IL-22 regulates MASTL expression in intestinal epithelial cells.

Microtubule-associated serine-threonine kinase-like (MASTL) has recently been identified as a oncogenic kinase given its overexpression in numerous cancers. Our group has shown that MASTL expression is upregulated in mouse models of sporadic CRC and colitis associated cancer (CAC). CAC is one of the most severe complications of chronic IBD, but a limited understanding of the mechanisms governing the switch from normal healing to neoplasia in IBD underscores the need for increased research in this area. However, MASTL expression in IBD patients and its molecular regulation in IBD and CAC have not been studied. This study reveals that MASTL is upregulated by the cytokine interleukin (IL)-22, which promotes proliferation and has important functions in colitis recovery; however, IL-22 can also promote tumorigenesis when chronically elevated. Upon reviewing the publicly available data, we found significantly elevated MASTL and IL-22 levels in the biopsies from late-stage ulcerative colitis patients compared to controls, and that MASTL upregulation was associated with high IL-22 expression. Our subsequent in vitro studies found that IL-22 increases MASTL expression in intestinal epithelial cell lines, facilitating IL-22- mediated cell proliferation and downstream survival signaling. Inhibition of AKT activation abrogated IL-22-induced MASTL upregulation. We further found an increased association of carbonic anhydrase IX (CAIX) with MASTL in IL-22-treated cells, which stabilized MASTL expression. Inhibition of CAIX prevented IL-22-induced MASTL expression and cell survival. Overall, we show that IL-22/AKT signaling increases MASTL expression to promote cell survival and proliferation. Further, CAIX stabilizes MASTL by associating with it in response to IL-22 stimulation.

Albendazole inhibits colon cancer progression and therapy resistance by targeting ubiquitin ligase RNF20.

BACKGROUND: The repurposing of FDA-approved drugs for anti-cancer therapies is appealing due to their established safety profiles and pharmacokinetic properties and can be quickly moved into clinical trials. Cancer progression and resistance to conventional chemotherapy remain the key hurdles in improving the clinical management of colon cancer patients and associated mortality. METHODS: High-throughput screening (HTS) was performed using an annotated library of 1,600 FDA-approved drugs to identify drugs with strong anti-CRC properties. The candidate drug exhibiting most promising inhibitory effects in in-vitro studies was tested for its efficacy using in-vivo models of CRC progression and chemoresistance and patient derived organoids (PTDOs). RESULTS: Albendazole, an anti-helminth drug, demonstrated the strongest inhibitory effects on the tumorigenic potentials of CRC cells, xenograft tumor growth and organoids from mice. Also, albendazole sensitized the chemoresistant CRC cells to 5-fluorouracil (5-FU) and oxaliplatin suggesting potential to treat chemoresistant CRC. Mechanistically, Albendazole treatment modulated the expression of RNF20, to promote apoptosis in CRC cells by delaying the G2/M phase and suppressing anti-apoptotic-Bcl2 family transcription. CONCLUSIONS: Albendazole, an FDA approved drug, carries strong therapeutic potential to treat colon cancers which are aggressive and potentially resistant to conventional chemotherapeutic agents. Our findings also lay the groundwork for further clinical testing.

Claudin-1 interacts with EPHA2 to promote cancer stemness and chemoresistance in colorectal cancer.

Therapy resistance is the primary problem in treating late-stage colorectal cancer (CRC). Claudins are frequently dysregulated in cancer, and several are being investigated as novel therapeutic targets and biomarkers. We have previously demonstrated that Claudin-1 (CLDN1) expression in CRC promotes epithelial-mesenchymal transition, metastasis, and resistance to anoikis. Here, we hypothesize that CLDN1 promotes cancer stemness and chemoresistance in CRC. We found that high CLDN1 expression in CRC is associated with cancer stemness and chemoresistance signaling pathways in patient datasets, and it promotes chemoresistance both in vitro and in vivo. Using functional stemness assays, proteomics, biophysical binding assays, and patient-derived organoids, we found that CLDN1 promotes properties of cancer stemness including CD44 expression, tumor-initiating potential, and chemoresistance through a direct interaction with ephrin type-A receptor 2 (EPHA2) tyrosine kinase. This interaction is dependent on the CLDN1 PDZ-binding motif, increases EPHA2 protein expression by inhibiting its degradation, and enhances downstream AKT signaling and CD44 expression to promote stemness and chemoresistance. These results suggest CLDN1 is a viable target for pharmacological intervention and/or biomarker development.

