The Epithelial-Mesenchymal Transcription Factor SNAI1 Represses Transcription of the Tumor Suppressor miRNA let-7 in Cancer.

We aimed to determine the mechanism of epithelial-mesenchymal transition (EMT)-induced stemness in cancer cells. Cancer relapse and metastasis are caused by rare stem-like cells within tumors. Studies of stem cell reprogramming have linked let-7 repression and acquisition of stemness with the EMT factor, SNAI1. The mechanisms for the loss of let-7 in cancer cells are incompletely understood. In four carcinoma cell lines from breast cancer, pancreatic cancer, and ovarian cancer and in ovarian cancer patient-derived cells, we analyzed stem cell phenotype and tumor growth via mRNA, miRNA, and protein expression, spheroid formation, and growth in patient-derived xenografts. We show that treatment with EMT-promoting growth factors or SNAI1 overexpression increased stemness and reduced let-7 expression, while SNAI1 knockdown reduced stemness and restored let-7 expression. Rescue experiments demonstrate that the pro-stemness effects of SNAI1 are mediated via let-7. In vivo, nanoparticle-delivered siRNA successfully knocked down SNAI1 in orthotopic patient-derived xenografts, accompanied by reduced stemness and increased let-7 expression, and reduced tumor burden. Chromatin immunoprecipitation demonstrated that SNAI1 binds the promoters of various let-7 family members, and luciferase assays revealed that SNAI1 represses let-7 transcription. In conclusion, the SNAI1/let-7 axis is an important component of stemness pathways in cancer cells, and this study provides a rationale for future work examining this axis as a potential target for cancer stem cell-specific therapies.

Nanomachines and Other Caps on Mesoporous Silica Nanoparticles for Drug Delivery.

Mesoporous silica nanoparticles (MSNs) are delivery vehicles that can carry cargo molecules and release them on command. The particles used in the applications reported in this Account are around 100 nm in diameter (about the size of a virus) and contain 2.5 nm tubular pores with a total volume of about 1 cm3/g. For the biomedical applications discussed here, the cargo is trapped in the pores until the particles are stimulated to release it. The challenges are to get the particles to the site of a disease and then to deliver the cargo on command. We describe methods to do both, and we illustrate the applicability of the particles to cure cancer and intracellular infectious disease. Our first steps were to design multifunctional nanoparticles with properties that allow them to carry and deliver hydrophobic drugs. Many important pharmaceuticals are hydrophobic and cannot reach the diseased sites by themselves. We describe how we modified MSNs to make them dispersible, imagable, and targetable and discuss in vitro studies. We then present examples of surface modifications that allow them to deliver large molecules such as siRNA. In vivo studies of siRNA delivery to treat triple-negative breast and ovarian cancers are presented. The next steps are to attach nanomachines and other types of caps that trap drug molecules but release them when stimulated. We describe nanomachines that respond autonomously (without human intervention) to stimuli specific to disease sites. A versatile type of machine is a nanovalve that is closed at neutral (blood) pH but opens upon acidification that occurs in endolysosomes of cancer cells. Another type of machine, a snap-top cap, is stimulated by reducing agents such as glutathione in the cytosol of cells. Both of these platforms were studied in vitro to deliver antibiotics to infected macrophages and in vivo to cure and kill the intracellular bacteria M. tuberculosis and F. tularensis. The latter is a tier 1 select agent of bioterrorism. Finally, we describe nanomachines for drug delivery that are controlled by externally administered light and magnetic fields. A futuristic dream for nanotherapy is the ability to control a nano-object everywhere in the body. Magnetic fields penetrate completely and have spatial selectivity governed by the size of the field-producing coil. We describe how to control nanovalves with alternating magnetic fields (AMFs) and superparamagnetic cores inside the MSNs. The AMF heats the cores, and temperature-sensitive caps release the cargo. In vitro studies demonstrate dose control of the therapeutic to cause apoptosis without overheating the cells. Nanocarriers have great promise for therapeutic applications, and MSNs that can carry drugs to the site of a disease to produce a high local concentration without premature release and off-target damage may have the capability of realizing this goal.

