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.

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 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.

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.

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.

Effects of a novel Nodal-targeting monoclonal antibody in melanoma.

Nodal is highly expressed in various human malignancies, thus supporting the rationale for exploring Nodal as a therapeutic target. Here, we describe the effects of a novel monoclonal antibody (mAb), 3D1, raised against human Nodal. In vitro treatment of C8161 human melanoma cells with 3D1 mAb shows reductions in anchorage-independent growth and vasculogenic network formation. 3D1 treated cells also show decreases of Nodal and downstream signaling molecules, P-Smad2 and P-ERK and of P-H3 and CyclinB1, with an increase in p27. Similar effects were previously reported in human breast cancer cells where Nodal expression was generally down-regulated; following 3D1 mAb treatment, both Nodal and P-H3 levels are reduced. Noteworthy is the reduced growth of human melanoma xenografts in Nude mice treated with 3D1 mAb, where immunostaining of representative tumor sections show diminished P-Smad2 expression. Similar effects both in vitro and in vivo were observed in 3D1 treated A375SM melanoma cells harboring the active BRAF(V600E) mutation compared to treatments with IgG control or a BRAF inhibitor, dabrafenib. Finally, we describe a 3D1-based ELISA for the detection of Nodal in serum samples from cancer patients. These data suggest the potential of 3D1 mAb for selecting and targeting Nodal expressing cancers.

A mouse model of human primitive neuroectodermal tumors resulting from microenvironmentally-driven malignant transformation of orthotopically transplanted radial glial cells.

There is growing evidence and a consensus in the field that most pediatric brain tumors originate from stem cells, of which radial glial cells constitute a subtype. Here we show that orthotopic transplantation of human radial glial (RG) cells to the subventricular zone of the 3rd ventricle--but not to other transplantation sites--of the brain in immunocompromised NOD-SCID mice, gives rise to tumors that have the hallmarks of CNS primitive neuroectodermal tumors (PNETs). The resulting mouse model strikingly recapitulates the phenotype of PNETs. Importantly, the observed tumorigenic transformation was accompanied by aspects of an epithelial to mesenchymal transition (EMT)-like process. It is also noteworthy that the tumors are highly invasive, and that they effectively recruit mouse endothelial cells for angiogenesis. These results are significant for several reasons. First, they show that malignant transformation of radial glial cells can occur in the absence of specific mutations or inherited genomic alterations. Second, they demonstrate that the same radial glial cells may either give rise to brain tumors or differentiate normally depending upon the microenvironment of the specific region of the brain to which the cells are transplanted. In addition to providing a prospect for drug screening and development of new therapeutic strategies, the resulting mouse model of PNETs offers an unprecedented opportunity to identify the cancer driving molecular alterations and the microenvironmental factors that are responsible for committing otherwise normal radial glial cells to a malignant phenotype.

Internalization by multiple endocytic pathways and lysosomal processing impact maspin-based therapeutics.

UNLABELLED: Patients with metastatic disease face high rates of mortality with a paucity of therapeutic options. Protein-based therapeutics provide advantages over traditional chemotherapy through increased specificity, decreased immune impairment, and more direct means of delivery. However, development is often hindered because of insufficient knowledge about protein processing by cells when exogenously applied. This study focuses on recombinant Maspin (rMaspin), a serine protease inhibitor (SERPINB5), which alters invasive properties when directly applied to cancer cells. Previous evidence suggests differences in the effects of rMaspin treatment when compared with endogenous reexpression, with little explanation for these discrepancies. A leading hypothesis is that exogenously applied rMaspin is subject to different regulatory and/or processing mechanisms in cancer cells when compared with endogenous expression. Therefore, a more detailed understanding of the mechanisms of internalization and subcellular trafficking of rMaspin is needed to guide future translational development. We describe the molecular trafficking of rMaspin in cytoplasmic vesicles of the endosomal/lysosomal pathway and characterize its uptake by multiple endocytic mechanisms. Time-lapse laser scanning confocal microscopy shows the uptake, in real time, of dye-labeled rMaspin in cancer cells. This study indicates that cellular processing of rMaspin plays a key role by affecting its biologic activity and highlights the need for new approaches aimed at increasing the availability of rMaspin when used to treat cancer. IMPLICATIONS: Novel characterization of internalization and subcellular trafficking of rMaspin provides new insights for future therapeutic development.

Melanoma tumor cell heterogeneity: a molecular approach to study subpopulations expressing the embryonic morphogen nodal.

As the frequency of melanoma increases, current treatment strategies are struggling to significantly impact patient survival. One of the critical issues in designing efficient therapies is understanding the composition of heterogeneous melanoma tumors in order to target cancer stem cells (CSCs) and drug-resistant subpopulations. In this review, we summarize recent findings pertinent to the reemergence of the embryonic Nodal signaling pathway in melanoma and its significance as a prognostic biomarker and therapeutic target. In addition, we offer a novel molecular approach to studying the functional relevance of Nodal-expressing subpopulations and their CSC phenotype.

