CVM-1118 (foslinanib), a 2-phenyl-4-quinolone derivative, promotes apoptosis and inhibits vasculogenic mimicry via targeting TRAP1.

CVM-1118 (foslinanib) is a phosphoric ester compound selected from 2-phenyl-4-quinolone derivatives. The NCI 60 cancer panel screening showed CVM-1125, the major active metabolite of CVM-1118, to exhibit growth inhibitory and cytotoxic effects at nanomolar range. CVM-1118 possesses multiple bioactivities, including inducing cellular apoptosis, cell cycle arrest at G2/M, as well as inhibiting vasculogenic mimicry (VM) formation. The TNF receptor associated protein 1 (TRAP1) was identified as the binding target of CVM-1125 using nematic protein organization technique (NPOT) interactome analysis. Further studies demonstrated CVM-1125 reduced the protein level of TRAP1 and impeded its downstream signaling by reduction of cellular succinate levels and destabilization of HIF-1α. The pharmacogenomic biomarkers associated with CVM-1118 were also examined by Whole Genome CRISPR Knock-Out Screening. Two hits (STK11 and NF2) were confirmed with higher sensitivity to the drug in cell knock-down experiments. Biological assays indicate that the mechanism of action of CVM-1118 is via targeting TRAP1 to induce mitochondrial apoptosis, suppress tumor cell growth, and inhibit vasculogenic mimicry formation. Most importantly, the loss-of-function mutations of STK11 and NF2 are potential biomarkers of CVM-1118 which can be applied in the selection of cancer patients for CVM-1118 treatment. CVM-1118 is currently in its Phase 2a clinical development.

Newly identified form of phenotypic plasticity of cancer: immunogenic mimicry.

Cancer plasticity is now a recognized new hallmark of cancer which is due to disturbances of cell differentiation programs. It is manifested not only in various forms like the best-known epithelial-mesenchymal transition (EMT) but also in vasculogenic and megakaryocytic mimicries regulated by EMT-specific or less-specific transcription factors such as HIF1a or STAT1/2. Studies in the past decades provided ample data that cancer plasticity can be manifested also in the expression of a vast array of immune cell genes; best-known examples are PDL1/CD274, CD47, or IDO, and we termed it immunogenic mimicry (IGM). However, unlike other types of plasticities which are epigenetically regulated, expression of IGM genes are frequently due to gene amplifications. It is important that the majority of the IGM genes are regulated by interferons (IFNs) suggesting that their protein expressions are regulated by the immune microenvironment. Most of the IGM genes have been shown to be involved in immune escape of cancers broadening the repertoire of these mechanisms and offering novel targets for immunotherapeutics.

Inorganic polyphosphate as an energy source in tumorigenesis.

Cancer cells have high demands for energy to maintain their exceedingly proliferative growth. However, the mechanism of energy expenditure in cancer is not well understood. We hypothesize that cancer cells might utilize energy-rich inorganic polyphosphate (polyP), as energetic reserve. PolyP is comprised of orthophosphates linked by phosphoanhydride bonds, as in ATP. Here, we show that polyP is highly abundant in several types of cancer cells, including brain tumor-initiating cells (BTICs), i.e., stem-like cells derived from a mouse brain tumor model that we have previously described. The polymer is avidly consumed during starvation of the BTICs. Depletion of ATP by inhibiting glycolysis and mitochondrial ATP-synthase (OXPHOS) further decreases the levels of polyP and alters morphology of the cells. Moreover, enzymatic hydrolysis of the polymer impairs the viability of cancer cells and significantly deprives ATP stores. These results suggest that polyP might be utilized as a source of phosphate energy in cancer. While the role of polyP as an energy source is established for bacteria, this finding is the first demonstration that polyP may play a similar role in the metabolism of cancer cells.

Heterogeneity of Melanoma with Stem Cell Properties.

