cancer cells (AC-1) by decreasing the expression of proteins associated with proliferation and cell cycle progression. While glyceollin + lapatinib treatment caused comparable inhibitory effects on the proliferation and migration in both cell lines, combination treatment selectively induced S and G2/M phase cell cycle arrest of the LTLT-Ca cells, which was mediated by decreased cyclin B1. This phenomenon may represent a unique opportunity to design novel combinatorial therapeutic approaches to target hormone-refractory breast tumors.
Subject(s)
Breast Neoplasms , Humans , Female , Letrozole/pharmacology , Breast Neoplasms/metabolism , Lapatinib/pharmacology , Cyclin B1/pharmacology , Nitriles/pharmacology , Triazoles/pharmacology , Drug Resistance, Neoplasm , Aromatase Inhibitors/pharmacology , Aromatase Inhibitors/therapeutic use , Estrogens/metabolism , Mitogen-Activated Protein Kinases , Cell Line, TumorABSTRACT
The Never in Mitosis Gene A (NIMA)-related kinases (NEKs) are a group of serine/threonine kinases that are involved in a wide array of cellular processes including cell cycle regulation, DNA damage repair response (DDR), apoptosis, and microtubule organization. Recent studies have identified the involvement of NEK family members in various diseases such as autoimmune disorders, malignancies, and developmental defects. Despite the existing literature exemplifying the importance of the NEK family of kinases, this family of protein kinases remains understudied. This report seeks to provide a foundation for investigating the role of different NEKs in malignancies. We do this by evaluating the 11 NEK family kinase gene expression associations with patients' overall survival (OS) from various cancers using the Kaplan-Meier Online Tool (KMPlotter) to correlate the relationship between mRNA expression of NEK1-11 in various cancers and patient survival. Furthermore, we use the Catalog of Somatic Mutations in Cancer (COSMIC) database to identify NEK family mutations in cancers of different tissues. Overall, the data suggest that the NEK family has varying associations with patient survival in different cancers with tumor-suppressive and tumor-promoting effects being tissue-dependent.
ABSTRACT
Research has led to the development of tailored treatment options for different cancers in different patients. Despite some treatments being able to provide remarkable responses, nearly all current treatments encounter the same issue: resistance. Here, we discuss our experiences with how breast cancers resist therapies. The focus of our discussion revolves around the cancer stem cell subpopulation and their mechanisms for resistance.
ABSTRACT
The significant increase in the incidence of obesity represents the next global health crisis. As a result, scientific research has focused on gaining deeper insights into obesity and adipose tissue biology. As a result of the excessive accumulation of adipose tissue, obesity results from hyperplasia and hypertrophy within the adipose tissue. The functional alterations in the adipose tissue are a confounding contributing factor to many diseases, including cancer. The increased incidence and aggressiveness of several cancers, including colorectal, postmenopausal breast, endometrial, prostate, esophageal, hematological, malignant melanoma, and renal carcinomas, result from obesity as a contributing factor. The increased morbidity and mortality of obesity-associated cancers are attributable to increased hormones, adipokines, and cytokines produced by the adipose tissue. The increased adipose tissue levels observed in obese patients result in more adipose stromal/stem cells (ASCs) distributed throughout the body. ASCs have been shown to impact cancer progression in vitro and in preclinical animal models. ASCs influence tumor biology via multiple mechanisms, including the increased recruitment of ASCs to the tumor site and increased production of cytokines and growth factors by ASCs and other cells within the tumor stroma. Emerging evidence indicates that obesity induces alterations in the biological properties of ASCs, subsequently leading to enhanced tumorigenesis and metastasis of cancer cells. As the focus of this review is the interaction and impact of ASCs on cancer, the presentation is limited to preclinical data generated on cancers in which there is a demonstrated role for ASCs, such as postmenopausal breast, colorectal, prostate, ovarian, multiple myeloma, osteosarcoma, cervical, bladder, and gastrointestinal cancers. Our group has investigated the interactions between obesity and breast cancer and the mechanisms that regulate ASCs and adipocytes in these different contexts through interactions between cancer cells, immune cells, and other cell types present in the tumor microenvironment (TME) are discussed. The reciprocal and circular feedback loop between obesity and ASCs and the mechanisms by which ASCs from obese patients alter the biology of cancer cells and enhance tumorigenesis will be discussed. At present, the evidence for ASCs directly influencing human tumor growth is somewhat limited, though recent clinical studies suggest there may be some link.