Onset of rosette formation during spontaneous neural differentiation of hESC and hiPSC colonies.

In vitro neural differentiation of human embryonic stem cells (hESCs) is an advantageous system for studying early neural development. The process of early neural differentiation in hESCs begins by initiation of primitive neuroectoderm, which is manifested by rosette formation, with consecutive differentiation into neural progenitors and early glial-like cells. In this study, we examined the involvement of early neural markers - OTX2, PAX6, Sox1, Nestin, NR2F1, NR2F2, and IRX2 - in the onset of rosette formation, during spontaneous neural differentiation of hESC and human induced pluripotent stem cell (hiPSC) colonies. This is in contrast to the conventional way of studying rosette formation, which involves induction of neuronal differentiation and the utilization of embryoid bodies. Here we show that OTX2 is highly expressed at the onset of rosette formation, when rosettes comprise no more than 3-5 cells, and that its expression precedes that of established markers of early neuronal differentiation. Importantly, the rise of OTX2 expression in these cells coincides with the down-regulation of the pluripotency marker OCT4. Lastly, we show that cells derived from rosettes that emerge during spontaneous differentiation of hESCs or hiPSCs are capable of differentiating into dopaminergic neurons in vitro, and into mature-appearing pyramidal and serotonergic neurons weeks after being injected into the motor cortex of NOD-SCID mice.

Tumor cell vasculogenic mimicry: from controversy to therapeutic promise.

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 distinction as a citation classic. Tumor cell vasculogenic mimicry (VM) refers to the plasticity of aggressive cancer cells forming de novo vascular networks, which thereby contribute to perfusion of rapidly growing tumors, transporting fluid from leaky vessels, and/or connecting with the constitutional endothelial-lined vasculature. The tumor cells capable of VM share a plastic, transendothelial phenotype, which may be induced by hypoxia. Since VM was introduced 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. Facilitating the functional plasticity of tumor cell VM are key proteins associated with vascular, stem cell, and hypoxia-related signaling pathways, each deserving serious consideration as potential therapeutic targets and diagnostic indicators of the aggressive, metastatic phenotype.

Maspin: molecular mechanisms and therapeutic implications.

Maspin, a non-inhibitory member of the serine protease inhibitor superfamily, has been characterized as a tumor suppressor gene in multiple cancer types. Among the established anti-tumor effects of Maspin are the inhibition of cancer cell invasion, attachment to extracellular matrices, increased sensitivity to apoptosis, and inhibition of angiogenesis. However, while significant experimental data support the role of Maspin as a tumor suppressor, clinical data regarding the prognostic implications of Maspin expression have led to conflicting results. This highlights the need for a better understanding of the context dependencies of Maspin in normal biology and how these are perturbed in the context of cancer. In this review, we outline the regulation and roles of Maspin in normal and developmental biology while discussing novel evidence and emerging theories related to its functions in cancer. We provide insight into the immense therapeutic potential of Maspin and the challenges related to its successful clinical translation.

Molecular pathways: vasculogenic mimicry in tumor cells: diagnostic and therapeutic implications.

Tumor cell vasculogenic mimicry (VM) describes the functional plasticity of aggressive cancer cells forming de novo vascular networks, thereby providing a perfusion pathway for rapidly growing tumors, transporting fluid from leaky vessels, and/or connecting with endothelial-lined vasculature. The underlying induction of VM seems to be related to hypoxia, which may also promote the plastic, transendothelial phenotype of tumor cells capable of VM. Since its introduction in 1999 as a novel paradigm for melanoma tumor perfusion, many studies have contributed new insights into the underlying molecular pathways supporting VM in a variety of tumors, including melanoma, glioblastoma, carcinomas, and sarcomas. In particular, critical VM-modulating genes are associated with vascular (VE-cadherin, EphA2, VEGF receptor 1), embryonic and/or stem cell (Nodal, Notch4), and hypoxia-related (hypoxia-inducible factor, Twist1) signaling pathways. Each of these pathways warrants serious scrutiny as potential therapeutic, vascular targets, and diagnostic indicators of plasticity, drug resistance, and the aggressive metastatic phenotype.

Cancer hallmarks in induced pluripotent cells: new insights.

Studies are beginning to emerge that demonstrate intriguing differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs). Here, we investigated the expression of key members of the Nodal embryonic signaling pathway, critical to the maintenance of pluripotency in hESCs. Western blot and real-time RT-PCR analyses reveal slightly lower levels of Nodal (a TGF-beta family member) and Cripto-1 (Nodal's co-receptor) and a dramatic decrease in Lefty (Nodal's inhibitor and TGF-beta family member) in hiPSCs compared with hESCs. The noteworthy drop in hiPSC's Lefty expression correlated with an increase in the methylation of Lefty B CpG island. Based on these findings, we addressed a more fundamental question related to the consequences of epigenetically reprogramming hiPSCs, especially with respect to maintaining a stable ESC phenotype. A global comparative analysis of 365 microRNAs (miRs) in two hiPSC versus four hESC lines ultimately identified 10 highly expressed miRs in hiPCSs with >10-fold difference, which have been shown to be cancer related. These data demonstrate cancer hallmarks expressed by hiPSCs, which will require further assessment for their impact on future therapies..