Protein kinase Cα-mediated phosphorylation of Twist1 at Ser-144 prevents Twist1 ubiquitination and stabilizes it.

Twist1 is a basic helix-loop-helix transcription factor that plays a key role in embryonic development, and its expression is down-regulated in adult cells. However, Twist1 is highly expressed during cancer development, conferring a proliferative, migratory, and invasive phenotype to malignant cells. Twist1 expression can be regulated post-translationally by phosphorylation or ubiquitination events. We report in this study a previously unknown and relevant Twist1 phosphorylation site that controls its stability. To identify candidate phosphorylation sites in Twist1, we first conducted an in silico analysis of the Twist1 protein, which yielded several potential sites. Because most of these sites were predicted to be phosphorylated by protein kinase C (PKC), we overexpressed PKCα in several cell lines and found that it phosphorylates Twist1 on Ser-144. Using a combination of immunoblotting, immunoprecipitation, protein overexpression, and CRISPR/Cas9-mediated PKCα knockout experiments, we observed that PKCα-mediated Twist1 phosphorylation at Ser-144 inhibits Twist1 ubiquitination and consequently stabilizes it. These results provide evidence for a direct association between PKCα and Twist1 and yield critical insights into the PKCα/Twist1 signaling axis that governs cancer aggressiveness.

p53-Pirh2 Complex Promotes Twist1 Degradation and Inhibits EMT.

Epithelial-mesenchymal transition (EMT) is a critical process involved in cancer metastasis and chemoresistance. Twist1 is a key EMT-inducing transcription factor, which is upregulated in multiple types of cancers and has been shown to promote tumor cell invasiveness and support tumor progression. Conversely, p53 is a tumor suppressor gene that is frequently mutated in cancers. This study demonstrates the ability of wild-type (WT) p53 to promote the degradation of Twist1 protein. By forming a complex with Twist1 and the E3 ligase Pirh2, WT p53 promotes the ubiquitination and proteasomal degradation of Twist1, thus inhibiting EMT and maintaining the epithelial phenotype. The ability of p53 to induce Twist1 degradation is abrogated when p53 is mutated. Consequently, the loss of p53-induced Twist1 degradation leads to EMT and the acquisition of a more invasive cancer phenotype.Implication: These data provide new insight into the metastatic process at the molecular level and suggest a signaling pathway that can potentially be used to develop new prognostic markers and therapeutic targets to curtail cancer progression.

Nanoparticle Delivery of TWIST Small Interfering RNA and Anticancer Drugs: A Therapeutic Approach for Combating Cancer.

Breast and ovarian cancer are the leading cause of cancer-related deaths in women in the United States with over 232,000 new Breast Cancer (BC) diagnoses expected in 2018 and almost 40,000 deaths and an estimated 239,000 new ovarian cancer (OC) cases and 152,000 deaths worldwide annually. OC is the most lethal gynecologic malignancy. This high mortality rate is due to tumor recurrence and metastasis, primarily caused by chemoresistant cancer stem-like cells (CSCs). Triple Negative Breast Cancer (TNBC) patients also become resistant to chemotherapy due to recurrence of CSCs. Currently, no ovarian or breast cancer therapies target CSC specifically. TWIST is overexpressed in the majority of chemoresistant cancers resulting in a low survival rate. Our long-term goal is to develop novel treatments for women with ovarian and breast cancer, specifically treatments that sensitize chemoresistant tumors. Despite successful initial surgery and chemotherapy, over 70% of advanced EOC will recur, and only 15-30% of recurrent disease will respond to chemotherapy (Cortez et al., 2017; Berezhnaya, 2010; Jackson et al., 2015). Moreover, drug resistance causes treatment failure in over 90% of patients with metastatic disease (Solmaz et al., 2015). Thus, recurrent metastatic disease is a major clinical challenge without effective therapy. One of the major challenges in the treatment of breast cancer is the presence of a subpopulation of cancer cells that are chemoresistant (CRC) and metastatic. Given that metastasis is the driving force behind mortality for breast and ovarian cancer patients, it is essential to identify the characteristics of these aberrant cancer cells that allow them to spread to distant sites in the body and develop into metastatic tumors. Understanding the metastatic mechanisms driving cancer cell dispersal will open the door to developing novel therapies that prevent metastasis and improve long-term outcomes for patients. In this chapter we assess the feasibility of targeting the Twist and EMT signaling pathways in breast and ovarian cancer. Additional discussions of the pathways that mediate epithelial-mesenchymal transition (EMT), a process that can give rise to chemoresistance. We review potential treatment strategies for targeting EMT and drug resistance as well as the problems that may arise with these targeted delivery therapeutic approaches. Finally, we examine recent advances in the field, including cancer stem cell targeted nanoparticle delivery and small interference RNA (siRNA) technology, and discuss the impact that these approaches may have on translating much needed therapeutic approaches into the clinic, for the benefit of patients battling this devastating disease.