Subject(s)
Breast Neoplasms , Colorectal Neoplasms , Adipose Tissue/metabolism , Adipose Tissue/pathology , Animals , Breast Neoplasms/pathology , Carcinogenesis/pathology , Cell Transformation, Neoplastic/metabolism , Colorectal Neoplasms/pathology , Cytokines/metabolism , Humans , Male , Obesity/complications , Obesity/metabolism , Stromal Cells/metabolism , Tumor MicroenvironmentABSTRACT
BACKGROUND: Triple-negative breast cancers (TNBCs) are clinically aggressive subtypes of breast cancer. TNBC is difficult to treat with targeted agents due to the lack of commonly targeted therapies within this subtype. Androgen receptor (AR) has been detected in 12-55% of TNBCs. AR stimulates breast tumor growth in the absence of estrogen receptor (ER), and it has become an emerging molecular target in TNBC treatment. METHODS: Ceritinib is a small molecule inhibitor of tyrosine kinase and it is used in the therapy of non-small lung cancer patients. Enzalutamide is a small molecule compound targeting the androgen receptor and it is used to treat prostate cancer. Combination therapy of these drugs were investigated using AR positive breast cancer mouse xenograft models. Also, combination treatment of ceritinib and paclitaxel investigated using AR- and AR low mouse xenograft and patient derived xenograft models. RESULTS: We screened 133 FDA approved drugs that have a therapeutic effect of AR+ TNBC cells. From the screen, we identified two drugs, ceritinib and crizotinib. Since ceritinib has a well- defined role in androgen independent AR signaling pathways, we further investigated the effect of ceritinib. Ceritinib treatment inhibited RTK/ACK/AR pathway and other downstream pathways in AR+ TNBC cells. The combination of ceritinib and enzalutamide showed a robust inhibitory effect on cell growth of AR+ TNBC cells in vitro and in vivo. Interestingly Ceritinib inhibits FAK-YB-1 signaling pathway that leads to paclitaxel resistance in all types of TNBC cells. The combination of paclitaxel and ceritinib showed drastic inhibition of tumor growth compared to a single drug alone. CONCLUSIONS: To improve the response of AR antagonist in AR positive TNBC, we designed a novel combinational strategy comprised of enzalutamide and ceritinib to treat AR+ TNBC tumors through the dual blockade of androgen-dependent and androgen-independent AR signaling pathways. Furthermore, we introduced a novel therapeutic combination of ceritinib and paclitaxel for AR negative or AR-low TNBCs and this combination inhibited tumor growth to a great extent. All agents used in our study are FDA-approved, and thus the proposed combination therapy will likely be useful in the clinic.
Subject(s)
Triple Negative Breast Neoplasms , Androgens/therapeutic use , Animals , Cell Line, Tumor , Humans , Mice , Paclitaxel/pharmacology , Paclitaxel/therapeutic use , Pyrimidines , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Sulfones , Triple Negative Breast Neoplasms/drug therapy , Triple Negative Breast Neoplasms/genetics , Triple Negative Breast Neoplasms/metabolismABSTRACT
Mitogen Activated Protein (MAP) kinases are a category of serine/threonine kinases that have been demonstrated to regulate intracellular events including stress responses, developmental processes, and cancer progression Although many MAP kinases have been extensively studied in various disease processes, MAP3K19 is an understudied kinase whose activities have been linked to lung disease and fibroblast development. In this manuscript, we use bioinformatics databases starBase, GEPIA, and KMPlotter, to establish baseline expressions of MAP3K19 in different tissue types and its correlation with patient survival in different cancers.