Discovery, synthesis and biological evaluation of a series of N-(phenylcarbamothioyl)-2-napthamides as inhibitors of Claudin-1.

Colorectal cancer (CRC) remains a leading cause of cancer-related deaths worldwide, despite advancements in diagnosis. The main reason for this is that many newly diagnosed CRC patients will suffer from metastasis to other organs. Thus, the development of new therapies is of critical importance. Claudin-1 protein is a component of tight junctions in epithelial cells, including those found in the lining of the colon. It plays a critical role in the formation and maintenance of tight junctions, which are essential for regulating the passage of molecules between cells. In CRC, claudin-1 is often overexpressed, leading to an increase in cell adhesion, which can contribute to the development and progression of the disease. Studies show that high levels of claudin-1 are associated with poor prognosis in CRC patients and targeting claudin-1 may have therapeutic potential for the treatment of CRC. Previously, we have identified a small molecule that inhibits claudin-1 dependent CRC progression. Reported herein are our lead optimization efforts around this scaffold to identify the key SAR components and the discovery of a key new compound that exhibits enhanced potency in SW620 cells.

Evaluation of potential inhibitory effects on acetylcholinesterase, pancreatic lipase, and cancer cell lines using raw leaves extracts of three fabaceae species.

The present study examined the biological potential and phytochemicals of Sophora mollis, Mucuna pruriens, and Indigofera atropurpurea methanolic leaf extracts. In vitro anti-acetylcholinesterase and anti-lipase assays were performed using different concentrations of plant extracts, and the IC50 values were determined. The cytotoxic potential of the selected plant extracts was assessed against HeLa, PC3, and 3T3 cell lines using an MTT assay. S. mollis leaf extract displayed the highest inhibition percentage (114.60% ± 19.95 at 1000 µg/mL) for the anti-acetylcholinesterase activity with a prominent IC50 value of 75.9 µg/mL. The anti-lipase potential was highest with the M. pruriens leaf extract (355.5 µg/mL IC50), followed by the S. mollis extract (862.7 µg/mL IC50). Among the cell lines tested, the cytotoxic potential of the I. atropurpurea extract (91.1 ppm IC50) against the PC3 cell line was promising. High-performance liquid chromatography revealed gallic acid, chlorogenic acid, caffeic acid, vanillic acid, rutin trihydrate, and quercetin dihydrate in varying concentrations in all plant species. The concentration of chlorogenic acid (69.09 ppm) was highest in M. pruriens, and the caffeic acid concentration (45.20 ppm) was higher in S. mollis. This paper reports the presence of bioactive therapeutic compounds in selected species of the Fabaceae family that could be micro-propagated, isolated, and utilized in pharmaceutical industries.

Identification and characterization of a first-generation inhibitor of claudin-1 in colon cancer progression and metastasis.

Colorectal cancer (CRC) is a leading cause of the cancer-related deaths worldwide. Thus, developing novel and targeted therapies for inhibiting CRC progression and metastasis is urgent. Several studies, including ours, have reported a causal role for an upregulated claudin-1 expression in promoting CRC metastasis through the activation of the Src and ß-catenin-signaling. In murine models of colon tumorigenesis, claudin-1 overexpression promotes oncogenic properties such as transformation and invasiveness. Conversely, the downregulation of claudin-1 inhibits colon tumorigenesis. Despite being a desirable target for cancer treatment, there are currently no known claudin-1 inhibitors with antitumor efficacy. Using a rigorous analytical design and implementing in- vitro and in-vivo testing and a brief medicinal chemistry campaign, we identified a claudin-1-specific inhibitor and named it I-6. Despite its high potency, I-6 was rapidly cleared in human liver microsomes. We, therefore, synthesized I-6 analogs and discovered a novel small molecule, PDS-0330. We determined that PDS0330 inhibits claudin-1-dependent CRC progression without exhibiting toxicity in in-vitro and in-vivo models of CRC and that it binds directly and specifically to claudin-1 with micromolar affinity. Further analyses revealed that PDS-0330 exhibits antitumor and chemosensitizer activities with favorable pharmacokinetic properties by inhibiting the association with metastatic oncogene Src. Overall, our data propose that PDS-0330 interferes with claudin-1/Src association to inhibit CRC progression and metastasis. Our findings are of direct clinical relevance and may open new therapeutic opportunities in colon cancer treatment and/or management by targeting claudin-1.