Pterostilbene Suppresses Ovarian Cancer Growth via Induction of Apoptosis and Blockade of Cell Cycle Progression Involving Inhibition of the STAT3 Pathway.

A growing body of evidence has demonstrated the promising anti-tumor effects of resveratrol in ovarian cancer cells, including its inhibitory effects on STAT3 activation. Nonetheless, the low bioavailability of resveratrol has reduced its attractiveness as a potential anti-cancer treatment. In contrast, pterostilbene, a stilbenoid and resveratrol analog, has demonstrated superior bioavailability, while possessing significant antitumor activity in multiple solid tumors. In this study, the therapeutic potential of pterostilbene was evaluated in ovarian cancer cells. Pterostilbene reduces cell viability in several different ovarian cancer cell lines by suppressing cell cycle progression and inducing apoptosis. Further molecular study has shown that pterostilbene effectively suppressed phosphorylation of STAT3, as well as STAT3 downstream genes that regulate cell cycle and apoptosis, indicating that inhibition of STAT3 pathway may be involved in its anti-tumor activity. The addition of pterostilbene to the commonly used chemotherapy cisplatin demonstrated synergistic antiproliferative activity in several ovarian cancer cell lines. Pterostilbene additionally inhibited cell migration in multiple ovarian cancer cell lines. The above results suggest that pterostilbene facilitates significant anti-tumor activity in ovarian cancer via anti-proliferative and pro-apoptotic mechanisms, possibly via downregulation of JAK/STAT3 pathway. Pterostilbene thus presents as an attractive non-toxic alternative for potential adjuvant or maintenance chemotherapy in ovarian cancer.

Tyrosine kinase receptor c-ros-oncogene 1 inhibition alleviates aberrant bone formation of TWIST-1 haploinsufficient calvarial cells from Saethre-Chotzen syndrome patients.

