Nodal Signaling As a Developmental Therapeutics Target in Oncology Aparna Kalyan1,2, Benedito A

Review Molecular Cancer Therapeutics Nodal Signaling as a Developmental Therapeutics Target in Oncology Aparna Kalyan1,2, Benedito A. Carneiro1,2, Sunandana Chandra1,2, Jason Kaplan1,2, Young Kwang Chae1,2, Maria Matsangou1,2, Mary J.C. Hendrix1,3, and Francis Giles1,2

Abstract

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

Introduction in carcinogenesis, highlighting potential therapeutic implications related to blocking this pathway. The tumor microenvironment is recognized as a key feature of cancer development, progression, and metastasis. Tumor and embryonic stem cells share similarities in their plasticity Nodal Signaling and innate signaling mechanisms, best highlighted in the The human Nodal gene, located on chromosome 10q22, Nodal family of receptors and its ligands, which are members mediates a signaling system that is highly regulated by transcrip- of the TGFb family. Nodal expression is normally seen during tional factors. Nodal propagates its signal by binding to a hetero- early development when undifferentiated embryonic progeni- dimeric complex between type I (ALK4/7) and type II (ActRIIB) tor cells progress toward endodermal and mesodermal differ- activin-like kinase receptors. This interaction leads to phosphor- entiation, including epithelial-to-mesenchymal transition ylation of ALK 4/7 by ActRIIB and activation of ALK4/7-induced (EMT; ref. 1) and establishment of left–right orientation (1). phosphorylation of cytoplasmic Smad2 and/or Smad3. This Recent understanding of cancer pathways has demonstrated phosphorylated complex then interacts with Smad4, resulting in that reactivation of the Nodal pathway occurs in aggressive the downstream formation of transcriptional complexes within epithelial-derived malignancies and has a crucial role in carci- the nucleus (1, 10, 11). The transcriptional complexes subse- nogenesis (2). Nodal signal transduction is mediated via type I quently initiate downstream gene transcription and translation, and type II serine–threonine kinase receptors and ultimately where other transcription factors, such as FoxH1 and Mixer, through the Smad2/Smad3 branch of the TGFb pathway promote developmental progression of EMT (Fig. 1; refs. 1–3, (1, 3–5). In normal development, Nodal signaling is essential 5, 12, 13). The ALK4/7 and ActIIRB receptors belong to the EGF – for the speciﬁcation of primary body axes and formation of Cripto-1, Frl-1, and Cryptic family (EGF-CFC family) and consist mesoderm and endoderm. However, particularly noteworthy is of cysteine-rich extracellular proteins attached to the plasma the recent discovery that Nodal signaling has been documented membrane through a glycosyl–phosphatidylinositol linkage in breast, prostate, and pancreatic cancers and melanoma, (1, 2, 14). Cripto-1, a coreceptor for Nodal, has direct interaction where expression levels are directly proportional to tumor with Alk4 (via its CFC domain) and Nodal (via its EGF domain). grade (2, 6–9). This review discusses the role of Nodal signaling The interaction between Cripto and Nodal helps propagate the downstream transmission of the Nodal signal. Furthermore, Cripto-1 interacts with Alk7 to augment the interaction between 1Developmental Therapeutics Program, Division of Hematology and Oncology, Alk7 and Nodal. This augmented effect is best illustrated in studies Northwestern University Feinberg School of Medicine, Olson Pavilion, Chicago, performed on Cripto-1–lacking mice, where the mice died at day Illinois. 2Robert H. Lurie Comprehensive Cancer Center of Northwestern Uni- 7.5 due to its inability to gastrulate, an occurrence that is depen- versity, Chicago, Illinois. 3Cancer Biology and Epigenomics Program, Stanley dent on Nodal signaling (15, 16). Nodal signaling has also been Manne Children's Research Institute, Anne and Robert H. Lurie Children's demonstrated to occur independent of Cripto-1. Example of this Hospital of Chicago, Chicago, Illinois. independence is best described by Reissman and colleagues who Corresponding Author: Aparna Kalyan, Northwestern University, 676 N St. Clair, showed that Nodal has the capacity to bind to Alk7 in the absence Suite 850, Chicago, IL 60611. Phone: 312-926-4291; Fax: 312-472-0564; E-mail: of Cripto-1, but that this binding is markedly ampliﬁed in the [email protected] presence of Cripto-1 (15–17). The binding of Nodal to Alk4 in the doi: 10.1158/1535-7163.MCT-16-0215 developing mouse embryo, independent of Cripto-1, resulted in 2017 American Association for Cancer Research. Nodal-associated genes to be transcribed (16, 18). More recently,

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Figure 1. Nodal signaling is initiated by binding to Cripto-1. This in turn causes phosphorylation of Smad2/3 ultimately leading to interaction with Smad4. Cerberus and Lefty innately antagonize Nodal. Once within the nucleus, activated Smad4 interacts with Fox-H1 (a winged transcription factor) or Mixer homeoproteins leading to transcription at the cellular level.