Subject(s)
Mitogen-Activated Protein Kinases , Neoplasms , Humans , MAP Kinase Kinase Kinases , MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/metabolism , Neoplasms/genetics , Phosphorylation , Protein Serine-Threonine Kinases/geneticsABSTRACT
Liver kinase B1 (LKB1) is a potent tumor suppressor that regulates cellular energy balance and metabolism as an upstream kinase of the AMP-activated protein kinase (AMPK) pathway. LKB1 regulates cancer cell invasion and metastasis in multiple cancer types, including breast cancer. In this study, we evaluated LKB1's role as a regulator of the tumor microenvironment (TME). This was achieved by seeding the MDA-MB-231-LKB1 overexpressing cell line onto adipose and tumor scaffolds, followed by the evaluation of tumor matrix-induced tumorigenesis and metastasis. Results demonstrated that the presence of tumor matrix enhanced tumorigenesis in both MDA-MB-231 and MDA-MB-231-LKB1 cell lines. Metastasis was increased in both MDA-MB-231 and -LKB1 cells seeded on the tumor scaffold. Endpoint analysis of tumor and adipose scaffolds revealed LKB1-mediated tumor microenvironment remodeling as evident through altered matrix protein production. The proteomic analysis determined that LKB1 overexpression preferentially decreased all major and minor fibril collagens (collagens I, III, V, and XI). In addition, proteins observed to be absent in tumor scaffolds in the LKB1 overexpressing cell line included those associated with the adipose matrix (COL6A2) and regulators of adipogenesis (IL17RB and IGFBP4), suggesting a role for LKB1 in tumor-mediated adipogenesis. Histological analysis of MDA-MB-231-LKB1-seeded tumors demonstrated decreased total fibril collagen and indicated decreased stromal cell presence. In accordance with this, in vitro condition medium studies demonstrated that the MDA-MB-231-LKB1 secretome inhibited adipogenesis of adipose-derived stem cells. Taken together, these data demonstrate a role for LKB1 in regulating the tumor microenvironment through fibril matrix remodeling and suppression of adipogenesis.
ABSTRACT
Melatonin, the primary hormone involved in circadian entrainment, plays a significant role in bone physiology. This study aimed to assess the role of MEK1/2 and MEK5 in melatonin-mediated actions in mouse and human mesenchymal stem cells (MSCs) and on bone using small-molecule inhibitors and CRISPR/Cas9 knockout approaches. Consistent with in vitro studies performed in mMSCs and hMSCs, nightly (25 mg/kg, i.p., 45 days) injections with PD184352 (MEK1/2 inhibitor) or Bix02189 (MEK5 inhibitor) or SC-1-151 (MEK1/2/5 inhibitor) demonstrated that MEK1/2 and MEK5 were the primary drivers underlying melatonin's actions on bone density, microarchitecture (i.e., trabecular number, separation, and connectivity density), and bone mechanical properties (i.e., ultimate stress) through increases in osteogenic (RUNX2, BMP-2, FRA-1, OPG) expression and decreases in PPARγ. Furthermore, CRISPR/Cas9 knockout of MEK1 or MEK5 in mMSCs seeded on PLGA scaffolds and placed into critical-size calvarial defects in Balb(c) mice (male and female) revealed that treatment with melatonin (15 mg/L; p.o., nightly, 90 days) mediates sex-specific actions of MEK1 and MEK5 in new bone formation. This study is the first to demonstrate a role for MEK1/2 and MEK5 in modulating melatonin-mediated actions on bone formation in vivo and in a sex-specific manner.
Subject(s)
Melatonin , Osteogenesis , Animals , Biomechanical Phenomena , Bone Density , Bone and Bones , Female , Humans , Male , Melatonin/pharmacology , Melatonin/physiology , MiceABSTRACT
The mitogen-activated protein kinase (MAPK) pathways are ubiquitous in cellular signaling and are essential for proper biological functions. Disruptions in this signaling axis can lead to diseases such as the development of cancer. In this review, we discuss members of the MAP3K family and correlate their mRNA expression levels to patient survival outcomes in different cancers. Furthermore, we highlight the importance of studying the MAP3K family due to their important roles in the larger, overall MAPK pathway, relationships with cancer progression, and the understudied status of these kinases.