Saethre-Chotzen syndrome (SCS), associated with TWIST-1 mutations, is characterized by premature fusion of cranial sutures. TWIST-1 haploinsufficiency, leads to alterations in suture mesenchyme cellular gene expression patterns, resulting in aberrant osteogenesis and craniosynostosis. We analyzed the expression of the TWIST-1 target, Tyrosine kinase receptor c-ros-oncogene 1 (C-ROS-1) in TWIST-1 haploinsufficient calvarial cells derived from SCS patients and calvaria of Twist-1del/+ mutant mice and found it to be highly expressed when compared to TWIST-1 wild-type controls. Knock-down of C-ROS-1 expression in TWIST-1 haploinsufficient calvarial cells derived from SCS patients was associated with decreased capacity for osteogenic differentiation in vitro. Furthermore, treatment of human SCS calvarial cells with the tyrosine kinase chemical inhibitor, Crizotinib, resulted in reduced C-ROS-1 activity and the osteogenic potential of human SCS calvarial cells with minor effects on cell viability or proliferation. Cultured human SCS calvarial cells treated with Crizotinib exhibited a dose-dependent decrease in alkaline phosphatase activity and mineral deposition, with an associated decrease in expression levels of Runt-related transcription factor 2 and OSTEOPONTIN, with reduced PI3K/Akt signalling in vitro. Furthermore, Crizotinib treatment resulted in reduced BMP-2 mediated bone formation potential of whole Twist-1del/+ mutant mouse calvaria organotypic cultures. Collectively, these results suggest that C-ROS-1 promotes osteogenic differentiation of TWIST-1 haploinsufficient calvarial osteogenic progenitor cells. Furthermore, the aberrant osteogenic potential of these cells is inhibited by the reduction of C-ROS-1. Therefore, targeting C-ROS-1 with a pharmacological agent, such as Crizotinib, may serve as a novel therapeutic strategy to alleviate craniosynostosis associated with aberrant TWIST-1 function.

Hyaluronic acid conjugated nanoparticle delivery of siRNA against TWIST reduces tumor burden and enhances sensitivity to cisplatin in ovarian cancer.

TWIST protein is critical to development and is activated in many cancers. TWIST regulates epithelial-mesenchymal transition, and is linked to angiogenesis, metastasis, cancer stem cell phenotype, and drug resistance. The majority of epithelial ovarian cancer (EOC) patients with metastatic disease respond well to first-line chemotherapy but most relapse with disease that is both metastatic and drug resistant, leading to a five-year survival rate under 20%. We are investigating the role of TWIST in mediating these relapses. We demonstrate TWIST-siRNA (siTWIST) and a novel nanoparticle delivery platform to reverse chemoresistance in an EOC model. Hyaluronic-acid conjugated mesoporous silica nanoparticles (MSN-HAs) carried siTWIST into target cells and led to sustained TWIST knockdown in vitro. Mice treated with siTWIST-MSN-HA and cisplatin exhibited specific tumor targeting and reduction of tumor burden. This platform has potential application for overcoming clinical challenges of tumor cell targeting, metastasis and chemoresistance in ovarian and other TWIST overexpressing cancers.

Pterostilbene, a natural phenolic compound, synergizes the antineoplastic effects of megestrol acetate in endometrial cancer.

Endometrial cancer is the most common gynecologic cancer in the United States and its incidence and mortality has been rising over the past decade. Few treatment options are available for patients with advanced and recurring endometrial cancers. Novel therapies, which are frequently toxic, are difficult to establish in this patient population which tends to be older and plagued by comorbidities such as diabetes mellitus and hypertension. Therefore, novel, non-toxic therapies are urgently needed. Megestrol acetate is a frequently used drug in endometrial cancer patients. However, its response rate is only 20-30%. To enhance the activity of megestrol acetate in endometrial cancer patients, we explored the potential of combining natural supplements with megestrol acetate and found that the addition of the natural phenolic compound, pterostilbene, to megestrol acetate resulted in a synergistic inhibition of cancer cell growth in vitro and an enhanced reduction of tumor growth in a xenograft mouse model. In addition, dual treatment led to attenuation of signaling pathways, as well as cell cycle and survival pathways. Our results demonstrated for the first time that the anti-tumor activity of megestrol acetate can be enhanced by combining with pterostilbene, providing an insight into the potential application of pterostilbene and megestrol acetate combination for the treatment of endometrial cancer.

Disruption of TWIST1-RELA binding by mutation and competitive inhibition to validate the TWIST1 WR domain as a therapeutic target.