Metastatic melanoma continues to present a significant challenge-with a cure rate of less than 10% and a median survival of 6-9 months. Despite noteworthy advances in the field, the heterogeneity of melanoma tumors, comprised of cell subpopulations expressing a cancer stem cell (CSC) phenotype concomitant with drug resistance markers presents a formidable challenge in the design of current therapies. Particularly vexing is the ability of distinct subpopulations of melanoma cells to resist standard-of-care treatments, resulting in relapse and progression to metastasis. Recent studies have provided new information and insights into the expression and function of CSC markers associated with the aggressive melanoma phenotype, such as the embryonic morphogen Nodal and CD133, together with a drug resistance marker ABCA1. This chapter highlights major findings that demonstrate the promise of targeting Nodal as a viable option to pursue in combination with standard-of-care therapy. In recognizing that aggressive melanoma tumors utilize multiple mechanisms to survive, we must consider a more strategic approach to effectively target heterogeneity, tumor cell plasticity, and functional adaptation and resistance to current therapies-to eliminate relapse, disease progression, and metastasis.

The Stem Cell Phenotype of Aggressive Breast Cancer Cells.

Aggressive cancer cells are characterized by their capacity to proliferate indefinitely and to propagate a heterogeneous tumor comprised of subpopulations with varying degrees of metastatic propensity and drug resistance properties. Particularly daunting is the challenge we face in the field of oncology of effectively targeting heterogeneous tumor cells expressing a variety of markers, especially those associated with a stem cell phenotype. This dilemma is especially relevant in breast cancer, where therapy is based on traditional classification schemes, including histological criteria, differentiation status, and classical receptor markers. However, not all patients respond in a similar manner to standard-of-care therapy, thereby necessitating the need to identify and evaluate novel biomarkers associated with the difficult-to-target stem cell phenotype and drug resistance. Findings related to the convergence of embryonic and tumorigenic signaling pathways have identified the embryonic morphogen Nodal as a promising new oncofetal target that is reactivated only in aggressive cancers, but not in normal tissues. The work presented in this paper confirms previous studies demonstrating the importance of Nodal as a cancer stem cell molecule associated with aggressive breast cancer, and advances the field by providing new findings showing that Nodal is not targeted by standard-of-care therapy in breast cancer patients. Most noteworthy is the linkage found between Nodal expression and the drug resistance marker ATP-binding cassette member 1 (ABCA1), which may provide new insights into developing combinatorial approaches to overcome drug resistance and disease recurrence.

Targeting PDGF-mediated recruitment of pericytes blocks vascular mimicry and tumor growth.

Aggressive tumor cells can adopt an endothelial cell-like phenotype and contribute to the formation of a tumor vasculature, independent of tumor angiogenesis. This adoptive mechanism is referred to as vascular mimicry and it is associated with poor survival in cancer patients. To what extent tumor cells capable of vascular mimicry phenocopy the angiogenic cascade is still poorly explored. Here, we identify pericytes as important players in vascular mimicry. We found that pericytes are recruited by vascular mimicry-positive tumor cells in order to facilitate sprouting and to provide structural support of the vascular-like networks. The pericyte recruitment is mediated through platelet-derived growth factor (PDGF)-B. Consequently, preventing PDGF-B signaling by blocking the PDGF receptors with either the small tyrosine kinase inhibitor imatinib or blocking antibodies inhibits vascular mimicry and tumor growth. Collectively, the current study identifies an important role for pericytes in the formation of vascular-like structures by tumor cells. Moreover, the mechanism that controls the pericyte recruitment provides therapeutic opportunities for patients with aggressive vascular mimicry-positive cancer types. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

9-ING-41, a small-molecule glycogen synthase kinase-3 inhibitor, is active in neuroblastoma.

Advanced stage neuroblastoma is a very aggressive pediatric cancer with limited treatment options and a high mortality rate. Glycogen synthase kinase-3ß (GSK-3ß) is a potential therapeutic target in neuroblastoma. Using immunohistochemical staining, we observed positive GSK-3ß expression in 67% of human neuroblastomas (34 of 51 cases). Chemically distinct GSK-3 inhibitors (AR-A014418, TDZD-8, and 9-ING-41) suppressed the growth of neuroblastoma cells, whereas 9-ING-41, a clinically relevant small-molecule GSK-3ß inhibitor with broad-spectrum preclinical antitumor activity, being the most potent. Inhibition of GSK-3 resulted in a decreased expression of the antiapoptotic molecule XIAP and an increase in neuroblastoma cell apoptosis. Mouse xenograft studies showed that the combination of clinically relevant doses of CPT-11 and 9-ING-41 led to greater antitumor effect than was observed with either agent alone. These data support the inclusion of patients with advanced neuroblastoma in clinical studies of 9-ING-41, especially in combination with CPT-11.