Antioxidant, Anticancer, and PXR-Dependent CYP3A4 Attributes of Schweinfurthia papilionacea (Burm.f.) Boiss., Tricholepis glaberrima DC. and Viola stocksii Boiss.

Present study established the biological potential of Schweinfurthia papilionacea, Tricholepis glaberrima and Viola stocksii extracts for their potential applications in drug formulations. Initially, FTIR was performed to ascertain functional groups and then plant extracts were prepared using five solvents depending on the polarity. Total phenolic contents were observed in the range of 36.36 ± 1.08 mg GAE/g to 95.55 ± 2.46 mg GAE/g while flavonoid contents were found in the range of 10.51 ± 0.25 mg QE/g to 22.17 ± 1.79 mg QE/g. Antioxidant activity was determined using TRP, CUPRAC, TAC and DPPH assays and was recorded highest in S. papilionacea followed by T. glaberrima extracts. TPC and TFC were found to be strongly correlated with TRP (r > 0.50), CUPRAC (r > 0.53) and DPPH (r = 0.31 and 0.72) assay while weakly correlated with TAC (r = 0.08 and 0.03) as determined by Pearson correlation analysis. Anticancer activity showed that S. papilionacea chloroform extracts possess highest cell viability (85.04 ± 4.24%) against HepG2 cell lines while T. glaberrima chloroform extracts exhibited highest activity (82.80 ± 2.68%) against HT144 cell lines. Afterwards, highest PXR activation was observed in T. glaberrima (3.49 ± 0.34 µg/mL fold) at 60 µg/mL and was correlated with increase in CYP3A4 activity (15.0 ± 3.00 µg/mL IC50 value). Furthermore, antimalarial activity revealed >47600 IC50 value against P. falciparum D6 and P. falciparum W2 and antimicrobial assay indicated highest activity (32 ± 2.80 mm) in S. papilionacea against C. neoformans. At the end, GC-MS analysis of n-hexane plant extracts showed 99.104% of total identified compounds in T. glaberrima and 94.31% in V. stocksii. In conclusion, present study provides insight about the different biological potentials of S. papilionacea and T. glaberrima extracts that rationalize the applications of these extracts in functional foods and herbal drugs for the management of oxidative-stress related diseases, antimicrobial infections and liver and skin cancer.

Chemical composition of Gastrocotyle hispida (Forssk.) bunge and Heliotropium crispum Desf. and evaluation of their multiple in vitro biological potentials.

Medicinal plants largely serve as a source of bioactive compounds in traditional medicines to cure various diseases. The present study was aimed at chemical composition, antioxidant, antimicrobial, cytotoxic and antihemolytic potential of five different extracts of G. hispida and H. crispum (Boraginaceae). G. hispida methanolic extract displayed highest number (eleven) of polyphenolic compounds by using high performance liquid chromatography (HPLC). Functional groups were identified by Fourier-transformed infrared spectroscopy (FTIR) and elements (Si, Fe, Ba, Mg, Ti, Ca, Mg and Cr) were observed by using laser-induced breakdown spectroscopy (LIBS) which were also highly expressed in G. hispida as compared to H. crispum. Antioxidant activity was determined via six assays and antibacterial activity was observed in decreasing order of methanol > ethanol > chloroform > ethyl acetate > n-Hexane in both species. Cytotoxic potential was investigated against brine shrimps and then liver (HepG2) and skin (HT144) cancer cell lines which was detected highest in the G. hispida ethanolic extract (50.76 % and 72.95 %). However, H. crispum chloroform extract revealed highest (31.869 µg/mL) antihemolytic activity and its methanolic extract indicated highest (13.5 %) alpha-amylase inhibitory potential. Altogether, results suggested that both species could be used effectively in food and drug industries owing to the presence of vital bioactive compounds and elements. In future, we recommend to isolate active compounds and to perform in vivo biological assays to further validate their potential biological applications.

Relationship between loci of control and health-promoting behaviors in Pakistani women with polycystic ovary syndrome: coping strategies as mediators.