BACKGROUND: Most cancer deaths result from tumor cells that have metastasized beyond their tissue of origin, or have developed drug resistance. Across many cancer types, patients with advanced stage disease would benefit from a novel therapy preventing or reversing these changes. To this end, we have investigated the unique WR domain of the transcription factor TWIST1, which has been shown to play a role in driving metastasis and drug resistance. METHODS: In this study, we identified evolutionarily well-conserved residues within the TWIST1 WR domain and used alanine substitution to determine their role in WR domain-mediated protein binding. Co-immunoprecipitation was used to assay binding affinity between TWIST1 and the NFκB subunit p65 (RELA). Biological activity of this complex was assayed using a dual luciferase assay system in which firefly luciferase was driven by the interleukin-8 (IL-8) promoter, which is upregulated by the TWIST1-RELA complex. Finally, in order to inhibit the TWIST1-RELA interaction, we created a fusion protein comprising GFP and the WR domain. Cell fractionation and proteasome inhibition experiments were utilized to elucidate the mechanism of action of the GFP-WR fusion. RESULTS: We found that the central residues of the WR domain (W190, R191, E193) were important for TWIST1 binding to RELA, and for increased activation of the IL-8 promoter. We also found that the C-terminal 245 residues of RELA are important for TWIST1 binding and IL-8 promoter activation. Finally, we found the GFP-WR fusion protein antagonized TWIST1-RELA binding and downstream signaling. Co-expression of GFP-WR with TWIST1 and RELA led to proteasomal degradation of TWIST1, which could be inhibited by MG132 treatment. CONCLUSIONS: These data provide evidence that mutation or inhibition of the WR domain reduces TWIST1 activity, and may represent a potential therapeutic modality.

Nanoparticle delivery of siRNA against TWIST to reduce drug resistance and tumor growth in ovarian cancer models.

Epithelial ovarian cancer (EOC) is the most deadly gynecologic malignancy on account of its late stage at diagnosis and frequency of drug resistant recurrences. Novel therapies to overcome these barriers are urgently needed. TWIST is a developmental transcription factor reactivated in cancers and linked to angiogenesis, metastasis, cancer stem cell phenotype, and drug resistance, making it a promising therapeutic target. In this work, we demonstrate the efficacy of TWIST siRNA (siTWIST) and two nanoparticle delivery platforms to reverse chemoresistance in EOC models. Polyamidoamine dendrimers and mesoporous silica nanoparticles (MSNs) carried siTWIST into target cells and led to sustained TWIST knockdown in vitro. Mice treated with cisplatin plus MSN-siTWIST exhibited lower tumor burden than mice treated with cisplatin alone, with most of the effect coming from reduction in disseminated tumors. This platform has potential application for overcoming the clinical challenges of metastasis and chemoresistance in EOC and other TWIST overexpressing cancers.

TWIST1 drives cisplatin resistance and cell survival in an ovarian cancer model, via upregulation of GAS6, L1CAM, and Akt signalling.

Epithelial ovarian cancer (EOC) is the most deadly gynaecologic malignancy due to late onset of symptoms and propensity towards drug resistance. Epithelial-mesenchymal transition (EMT) has been linked to the development of chemoresistance in other cancers, yet little is known regarding its role in EOC. In this study, we sought to determine the role of the transcription factor TWIST1, a master regulator of EMT, on cisplatin resistance in an EOC model. We created two Ovcar8-derived cell lines that differed only in their TWIST1 expression. TWIST1 expression led to increased tumour engraftment in mice, as well as cisplatin resistance in vitro. RNA sequencing analysis revealed that TWIST1 expression resulted in upregulation of GAS6 and L1CAM and downregulation of HMGA2. Knockdown studies of these genes demonstrated that loss of GAS6 or L1CAM sensitized cells to cisplatin, but that loss of HMGA2 did not give rise to chemoresistance. TWIST1, in part via GAS6 and L1CAM, led to higher expression and activation of Akt upon cisplatin treatment, and inhibition of Akt activation sensitized cells to cisplatin. These results suggest TWIST1- and EMT-driven increase in Akt activation, and thus tumour cell proliferation, as a potential mechanism of drug resistance in EOC.