Characterization of brain tumor initiating cells isolated from an animal model of CNS primitive neuroectodermal tumors.

CNS Primitive Neuroectodermal tumors (CNS-PNETs) are members of the embryonal family of malignant childhood brain tumors, which remain refractory to current therapeutic treatments. Current paradigm of brain tumorigenesis implicates brain tumor-initiating cells (BTIC) in the onset of tumorigenesis and tumor maintenance. However, despite their significance, there is currently no comprehensive characterization of CNS-PNETs BTICs. Recently, we described an animal model of CNS-PNET generated by orthotopic transplantation of human Radial Glial (RG) cells - the progenitor cells for adult neural stem cells (NSC) - into NOD-SCID mice brain and proposed that BTICs may play a role in the maintenance of these tumors. Here we report the characterization of BTIC lines derived from this CNS-PNET animal model. BTIC's orthotopic transplantation generated highly aggressive tumors also characterized as CNS-PNETs. The BTICs have the hallmarks of NSCs as they demonstrate self-renewing capacity and have the ability to differentiate into astrocytes and early migrating neurons. Moreover, the cells demonstrate aberrant accumulation of wild type tumor-suppressor protein p53, indicating its functional inactivation, highly up-regulated levels of onco-protein cMYC and the BTIC marker OCT3/4, along with metabolic switch to glycolysis - suggesting that these changes occurred in the early stages of tumorigenesis. Furthermore, based on RNA- and DNA-seq data, the BTICs did not acquire any transcriptome-changing genomic alterations indicating that the onset of tumorigenesis may be epigenetically driven. The study of these BTIC self-renewing cells in our model may enable uncovering the molecular alterations that are responsible for the onset and maintenance of the malignant PNET phenotype.

Targeting the Stem Cell Properties of Adult Breast Cancer Cells: Using Combinatorial Strategies to Overcome Drug Resistance.

PURPOSE OF REVIEW: Cancer is a major public health problem worldwide. In aggressive cancers, which are heterogeneous in nature, there exists a paucity of targetable molecules that can be used to predict outcome and response to therapy in patients, especially those in the high risk category with a propensity to relapse following chemotherapy. This review addresses the challenges pertinent to treating aggressive cancer cells with inherent stem cell properties, with a special focus on triple-negative breast cancer (TNBC). RECENT FINDINGS: Plasticity underlies the cancer stem cell (CSC) phenotype in aggressive cancers like TNBC. Progenitors and CSCs implement similar signaling pathways to sustain growth, and the convergence of embryonic and tumorigenic signaling pathways has led to the discovery of novel oncofetal targets, rigorously regulated during normal development, but aberrantly reactivated in aggressive forms of cancer. SUMMARY: Translational studies have shown that Nodal, an embryonic morphogen, is reactivated in aggressive cancers, but not in normal tissues, and underlies tumor growth, invasion, metastasis and drug resistance. Front-line therapies do not inhibit Nodal, but when a combinatorial approach is used with an agent such as doxorubicin followed by anti-Nodal antibody therapy, significant decreases in cell growth and viability occur. These findings are of special interest in the development of new therapeutic interventions that target the stem cell properties of cancer cells to overcome drug resistance and metastasis.

Nodal Signaling as a Developmental Therapeutics Target in Oncology.

The tumor microenvironment is a vital feature of oncogenesis and tumor progression. There are several parallels between cancer cells and early developmental stem cells, including their plasticity and signaling mechanisms. In early fetal development, Nodal is expressed for endodermal and mesodermal differentiation. This expression has been shown reemerge in the setting of epithelial cancers, such as breast and melanoma. High Nodal expression correlates to an aggressive tumor grade in these malignancies. Nodal signal begins with its interaction with its coreceptor, Cripto-1, leading to activation of Smad2/Smad3 and ultimately downstream transcription and translation. Lefty is the natural inhibitor of Nodal and controls Nodal signaling during fetal development. However, cancer cells lack the presence of Lefty, thus leading to uncontrolled tumor growth. Given this understanding, inhibition of the Nodal pathway offers a new novel therapeutic target in oncology. Mol Cancer Ther; 16(5); 787-92. ©2017 AACR.