BACKGROUND/OBJECTIVES: Understanding the factors that promote healthy lifestyle behaviors in women with polycystic ovary syndrome is of substantial importance. Health-promoting lifestyle behaviors (HPLB) have been observed to be effective in managing various symptoms related to PCOS. This study aimed to examine the relationship between loci of control and health-promoting lifestyle behaviors in Pakistani women with polycystic ovary syndrome and the mediating role of coping strategies. METHOD: A correlational study was carried out with 145 unmarried women with polycystic ovary syndrome diagnosed by a gynecologist using the Rotterdam Criteria of 2003 (M age = 24.75 years). Participants were recruited from public sector hospitals in Lahore, Punjab, Pakistan and a series of hierarchical regression analyses were used to analyze results. RESULTS: Findings suggest that women with internal and powerful others locus of control use more active practical coping strategies and less active distractive coping strategies. These women also get more involved in health-promoting behaviors. On the other hand, those with a high level of chance locus of control use less active practical coping strategies and more active distractive coping strategies. In turn, they engage less in health-promoting behaviors. CONCLUSION: Health professionals should consider the effects of different types of locus of control and coping strategies when planning interventions for women with polycystic ovary syndrome.

MASTL regulates EGFR signaling to impact pancreatic cancer progression.

Pancreatic cancer (PC) remains a major cause of cancer-related deaths primarily due to its inherent potential of therapy resistance. Checkpoint inhibitors have emerged as promising anti-cancer agents when used in combination with conventional anti-cancer therapies. Recent studies have highlighted a critical role of the Greatwall kinase (microtubule-associated serine/threonine-protein kinase-like (MASTL)) in promoting oncogenic malignancy and resistance to anti-cancer therapies; however, its role in PC remains unknown. Based on a comprehensive investigation involving PC patient samples, murine models of PC progression (Kras;PdxCre-KC and Kras;p53;PdxCre-KPC), and loss and gain of function studies, we report a previously undescribed critical role of MASTL in promoting cancer malignancy and therapy resistance. Mechanistically, MASTL promotes PC by modulating the epidermal growth factor receptor protein stability and, thereupon, kinase signaling. We further demonstrate that combinatorial therapy targeting MASTL promotes the efficacy of the cell-killing effects of Gemcitabine using both genetic and pharmacological inhibitions. Taken together, this study identifies a key role of MASTL in promoting PC progression and its utility as a novel target in promoting sensitivity to the anti-PC therapies.

Targeting Wnt Signaling in Endometrial Cancer.

This review presents new findings on Wnt signaling in endometrial carcinoma and implications for possible future treatments. The Wnt proteins are essential mediators in cell signaling during vertebrate embryo development. Recent biochemical and genetic studies have provided significant insight into Wnt signaling, in particular in cell cycle regulation, inflammation, and cancer. The role of Wnt signaling is well established in gastrointestinal and breast cancers, but its function in gynecologic cancers, especially in endometrial cancers, has not been well elucidated. Development of a subset of endometrial carcinomas has been attributed to activation of the APC/ß-catenin signaling pathway (due to ß-catenin mutations) and downregulation of Wnt antagonists by epigenetic silencing. The Wnt pathway also appears to be linked to estrogen and progesterone, and new findings implicate it in mTOR and Hedgehog signaling. Therapeutic interference of Wnt signaling remains a significant challenge. Herein, we discuss the Wnt-activating mechanisms in endometrial cancer and review the current advances and challenges in drug discovery.

Pancreatic Cancer and Therapy: Role and Regulation of Cancer Stem Cells.

Despite significant improvements in clinical management, pancreatic cancer (PC) remains one of the deadliest cancer types, as it is prone to late detection with extreme metastatic properties. The recent findings that pancreatic cancer stem cells (PaCSCs) contribute to the tumorigenesis, progression, and chemoresistance have offered significant insight into the cancer malignancy and development of precise therapies. However, the heterogeneity of cancer and signaling pathways that regulate PC have posed limitations in the effective targeting of the PaCSCs. In this regard, the role for K-RAS, TP53, Transforming Growth Factor-ß, hedgehog, Wnt and Notch and other signaling pathways in PC progression is well documented. In this review, we discuss the role of PaCSCs, the underlying molecular and signaling pathways that help promote pancreatic cancer development and metastasis with a specific focus on the regulation of PaCSCs. We also discuss the therapeutic approaches that target different PaCSCs, intricate mechanisms, and therapeutic opportunities to eliminate heterogeneous PaCSCs populations in pancreatic cancer.

MASTL: A novel therapeutic target for Cancer Malignancy.