15-Methylene-Eburnamonine Kills Leukemic Stem Cells and Reduces Engraftment in a Humanized Bone Marrow Xenograft Mouse Model of Leukemia.

Recent studies suggest that leukemia stem cells (LSCs) play a critical role in the initiation, propagation, and relapse of leukemia. Herein we show that (-)-15-methylene-eburnamonine, a derivative of the alkaloid (-)-eburnamonine, is cytotoxic against acute and chronic lymphocytic leukemias (ALL and CLL) and acute myelogenous leukemia (AML). The agent also decreases primary LSC frequency in vitro. The cytotoxic effects appear to be mediated via the oxidative stress pathways. Furthermore, we show that the compound kills AML, ALL, and CLL stem cells. By the use of a novel humanized bone marrow murine model of leukemia (huBM/NSG), it was found to decrease progenitor cell engraftment.

Twist-1 Enhances Bone Marrow Mesenchymal Stromal Cell Support of Hematopoiesis by Modulating CXCL12 Expression.

Twist-1 encodes a basic helix-loop-helix transcription factor, known to contribute to mesodermal and skeletal tissue development. We have reported previously that Twist-1 maintains multipotent human bone marrow-derived mesenchymal stem/stromal cells (BMSC) in an immature state, enhances their life-span, and influences cell fate determination. In this study, human BMSC engineered to express high levels of Twist-1 were found to express elevated levels of the chemokine, CXCL12. Analysis of the CXCL12 proximal promoter using chromatin immunoprecipitation analysis identified several E-box DNA sites bound by Twist-1. Functional studies using a luciferase reporter construct showed that Twist-1 increased CXCL12 promoter activity in a dose dependent manner. Notably, Twist-1 over-expressing BMSC exhibited an enhanced capacity to maintain human CD34 + hematopoietic stem cells (HSC) in long-term culture-initiating cell (LTC-IC) assays. Moreover, the observed increase in HSC maintenance by Twist-1 over-expressing BMSC was blocked in the presence of the CXCL12 inhibitor, AMD3100. Supportive studies, using Twist-1 deficient heterozygous mice demonstrated a significant decrease in the frequency of stromal progenitors and increased numbers of osteoblasts within the bone. These observations correlated to a decreased incidence in the number of clonogenic stromal progenitors (colony forming unit-fibroblasts) and lower levels of CXCL12 in Twist-1 mutant mice. Furthermore, Twist-1 deficient murine stromal feeder layers, exhibited a significant decrease in CXCL12 levels and lower numbers of hematopoietic colonies in LTC-IC assays, compared with wild type controls. These findings demonstrate that Twist-1, which maintains BMSC at an immature state, endows them with an increased capacity for supporting hematopoiesis via direct activation of CXCL12 gene expression.

Mesoporous silica nanoparticle delivery of chemically modified siRNA against TWIST1 leads to reduced tumor burden.

Growth and progression of solid tumors depend on the integration of multiple pro-growth and survival signals, including the induction of angiogenesis. TWIST1 is a transcription factor whose reactivation in tumors leads to epithelial to mesenchymal transition (EMT), including increased cancer cell stemness, survival, and invasiveness. Additionally, TWIST1 drives angiogenesis via activation of IL-8 and CCL2, independent of VEGF signaling. In this work, results suggest that chemically modified siRNA against TWIST1 reverses EMT both in vitro and in vivo. siRNA delivery with a polyethyleneimine-coated mesoporous silica nanoparticle (MSN) led to reduction of TWIST1 target genes and migratory potential in vitro. In mice bearing xenograft tumors, weekly intravenous injections of the siRNA-nanoparticle complexes resulted in decreased tumor burden together with a loss of CCL2 suggesting a possible anti-angiogenic response. Therapeutic use of TWIST1 siRNA delivered via MSNs has the potential to inhibit tumor growth and progression in many solid tumor types. FROM THE CLINICAL EDITOR: Tumor progression and metastasis eventually lead to patient mortality in the clinical setting. In other studies, it has been found that TWIST1, a transcription factor, if reactivated in tumors, would lead to downstream events including angiogenesis and result in poor prognosis in cancer patients. In this article, the authors were able to show that when siRNA against TWIST1 was delivered via mesoporous silica nanoparticle, there was tumor reduction in an in-vivo model. The results have opened up a new avenue for further research in this field.