Vasculogenic mimicry in small cell lung cancer.

Small cell lung cancer (SCLC) is characterized by prevalent circulating tumour cells (CTCs), early metastasis and poor prognosis. We show that SCLC patients (37/38) have rare CTC subpopulations co-expressing vascular endothelial-cadherin (VE-cadherin) and cytokeratins consistent with vasculogenic mimicry (VM), a process whereby tumour cells form 'endothelial-like' vessels. Single-cell genomic analysis reveals characteristic SCLC genomic changes in both VE-cadherin-positive and -negative CTCs. Higher levels of VM are associated with worse overall survival in 41 limited-stage patients' biopsies (P<0.025). VM vessels are also observed in 9/10 CTC patient-derived explants (CDX), where molecular analysis of fractionated VE-cadherin-positive cells uncovered copy-number alterations and mutated TP53, confirming human tumour origin. VE-cadherin is required for VM in NCI-H446 SCLC xenografts, where VM decreases tumour latency and, despite increased cisplatin intra-tumour delivery, decreases cisplatin efficacy. The functional significance of VM in SCLC suggests VM regulation may provide new targets for therapeutic intervention.

Lefty Glycoproteins in Human Embryonic Stem Cells: Extracellular Delivery Route and Posttranslational Modification in Differentiation.

Lefty is a member of transforming growth factor-beta (TGF-ß) superfamily and a potent antagonist of the TGF-ß/Nodal/Activin signaling pathway. Lefty is critical in sustaining self-renewal/pluripotency status, and implicated in the differentiation of embryonic stem cells (ESCs). However, emerging studies depict Lefty as a multifaceted protein involved in myriad cellular events. Lefty proteins (human Lefty A and B) are secreted glycoproteins, but their mode of secretion and the significance of their "glycan" moiety remain mostly unexplored. By employing an in vitro system of human ESCs (hESCs), we observed that Lefty protein(s) are encased in exosomes for extracellular release. The exosomal- and cell-associated Lefty diverge in their proteolytic processing, and possess N-glycan structures of high mannose and complex nature. Differentiation of hESCs to mesenchymal cells (MSCs) or neuronal progenitor cells (NPCs) entails distinct changes in the Lefty A/Lefty B gene(s), and protein expression. Specifically, the proteolytic cleavage and N-glycan composition of the cell-associated and exosomal Lefty differ in the differentiated progenies. These modifications affected Lefty's inhibitory effect on Nodal signaling in aggressive melanoma cells. The microheterogeneity in the processing and glycosylation of Lefty protein(s) between hESCs, MSCs, and NPCs could present efficient means of diversifying the endogenous functions of Lefty. Whether Lefty's diverse functions in embryonic patterning, as well as its diffusion range in the extracellular environment, are similarly affected remains to be determined. Our studies underscore the potential relevance of Lefty-packaged exosomes for combating debilitating diseases such as cancer.

Translational significance of Nodal, Cripto-1 and Notch4 in adult nevi.

The TGF-ß associated growth factor Nodal is highly expressed in aggressive metastatic melanoma. Determining the risk for melanomagenesis from Nodal expression in nevi prior to the development of melanoma may be useful for both the screening and prevention of melanoma. Tissue sections of human adult nevi with or without a history of melanoma were stained by immunohistochemistry (IHC) for Nodal, the Nodal co-receptor Cripto-1, and Notch4, which have previously been shown to be associated with Nodal expression in melanoma. The degree of Nodal, Cripto-1 and Notch4 staining was scored and correlated with available clinical data. Median IHC scores for Nodal, Cripto-1 and Notch4 expression were significantly higher in nevi removed from patients who eventually developed melanoma compared with nevi from patients with no history of melanoma. In addition, the degree of Nodal expression in nevi from patients who eventually developed melanoma correlated significantly with the Breslow depth of the melanoma. Expression of Nodal and components of its signaling pathway in nevi may represent a biomarker for selecting a unique subset of patients requiring increased surveillance for screening and prevention of melanoma.