Targeting mitotic kinases is an emerging anticancer approach with promising preclinical outcomes. Microtubule-associated serine/threonine kinase like (MASTL), also known as Greatwall (Gwl), is an important mitotic kinase that regulates mitotic progression of normal or transformed cells by blocking the activity of tumor suppressor protein phosphatase 2A (PP2A). MASTL upregulation has now been detected in multiple cancer types and associated with aggressive clinicopathological features. Apart, an aberrant MASTL activity has been implicated in oncogenic transformation through the development of chromosomal instability and alteration of key oncogenic signaling pathways. In this regard, recent publications have revealed potential role of MASTL in the regulation of AKT/mTOR and Wnt/ß-catenin signaling pathways, which may be independent of its regulation of PP2A-B55 (PP2A holoenzyme containing a B55-family regulatory subunit). Taken together, MASTL kinase has emerged as a novel target for cancer therapeutics, and hence development of small molecule inhibitors of MASTL may significantly improve the clinical outcomes of cancer patients. In this article, we review the role of MASTL in cancer progression and the current gaps in this knowledge. We also discuss potential efficacy of MASTL expression for cancer diagnosis and therapy.

Upregulated Expression of Calcium-Dependent Annexin A6: A Potential Biomarker of Ovarian Carcinoma.

PURPOSE: An early and accurate diagnosis of ovarian carcinoma (OC) may reduce morbidity and mortality of the patients. To improve the clinical outcome in OC patients, the present study is aimed at identifying robust biomarkers for early OC diagnosis. EXPERIMENTAL DESIGN: In order to look for early-stage protein markers, a systematic protein profiling approach involving 2-dimensional electrophoresis coupled with mass spectrometric analyses of human malignant and non-malignant ovarian biopsy samples, is performed. RESULTS: Six 2D gel spots, corresponding to five proteins, display statistically significant differential expression in the tumor tissues compared to benign controls (FDR ≤ 0.05; PMF score ≥ 79). Ingenuity pathway analysis predicts two proteins, that is, Ca2+ -dependent membrane-binding protein annexin A6 (AnxA6) and the metabolic enzyme l-lactate dehydrogenase A chain, as potential predictive biomarkers. Increased expression of AnxA6 is further ascertained by Western blot and enzyme linked immunosorbent assay in the resected tissues and the plasma samples. The expression is found markedly increasing particularly in the advanced stage tumors. CONCLUSIONS AND CLINICAL RELEVANCE: The significant upregulation of AnxA6 in OC, reported for the first time, is likely to provide insight into the mechanism of OC progression, which may lead to the design of potential diagnostic and therapeutic strategies.

Simultaneous Multi-Organ Metastases from Chemo-Resistant Triple-Negative Breast Cancer Are Prevented by Interfering with WNT-Signaling.

Triple-negative breast cancers (TNBCs), which lack specific targeted therapy options, evolve into highly chemo-resistant tumors that metastasize to multiple organs simultaneously. We have previously shown that TNBCs maintain an activated WNT10B-driven network that drives metastasis. Pharmacologic inhibition by ICG-001 decreases ß-catenin-mediated proliferation of multiple TNBC cell lines and TNBC patient-derived xenograft (PDX)-derived cell lines. In vitro, ICG-001 was effective in combination with the conventional cytotoxic chemotherapeutics, cisplatin and doxorubicin, to decrease the proliferation of MDA-MB-231 cells. In contrast, in TNBC PDX-derived cells doxorubicin plus ICG-001 was synergistic, while pairing with cisplatin was not as effective. Mechanistically, cytotoxicity induced by doxorubicin, but not cisplatin, with ICG-001 was associated with increased cleavage of PARP-1 in the PDX cells only. In vivo, MDA-MB-231 and TNBC PDX orthotopic primary tumors initiated de novo simultaneous multi-organ metastases, including bone metastases. WNT monotherapy blocked multi-organ metastases as measured by luciferase imaging and histology. The loss of expression of the WNT10B/ß-catenin direct targets HMGA2, EZH2, AXIN2, MYC, PCNA, CCND1, transcriptionally active ß-catenin, SNAIL and vimentin both in vitro and in vivo in the primary tumors mechanistically explains loss of multi-organ metastases. WNT monotherapy induced VEGFA expression in both tumor model systems, whereas increased CD31 was observed only in the MDA-MB-231 tumors. Moreover, WNT-inhibition sensitized the anticancer response of the TNBC PDX model to doxorubicin, preventing simultaneous metastases to the liver and ovaries, as well as to bone. Our data demonstrate that WNT-inhibition sensitizes TNBC to anthracyclines and treats multi-organ metastases of TNBC.