Development and cancer: at the crossroads of Nodal and Notch signaling.

Aggressive tumor cells express a plastic, multipotent phenotype similar to embryonic stem cells. However, the absence of major regulatory checkpoints in these tumor cells allows aberrant activation of embryonic signaling pathways, which seems to contribute to their plastic phenotype. Emerging evidence showing the molecular cross-talk between two major stem cell signaling pathways Nodal and Notch suggests a promising therapeutic strategy that could target aggressive tumor cells on the basis of their unique plasticity, and provide new insights into the mechanisms underlying the re-emergence of developmental signaling pathways during tumor progression.

Emerging roles of nodal and Cripto-1: from embryogenesis to breast cancer progression.

Breast carcinoma cells and embryonic progenitors similarly implement stem cell-associated signaling pathways to sustain continued growth and plasticity. Indeed, recent studies have implicated signaling pathways, including those associated with the Notch, and Transforming Growth Factor-Beta (TGF-beta) superfamilies, as instrumental to both embryological development and breast cancer progression. In particular, Nodal, an embryonic morphogen belonging to the TGF-beta superfamily, and its co-receptor, Cripto-1, are requisite to both embryogenesis and mammary gland maturation. Moreover, these developmental proteins have been shown to promote breast cancer progression. Here, we review the role of Nodal and its co-receptor Cripto-1 during development and we describe how this signaling pathway may be involved in breast cancer tumorigenesis. Moreover, we emphasize the potential utility of this signaling pathway as a novel target for the treatment and diagnosis of breast cancer.

Reprogramming multipotent tumor cells with the embryonic neural crest microenvironment.

The embryonic microenvironment is an important source of signals that program multipotent cells to adopt a particular fate and migratory path, yet its potential to reprogram and restrict multipotent tumor cell fate and invasion is unrealized. Aggressive tumor cells share many characteristics with multipotent, invasive embryonic progenitors, contributing to the paradigm of tumor cell plasticity. In the vertebrate embryo, multiple cell types originate from a highly invasive cell population called the neural crest. The neural crest and the embryonic microenvironments they migrate through represent an excellent model system to study cell diversification during embryogenesis and phenotype determination. Recent exciting studies of tumor cells transplanted into various embryo models, including the neural crest rich chick microenvironment, have revealed the potential to control and revert the metastatic phenotype, suggesting further work may help to identify new targets for therapeutic intervention derived from a convergence of tumorigenic and embryonic signals. In this mini-review, we summarize markers that are common to the neural crest and highly aggressive human melanoma cells. We highlight advances in our understanding of tumor cell behaviors and plasticity studied within the chick neural crest rich microenvironment. In so doing, we honor the tremendous contributions of Professor Elizabeth D. Hay toward this important interface of developmental and cancer biology.

Human embryonic stem cell microenvironment suppresses the tumorigenic phenotype of aggressive cancer cells.

Embryonic stem cells sustain a microenvironment that facilitates a balance of self-renewal and differentiation. Aggressive cancer cells, expressing a multipotent, embryonic cell-like phenotype, engage in a dynamic reciprocity with a microenvironment that promotes plasticity and tumorigenicity. However, the cancer-associated milieu lacks the appropriate regulatory mechanisms to maintain a normal cellular phenotype. Previous work from our laboratory reported that aggressive melanoma and breast carcinoma express the embryonic morphogen Nodal, which is essential for human embryonic stem cell (hESC) pluripotency. Based on the aberrant expression of this embryonic plasticity gene by tumor cells, this current study tested whether these cells could respond to regulatory cues controlling the Nodal signaling pathway, which might be sequestered within the microenvironment of hESCs, resulting in the suppression of the tumorigenic phenotype. Specifically, we discovered that metastatic tumor cells do not express the inhibitor to Nodal, Lefty, allowing them to overexpress this embryonic morphogen in an unregulated manner. However, exposure of the tumor cells to a hESC microenvironment (containing Lefty) leads to a dramatic down-regulation in their Nodal expression concomitant with a reduction in clonogenicity and tumorigenesis accompanied by an increase in apoptosis. Furthermore, this ability to suppress the tumorigenic phenotype is directly associated with the secretion of Lefty, exclusive to hESCs, because it is not detected in other stem cell types, normal cell types, or trophoblasts. The tumor-suppressive effects of the hESC microenvironment, by neutralizing the expression of Nodal in aggressive tumor cells, provide previously unexplored therapeutic modalities for cancer treatment.