Transgene Reactivation in Induced Pluripotent Stem Cell Derivatives and Reversion to Pluripotency of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells.

Induced pluripotent stem cells (iPSCs) have enormous potential in regenerative medicine and disease modeling. It is now felt that clinical trials should be performed with iPSCs derived with nonintegrative constructs. Numerous studies, however, including those describing disease models, are still being published using cells derived from iPSCs generated with integrative constructs. Our experimental work presents the first evidence of spontaneous transgene reactivation in vitro in several cellular types. Our results show that the transgenes were predominantly silent in parent iPSCs, but in mesenchymal and endothelial iPSC derivatives, the transgenes experienced random upregulation of Nanog and c-Myc. Additionally, we provide evidence of spontaneous secondary reprogramming and reversion to pluripotency in mesenchymal stem cells derived from iPSCs. These findings strongly suggest that the studies, which use cellular products derived from iPSCs generated with retro- or lentiviruses, should be evaluated with consideration of the possibility of transgene reactivation. The in vitro model described here provides insight into the earliest events of culture transformation and suggests the hypothesis that reversion to pluripotency may be responsible for the development of tumors in cell replacement experiments. The main goal of this work, however, is to communicate the possibility of transgene reactivation in retro- or lenti-iPSC derivatives and the associated loss of cellular fidelity in vitro, which may impact the outcomes of disease modeling and related experimentation.

Nodal expression in triple-negative breast cancer: Cellular effects of its inhibition following doxorubicin treatment.

Triple-negative breast cancer (TNBC) represents an aggressive cancer subtype characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). The independence of TNBC from these growth promoting factors eliminates the efficacy of therapies which specifically target them, and limits TNBC patients to traditional systemic neo/adjuvant chemotherapy. To better understand the growth advantage of TNBC - in the absence of ER, PR and HER2, we focused on the embryonic morphogen Nodal (associated with the cancer stem cell phenotype), which is re-expressed in aggressive breast cancers. Most notably, our previous data demonstrated that inhibition of Nodal signaling in breast cancer cells reduces their tumorigenic capacity. Furthermore, inhibiting Nodal in other cancers has resulted in improved effects of chemotherapy, although the mechanisms for this remain unknown. Thus, we hypothesized that targeting Nodal in TNBC cells in combination with conventional chemotherapy may improve efficacy and represent a potential new strategy. Our preliminary data demonstrate that Nodal is highly expressed in TNBC when compared to invasive hormone receptor positive samples. Treatment of Nodal expressing TNBC cell lines with a neutralizing anti-Nodal antibody reduces the viability of cells that had previously survived treatment with the anthracycline doxorubicin. We show that inhibiting Nodal may alter response mechanisms employed by cancer cells undergoing DNA damage. These data suggest that development of therapies which target Nodal in TNBC may lead to additional treatment options in conjunction with chemotherapy regimens - by altering signaling pathways critical to cellular survival.

Melanocytes Affect Nodal Expression and Signaling in Melanoma Cells: A Lesson from Pediatric Large Congenital Melanocytic Nevi.