Natural Products Mediated Targeting of Virally Infected Cancer.

The role of viral infection in developing cancer was determined in the start of 20th century. Until now, 8 different virus-associated cancers have been discovered and most of them progressed in immunosuppressed individuals. The aim of the present study is to look into the benefits of natural products in treating virally infected cancers. The study focuses on bioactive compounds derived from natural sources. Numerous pharmaceutical agents have been identified from plants (vincristine, vinblastine, stilbenes, combretastatin, and silymarin), marine organisms (bryostatins, cephalostatin, ecteinascidins, didemnin, and dolastatin), insects (cantharidin, mastoparan, parectadial, and cecropins), and microorganisms (vancomycin, rhizoxin, ansamitocins, mitomycin, and rapamycin). Beside these, various compounds have been observed from fruits and vegetables which can be utilized in anticancer therapy. These include curcumin in turmeric, resveratrol in red grapes, S-allyl cysteine in allium, allicin in garlic, catechins in green tea, and ß-carotene in carrots. The present study addresses various types of virally infected cancers, their mechanism of action, and the role of different cell surface molecules elicited during viral binding and entry into the target cell along with the anticancer drugs derived from natural products by targeting screening of bioactive compounds from natural sources.

Increased trend of unnecessary use of radiological diagnostic modalities in Pakistan: radiologists perspective.

BACKGROUND: Over the past few years, a significant overuse of radiological investigations influenced the quality and cost of healthcare of the country as it may lead to non-compliance of the patient due to non-affordability and also may harm the patient in terms of radiation hazards. Pakistan, being a low-income, resource-constraint country, is facing financial impact on families as well as health system due to multiple reasons. OBJECTIVES: The purpose of study is to identify reasons of unnecessary use of radiological diagnostic modalities in Pakistani hospitals as perceived by radiologists. METHODS: A cross-sectional study was conducted on a total of all 105 radiologists, having at least 1 year experience of working in radiology, working in five tertiary care hospitals in Lahore. A self-constructed, self-administered, pretested 5-point Likert scale opinion-based questionnaire was administered after taking informed consent. It includes questions about excessive radiological use that may be attributed to the physicians, investigations, patients and other non-categorized causes. Results were analyzed using SPSS version 23. RESULTS: Since the assessment forms were handed over and collected from the radiologists in person, the response rate was 100%. Of a total of 105 respondents, 78 (74.28%) respondentsagreed that there is an actual increase, 25 (23.80%) respondents disagreed and 2 (1.90%) respondents were unsure. Most important reasons for increased usage of radiological investigations are 'need of accuracy of diagnosis' (P = 0.009), 'trend of physicians to repeat tests in order to confirm preset diagnoses' (P-value = 0.03), 'lack of knowledge about proper usage of radiological advances' (P-value = 0.005) and 'lack of proper clinical examination' (P-value = 0.04). CONCLUSION: Unnecessary use of radiological investigations is actually there as perceived by radiologists, which is attributed to inadequate knowledge, attitude and training of physicians to refer patients to radiological resources. This research can be a stepping stone for future researchers as it can be used for elaborating these causes individually and finding ways as to how each of these causes can be controlled and minimized to bring about a decline in excessive usage of these modalities for the betterment of the patients.

The WNT10B Network Is Associated with Survival and Metastases in Chemoresistant Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) commonly develops resistance to chemotherapy, yet markers predictive of chemoresistance in this disease are lacking. Here, we define WNT10B-dependent biomarkers for ß-CATENIN/HMGA2/EZH2 signaling predictive of reduced relapse-free survival. Concordant expression of HMGA2 and EZH2 proteins is observed in MMTV-Wnt10bLacZ transgenic mice during metastasis, and Hmga2 haploinsufficiency decreased EZH2 protein expression, repressing lung metastasis. A novel autoregulatory loop interdependent on HMGA2 and EZH2 expression is essential for ß-CATENIN/TCF-4/LEF-1 transcription. Mechanistically, both HMGA2 and EZH2 displaced Groucho/TLE1 from TCF-4 and served as gatekeepers for K49 acetylation on ß-CATENIN, which is essential for transcription. In addition, we discovered that HMGA2-EZH2 interacts with the PRC2 complex. Absence of HMGA2 or EZH2 expression or chemical inhibition of Wnt signaling in a chemoresistant patient-derived xenograft (PDX) model of TNBC abolished visceral metastasis, repressing AXIN2, MYC, EZH2, and HMGA2 expression in vivo. Combinatorial therapy of a WNT inhibitor with doxorubicin synergistically activated apoptosis in vitro, resensitized PDX-derived cells to doxorubicin, and repressed lung metastasis in vivo. We propose that targeting the WNT10B biomarker network will provide improved outcomes for TNBC. SIGNIFICANCE: These findings reveal targeting the WNT signaling pathway as a potential therapeutic strategy in triple-negative breast cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/5/982/F1.large.jpg.