RNA-based TWIST1 inhibition via dendrimer complex to reduce breast cancer cell metastasis.

Breast cancer is the leading cause of cancer-related deaths among women in the United States, and survival rates are lower for patients with metastases and/or triple-negative breast cancer (TNBC; ER, PR, and Her2 negative). Understanding the mechanisms of cancer metastasis is therefore crucial to identify new therapeutic targets and develop novel treatments to improve patient outcomes. A potential target is the TWIST1 transcription factor, which is often overexpressed in aggressive breast cancers and is a master regulator of cellular migration through epithelial-mesenchymal transition (EMT). Here, we demonstrate an siRNA-based TWIST1 silencing approach with delivery using a modified poly(amidoamine) (PAMAM) dendrimer. Our results demonstrate that SUM1315 TNBC cells efficiently take up PAMAM-siRNA complexes, leading to significant knockdown of TWIST1 and EMT-related target genes. Knockdown lasts up to one week after transfection and leads to a reduction in migration and invasion, as determined by wound healing and transwell assays. Furthermore, we demonstrate that PAMAM dendrimers can deliver siRNA to xenograft orthotopic tumors and siRNA remains in the tumor for at least four hours after treatment. These results suggest that further development of dendrimer-based delivery of siRNA for TWIST1 silencing may lead to a valuable adjunctive therapy for patients with TNBC.

The histone demethylase jumonji coordinates cellular senescence including secretion of neural stem cell-attracting cytokines.

UNLABELLED: Jumonji domain-containing protein 3 (JMJD3/KDM6B) demethylates lysine 27 on histone H3 (H3K27me3), a repressive epigenetic mark controlling chromatin organization and cellular senescence. To better understand the functional consequences of JMJD3 its expression was investigated in brain tumor cells. Querying patient expression profile databases confirmed JMJD3 overexpression in high-grade glioma. Immunochemical staining of two glioma cell lines, U251 and U87, indicated intrinsic differences in JMJD3 expression levels that were reflected in changes in cell phenotype and variations associated with cellular senescence, including senescence-associated ß-galactosidase (SA-ß-gal) activity and the senescence-associated secretory phenotype (SASP). Overexpressing wild-type JMJD3 (JMJD3wt) activated SASP-associated genes, enhanced SA-ß-gal activity, and induced nuclear blebbing. Conversely, overexpression of a catalytically inactive dominant negative mutant JMJD3 (JMJD3mut) increased proliferation. In addition, a large number of transcripts were identified by RNA-seq as altered in JMJD3 overexpressing cells, including cancer- and inflammation-related transcripts as defined by Ingenuity Pathway Analysis. These results suggest that expression of the SASP in the context of cancer undermines normal tissue homeostasis and contributes to tumorigenesis and tumor progression. These studies are therapeutically relevant because inflammatory cytokines have been linked to homing of neural stem cells and other stem cells to tumor loci. IMPLICATIONS: This glioma study brings together actions of a normal epigenetic mechanism (JMJD3 activity) with dysfunctional activation of senescence-related processes, including secretion of SASP proinflammatory cytokines and stem cell tropism toward tumors.

Targeting the Twist and Wnt signaling pathways in metastatic breast cancer.