Expression of Nodal, a Transforming Growth Factor-beta (TGF-ß) related growth factor, is associated with aggressive melanoma. Nodal expression in adult dysplastic nevi may predict the development of aggressive melanoma in some patients. A subset of pediatric patients diagnosed with giant or large congenital melanocytic nevi (LCMN) has shown increased risk for development of melanoma. Here, we investigate whether Nodal expression can help identify the rare cases of LCMN that develop melanoma and shed light on why the majority of these patients do not. Immunohistochemistry (IHC) staining results show varying degree of Nodal expression in pediatric dysplastic nevi and LCMN. Moreover, median scores from Nodal IHC expression analysis were not significantly different between these two groups. Additionally, none of the LCMN patients in this study developed melanoma, regardless of Nodal IHC levels. Co-culture experiments revealed reduced tumor growth and lower levels of Nodal and its signaling molecules P-SMAD2 and P-ERK1/2 when melanoma cells were grown in vivo or in vitro with normal melanocytes. The same was observed in melanoma cells cultured with melanocyte conditioned media containing pigmented melanocyte derived melanosomes (MDM). Since MDM contain molecules capable of inactivating radical oxygen species, to investigate potential anti-oxidant effect of MDM on Nodal expression and signaling in melanoma, melanoma cells were treated with either N-acetyl-l-cysteine (NAC), a component of the anti-oxidant glutathione or synthetic melanin, which in addition to providing pigmentation can also exert free radical scavenging activity. Melanoma cells treated with NAC or synthetic melanin showed reduced levels of Nodal, P-SMAD2 and P-ERK1/2 compared to untreated melanoma cells. Thus, the potential role for Nodal in melanoma development in LCMN is less evident than in adult dysplastic nevi possibly due to melanocyte cross-talk in LCMN capable of offsetting or delaying the pro-melanoma effects of Nodal via anti-oxidant effects of MDM.

Plasticity underlies tumor progression: role of Nodal signaling.

The transforming growth factor beta (TGFß) superfamily member Nodal is an established regulator of early embryonic development, with primary roles in endoderm induction, left-right asymmetry, and primitive streak formation. Nodal signals through TGFß family receptors at the plasma membrane and induces signaling cascades leading to diverse transcriptional regulation. While conceptually simple, the regulation of Nodal and its molecular effects are profoundly complex and context dependent. Pioneering work by developmental biologists has characterized the signaling pathways, regulatory components, and provided detailed insight into the mechanisms by which Nodal mediates changes at the cellular and organismal levels. Nodal is also an important factor in maintaining pluripotency of embryonic stem cells through regulation of core transcriptional programs. Collectively, this work has led to an appreciation for Nodal as a powerful morphogen capable of orchestrating multiple cellular phenotypes. Although Nodal is not active in most adult tissues, its reexpression and signaling have been linked to multiple types of human cancer, and Nodal has emerged as a driver of tumor growth and cellular plasticity. In vitro and in vivo experimental evidence has demonstrated that inhibition of Nodal signaling reduces cancer cell aggressive characteristics, while clinical data have established associations with Nodal expression and patient outcomes. As a result, there is great interest in the potential targeting of Nodal activity in a therapeutic setting for cancer patients that may provide new avenues for suppressing tumor growth and metastasis. In this review, we evaluate our current understanding of the complexities of Nodal function in cancer and highlight recent experimental evidence that sheds light on the therapeutic potential of its inhibition.

Tumor cell vascular mimicry: Novel targeting opportunity in melanoma.

In 1999, the American Journal of Pathology published an article, entitled "Vascular channel formation by human melanoma cells in vivo and in vitro: vasculogenic mimicry" by Maniotis and colleagues, which ignited a spirited debate for several years and earned the journal's distinction of a "citation classic" (Maniotis et al., 1999). Tumor cell vasculogenic mimicry (VM), also known as vascular mimicry, describes the plasticity of aggressive cancer cells forming de novo vascular networks and is associated with the malignant phenotype and poor clinical outcome. The tumor cells capable of VM share the commonality of a stem cell-like, transendothelial phenotype, which may be induced by hypoxia. Since its introduction as a novel paradigm for melanoma tumor perfusion, many studies have contributed new findings illuminating the underlying molecular pathways supporting VM in a variety of tumors, including carcinomas, sarcomas, glioblastomas, astrocytomas, and melanomas. Of special significance is the lack of effectiveness of angiogenesis inhibitors on tumor cell VM, suggesting a selective resistance by this phenotype to conventional therapy. Facilitating the functional plasticity of tumor cell VM are key proteins associated with vascular, stem cell, extracellular matrix, and hypoxia-related signaling pathways--each deserving serious consideration as potential therapeutic targets and diagnostic indicators of the aggressive, metastatic phenotype. This review highlights seminal findings pertinent to VM, including the effects of a novel, small molecular compound, CVM-1118, currently under clinical development to target VM, and illuminates important molecular pathways involved in the suppression of this plastic, aggressive phenotype, using melanoma as a model.