Subject(s)
MAP Kinase Signaling System , Neoplasms , Humans , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Neoplasms/genetics , Phosphorylation , Signal Transduction/geneticsABSTRACT
Adipose tissue is characterized as an endocrine organ that acts as a source of hormones and paracrine factors. In diseases such as cancer, endocrine and paracrine signals from adipose tissue contribute to cancer progression. Young individuals with estrogen receptor-alpha positive (ER-α+) breast cancer (BC) have an increased resistance to endocrine therapies, suggesting that alternative estrogen signaling is activated within these cells. Despite this, the effects of stromal age on the endocrine response in BC are not well defined. To identify differences between young and aged ER-α+ breast tumors, RNA sequencing data were obtained from The Cancer Genome Atlas. Analysis revealed enrichment of matrix and paracrine factors in young (≤40 years old) patients compared to aged (≥65 years old) tumor samples. Adipose-derived stromal/stem cells (ASCs) from noncancerous lipoaspirate of young and aged donors were evaluated for alterations in matrix production and paracrine secreted factors to determine if the tumor stroma could alter estrogen signaling. Young and aged ASCs demonstrated comparable proliferation, differentiation, and matrix production, but exhibited differences in the expression levels of inflammatory cytokines (Interferon gamma, interleukin [IL]-8, IL-10, Tumor necrosis factor alpha, IL-2, and IL-6). Conditioned media (CM)-based experiments showed that young ASC donor age elevated endocrine response in ER-α+ BC cell lines. MCF-7 ER-α+ BC cell line treated with secreted factors from young ASCs had enhanced ER-α regulated genes (PGR and SDF-1) compared to MCF-7 cells treated with aged ASC CM. Western blot analysis demonstrated increased activation levels of p-ER ser-167 in the MCF-7 cell line treated with young ASC secreted factors. To determine if ER-α+ BC cells heightened the cytokine release in ASCs, ASCs were stimulated with MCF-7-derived CM. Results demonstrated no change in growth factors or cytokines when treated with the ER-α+ secretome. In contrast to ER-α+ CM, the ER-α negative MDA-MB-231 derived CM demonstrated increased stimulation of pro-inflammatory cytokines in ASCs. While there was no observed change in the release of selected paracrine factors, MCF-7 cells did induce matrix production and a pro-adipogenic lineage commitment. The adipogenesis was evident by increased collagen content through Sirius Red/Fast Green Collagen stain, lipid accumulation evident by Oil Red O stain, and significantly increased expression in PPARγ mRNA expression. The data from this study provide evidence suggesting more of a subtype-dependent than an age-dependent difference in stromal response to BC, suggesting that this signaling is not heightened by reciprocal signals from ER-α+ BC cell lines. These results are important in understanding the mechanisms of estrogen signaling and the dynamic and reciprocal nature of cancer cell-stromal cell crosstalk that can lead to tumor heterogeneity and variance in response to therapy.
Subject(s)
Breast Neoplasms , Adult , Aged , Female , Humans , Adipose Tissue , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Proliferation , Culture Media, Conditioned/pharmacology , Estrogens/metabolism , Interferon-gamma/metabolism , Interleukin-10/metabolism , Interleukin-2/metabolism , Interleukin-6/metabolism , PPAR gamma/metabolism , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , RNA, Messenger/metabolism , Stem Cells , Stromal Cells/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Desmoplastic Small Round Cell Tumor (DSRCT) is a rare and aggressive malignant cancer caused by a chromosomal translocation t(11;22)(p13;q12) that produces an oncogenic transcription factor, EWSR1-WT1. EWSR1-WT1 is essential for the initiation and progression of DSRCT. However, the precise mechanism by which EWSR1-WT1 drives DSRCT oncogenesis remains unresolved. Through our integrative gene expression analysis, we identified Salt Inducible Kinase 1 (SIK1) as a direct target of EWSR1-WT1. SIK1 as a member of the AMPK related kinase is involved in many biological processes. We showed that depletion of SIK1 causes inhibition of tumor cell growth, similar to the growth inhibition observed when EWSR1-WT1 is depleted. We further showed that silencing SIK1 leads to cessation of DNA replication in DSRCT cells and inhibition of tumor growth in vivo. Lastly, combined inhibition of SIK1 and CHEK1with small molecule inhibitors, YKL-05-099 and prexasertib, respectively, showed enhanced cytotoxicity in DSRCT cells compared to inhibition of either kinases alone. This work identified SIK1 as a new potential therapeutic target in DSRCT and the efficacy of SIK1 inhibition may be improved when combined with other intervention strategies.