this phenomenon has garnered more support by studies of endoderm formation. Mice that lack Nodal fail to form a primitive murine knockout models where Nodal signaling and axis spec- streak and lack endoderm and mesoderm (13, 16). Nodal plays a ification occurred without Cripto-1 (19). crucial role in neural development as Nodal signaling is required Multiple mechanisms exist to regulate Nodal, resulting in for anterior neural tissue generation, its subsequent maintenance, positive and negative feedback loops for this signaling pathway. and patterning (1). Nodal antagonists, Lefty1 and Cerberus-1, are Nodal has the capacity to upregulate its own transcription in a essential in anterior neural patterning by ensuring enlarged prim- positive feedback loop (15). Conversely, innate inhibitors of this itive streaks do not occur from uncontrolled Nodal signal (1). morphogen ultimately regulate its levels. Embryonic stem cells Prior to gastrulation, Nodal acts as the initiator for axis formation. secrete Lefty A, Lefty B, and Cerberus, which, in embryological During embryogenesis, Cripto-1 levels are found in increasing development, have been demonstrated to spatially and tempo- levels in the primitive streak, as cells undergo EMT in the meso- rally antagonize the Nodal signal (14, 20) by direct interaction derm (22). with the EGF-CFC receptors and Nodal ligands, thereby blocking In embryonic development, Nodal signaling is crucial in left– the formation of receptor complexes (1). Lefty genes are down- right (L-R) axis specification, where it regulates the transmission stream targets of Nodal signaling and are the most potent neg- of left-sided information to the lateral plate mesoderm (1, 23). ative-feedback loop signal for this pathway (1, 14, 20). Specifi- Prior to gastrulation, Nodal is symmetrically expressed on the cally, Lefty A and B inhibit the Nodal signal by associating with right and left sides. However, Nodal expression is limited to the Nodal and/or Cripto-1 to antagonize ALK activation (1, 4, 15). left side during segmentation (1, 16). Restriction to the left side is The soluble cysteine-rich Cerberus family of receptors blocks essential for the L-R asymmetry. All vertebrates have been shown Nodal signal by direct interaction with the ligands. to have asymmetric Nodal expression, causing tissue specific laterality during embryogenesis (1, 23). Nodal in Fetal Development Nodal signaling plays a central role in embryogenesis, partic- Nodal as an Oncoprotein ularly with mesoderm formation in conjunction with the Wnt Recent advances have implicated the commonality in signaling signal (1, 21). Endodermal formation also utilizes the Nodal pathways between aggressive cancer cell lines and embryonic stem pathway by interacting with Mixer homeoproteins, leading to cells. These similarities include proliferative potential, cellular induction of endoderm-specific genes (1, 21). Studies in zebrafish plasticity, and the genes that are involved in maintaining the have shown that the presence of Nodal is vital for mesoderm and pluripotency/plasticity. The microenvironment plays a crucial