Breast cancer is the leading cause of cancer-related deaths in the United States with over 232,000 new diagnoses expected in 2014 and almost 40,000 deaths. While treatment of primary breast cancer is often well-managed with surgery and radiation, metastatic breast cancer (MBC) that has spread to the brain, bones, liver, and lungs is often incurable. One of the major challenges in the treatment of breast cancer is the presence of a subpopulation of cancer cells that are chemoresistant and metastatic. Given that metastasis is the driving force behind mortality for breast cancer patients, it is essential to identify the characteristics of these aberrant cancer cells that allow them to spread to distant sites in the body and develop into metastatic tumors. Understanding the metastatic mechanisms driving cancer cell dispersal will open the door to developing novel therapies that prevent metastasis and improve long-term outcomes for patients. In this review we assess the feasibility of targeting the Twist and Wnt signaling pathways in breast cancer. These pathways mediate epithelial-mesenchymal transition (EMT), a process that can give rise to chemoresistance. We review potential treatment strategies for targeting EMT and drug resistance as well as the problems that may arise with these targeted delivery therapeutic approaches. Finally, we examine recent advances in the field, including nanoparticle delivery and small interfering RNA (siRNA) technology, and discuss the impact that these approaches may have on translating much needed therapeutic approaches into the clinic, for the benefit of patients battling MBC.

A reconstructed metastasis model to recapitulate the metastatic spread in vitro.

Metastasis remains a leading cause of morbidity and mortality from solid tumors. Lack of comprehensive systems to study the progression of metastasis contributes to the low success of treatment. We developed a novel three-dimensional in vitro reconstructed metastasis (rMet) model that incorporates extracellular matrix (ECM) elements characteristic of the primary (breast, prostate, or lung) and metastatic (bone marrow, BM) sites. A cytokine-rich liquid interphase separates the primary and distant sites, further recapitulating circulation. Similar to main events underlying the metastatic cascade, the rMet model fractionated human tumor cell lines into sub-populations with distinct invasive and migratory abilities: (i) a primary tumor-like fraction mainly consisting of non-migratory spheroids; (ii) an invasive fraction that invaded through the primary tumor ECM, but failed to acquire anchorage-independence and reach the BM; and (iii) a highly migratory BM-colonizing population that invaded the primary ECM, survived in the "circulation-like" media, and successfully invaded and proliferated within BM ECM. BM-colonizing fractions successfully established metastatic bone lesions in vivo, whereas the tumor-like spheroids failed to engraft the bones, showing the ability of the rMet model to faithfully select for highly aggressive sub-populations with a propensity to colonize a metastatic site. By applying the rMet model to study real-time ECM remodeling, we show that tumor cells secrete collagenolytic enzymes for invading the primary site ECM but not for entering the BM ECM, indicating possible differences in ECM remodeling mechanisms at primary tumor versus metastatic sites.

Dkk-1 and IL-7 in plasma of patients with multiple myeloma prevent differentiation of mesenchymal stem cells into osteoblasts.

Bone disease is the leading cause of morbidity associated with multiple myeloma (MM). Lytic bone lesions have been detected in 90% of patients diagnosed with MM and present a great therapeutic challenge. After the removal of the tumor burden, the bone lesions persist and the bone remodeling homeostasis is not restored even in patients in clinical remission. To determine whether systemic factors generated by malignant MM cells can skew the osteoblast (OB) differentiation program of normal mesenchymal stem cells (MSCs), we generated an immortalized bone marrow MSC line (hTERT-MSC). The hTERT-MSCs were exposed to plasma from healthy donors and patients with MM. Cells grown in media supplemented with plasma from MM patients failed to differentiate into OBs, while the hTERT-MSCs grown in the presence of normal human plasma generated OB clusters that mineralized calcium, expressed Runx2, and were positive for alkaline phosphatase, fibronectin, collagen I, osteocalcin, and osteopontin. Blocking Dickkopf-1 (Dkk-1) and interleukin-7 (IL-7) in MM plasma restored proper OB differentiation of hTERT-MSCs. Finally, we show that hTERT-MSCs cultured in the presence of MM plasma adopt a cancer-associated stroma phenotype. Thus, we show, that systemic factors present in the plasma of patients with MM affect the behavior of non-malignant MSCs and contribute to the sustained bone disease reported in MM.