Activation of Estrogen Receptor Alpha by Decitabine Inhibits Osteosarcoma Growth and Metastasis.

Osteosarcoma is a malignant tumor in the bone, which originates from normal osteoblasts or osteoblast precursors. Normal osteoblasts express estrogen receptor alpha (ERα); however, osteosarcomas do not express ERα due to promoter DNA methylation. Here we show that treatment of 143B osteosarcoma cells with decitabine (DAC, 5-Aza-2'-deoxycytidine) induces expression of ERα and leads to decreased proliferation and concurrent induction of osteoblast differentiation. DAC exposure reduced protein expression of metastasis-associated markers VIMENTIN, SLUG, ZEB1, and MMP9, with a concurrent decrease in mRNA expression of known stem cell markers SOX2, OCT4, and NANOG. Treatment with 17ß-estradiol (E2) synergized with DAC to reduce proliferation. Overexpression of ERα inhibited proliferation and induced osteoblast differentiation, whereas knockout of ERα by CRISPR/Cas9 prevented the effects of DAC. In an orthotopic model of osteosarcoma, DAC inhibited tumor growth and metastasis of 143B cells injected into the tibia of NOD SCID gamma mice. Furthermore, ERα overexpression reduced tumor growth and metastasis, and ERα knockout prevented the effects of DAC in vivo. Together, these experiments provide preclinical evidence that the FDA-approved DNA methylation inhibitor DAC may be repurposed to treat patients with osteosarcoma based on its efficacy to decrease proliferation, to induce osteoblast differentiation, and to reduce metastasis to visceral organs.Significance: These findings describe the effects of DNA methyltransferase inhibition on ERα and its potential role as a tumor suppressor in osteosarcoma.See related commentary by Roberts, p. 1034 See related article by El Ayachi and colleagues; Cancer Res 79(5);982-93.

GATA4 represses RANKL in osteoblasts via multiple long-range enhancers to regulate osteoclast differentiation.

GATA4 is a transcription factor that is responsible for tissue-specific gene regulation in many tissues, and more recent studies showed that it is necessary for osteoblast differentiation. Previously, we showed that in vivo deletion of Gata4 using Cre-recombinase under the control of the Col1a1 2.3 kb promoter, showed significantly reduced trabecular bone properties. To understand the role of GATA4 in more differentiated cells, GATA4fl/fl mice were crossed with mice expressing Cre-recombinase under the control of the osteocalcin promoter. MicroCT analysis of trabecular bone properties of the femur and tibia from 14-week-old female osteocalcin-Cre/GATA4fl/fl (OCN-cKO) mice showed a significant reduction in percentage bone volume, a decrease in trabecular number and an increase in trabecular spacing. In vivo, histomorphometric analysis revealed a decrease in the number of osteoblasts and an increase in the number of osteoclasts in the tibiae of OCN-cKO mice. In vivo and in vitro systems correlated a decrease in Gata4 mRNA with increased RANKL gene expression. To determine if RANKL is a direct target of GATA4, chromatin immunoprecipitation (ChIP)-sequencing was performed, and it demonstrated that GATA4 is recruited to seven enhancers near RANKL. Furthermore, when Gata4 is knocked down, the chromatin at the RANKL region is further opened, as detected by a reduction in histone 3 lysine 27 trimethylation (H3K27me3) and an increase in histone 3 lysine 4 dimethylation (H3K4me2) in the RANKL locus. In vitro, TRAP staining of cells from bone marrow cultures from Gata4 knockout cells show that the increased levels of RANKL are sufficient for osteoclast formation. Together, the data suggest that GATA4 directly represses RANKL expression via seven cis-regulatory regions and plays an important role in maintaining proper bone development and osteoclast formation.