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role in the phenotypic characteristics shared between stem cells suggest that the embryonic stem cell microenvironment plays a and cancer cells. Normal embryogenic stem cells have checkpoints role in reducing melanoma tumorigenicity, likely via Nodal that tightly regulate the signaling cascades, which prevent unre- inhibition by Lefty secreted by hESCs. stricted growth, proliferation, and survival. The lack of these Recent studies have evaluated the downregulation of Nodal checkpoints in cancer cell lines results in tumor progression and expression in both in vitro and in vivo models of melanoma, while potential for metastases. However, in malignant breast cancer or subsequently measuring the outcome on tumor cell phenotype, melanoma cells, the absence of the inhibitor molecule Lefty leads biological function, tumorigenesis, and metastatic potential. to Nodal signaling and the Nodal-driven plasticity program to These studies illustrate that downregulation of Nodal causes: proceed unrestricted (2, 8). (i) suppression of the plastic phenotype; (ii) upregulation of Changes to Nodal expression in aggressive melanoma cell lines melanocyte pathway genes; (iii) decreased invasive and prolifer- following exposure to the embryonic stem cell microenvironment ative capacity in vitro; and (iv) significant reduction in tumorige- have been evaluated. Nodal overexpression is seen in embryonic nicity in vivo (13, 26, 27). In mouse models of pulmonary stem cells as well as melanoma cell lines. Nodal expression, as metastases from melanoma treated with an anti-Nodal antibody, determined by mRNA and protein levels, is significantly down- targeted apoptosis was induced in the lung metastases. In an regulated (by up to 80%) following exposure of melanoma cell orthotopic model, melanoma palpable tumors were injected lines to the embryonic stem cell microenvironment, due to the intratumorally with Lefty, the natural potent inhibitor of Nodal, presence of Lefty within the human embryonic stem cell (hESC) resulting in apoptosis of Nodal-positive tumor cells (26, 27). extracellular matrix (14, 24). Interestingly, the levels remained More recently, an mAb (named 3D1), was developed to target suppressed for several days even after the melanoma cells are Nodal at region 46–67, a region that has been recognized as the removed from the embryonic stem cell microenvironment. When binding site of Cripto-1 (coreceptor for Nodal; ref. 29). In vitro Lefty expression is downregulated in hESCs, followed by culturing studies with melanoma cell lines demonstrated that 3D1 inhibits melanoma cells on the Lefty-depleted microenvironment, Nodal the Nodal–Cripto-1 binding and its downstream Smad2/3 phos- expression in the tumor cells is uninhibited (14). phorylation (29, 30). Furthermore, 3D1 mAb treatment reduces the vasculogenic network formation in melanoma cell lines (30). Nodal in Melanoma These results mimic those seen when breast cancer cell lines are treated with 3D1. Collectively, these findings implicate Nodal as a The embryonic signaling pathways in melanoma demonstrate potential new therapeutic target supported by compelling proof- that the morphogen Nodal is reexpressed in the aggressive phe- of-principle evidence. notype of melanoma (9, 13, 25, 26). IHC has shown that Nodal is absent in normal melanocytes, weakly expressed in primary melanoma, but was present in 60% of cutaneous melanoma Nodal in Breast Cancer metastases (9, 13, 14, 26, 27). Furthermore, Western blot analyses Nodal and its coreceptor Cripto-1 are important in the matu- demonstrated that 45% of melanoma metastases were positive for ration and differentiation of mammary glands (16). Nodal has Nodal, while its expression was lacking in normal skin melano- been extensively studied in breast cancer, where levels of Nodal cytes (27). These results elegantly demonstrated for the first time expression correlate with tumor stage. Studies by Hendrix and that there was a positive correlation between Nodal expression colleagues established that expression of Nodal in aggressive and melanoma progression. breast cancer cell lines is proportional to tumor grade (14). In In addition, treatment of aggressive melanoma cell lines a series of 20 triple-negative breast cancer biopsies, Nodal expres- (C8161) with morpholino oligonucleotides directed against sion by IHC was elevated, whereas it was almost undetectable in Nodal (MOnodal) resulted in reduced phosphorylation of SMAD2 patients with benign breast lesions (2). In evaluating the func- (27). Treatment with an ALK4/5/7 inhibitor abrogates Nodal tional requirement of Nodal signaling, knockdown models with expression in embryonic stem cells. When aggressive melanoma highly aggressive breast cancer cell lines demonstrated decreased cell lines are treated with the same ALK inhibitor, there was a Nodal protein and decreased Smad3 phosphorylation, confirm- significant reduction in the ability of the metastatic cells to invade ing that Nodal signaling is essential for Smad3 phosphorylation the extracellular matrix. These results were corroborated in ortho- in breast cancer (2). Nodal knockdown has also resulted in growth topic Nodal knockout mouse models, where there was a reduction suppression, decreased cell viability, increased apoptosis, and loss in tumorigenicity of the metastatic cell lines (27). of self-renewal ability for breast cancer cells (2). Taken together, Postovit and colleagues utilized an in vitro three-dimensional this evidence supports the notion that Nodal signaling is vital in (3D) model to determine whether the embryonic stem cell cell proliferation, invasion, and self-renewal, all of which are key microenvironment could alter the metastatic melanoma pheno- elements of triple-negative breast cancer. Disease progression in type (14, 28). In this model, embryonic stem cells "enriched" a 3D breast cancer cell lines was further evaluated in in vivo xenograft matrix. Melanoma cells were then exposed to this enriched models. Cell lines with Nodal knockdown had dramatically extracellular microenvironment after removal of the embryonic slower tumor progression compared with controls (2). stem cells. Postovit and colleagues were able to demonstrate that Breast cancer is characterized by its heterogeneity. Clinically, exposure of aggressive melanoma cells to the embryonic stem cell several validated grading systems characterize the natural history microenvironment resulted not only in reinduction of Melan-A (a of breast cancer. One such example, the Nottingham grading melanocyte-specific marker), but in an 87% reduction in Nodal system, utilizes nuclear pleomorphism, tubular formation, and expression and corresponding tumorigenicity (14, 15). In ortho- mitoses to classify the nature of breast cancer. In this grading topic mouse models, these microenvironment-exposed melano- system, features of poor differentiation, high mitoses, and ma cell lines demonstrated reduced tumor growth, proliferation, increased nuclear pleomorphism characterize aggressive disease. and increased apoptosis (14, 15). Taken together, these findings The heterogeneity of breast cancer makes utilization of such

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grading systems, hormonal receptor status, or HER2 status as a evidence suggests that pancreatic adenocarcinomas may harbor a clinical marker difficult in all clinical scenarios. Recent interest in population of putative cancer stem cells (CSC), which have identifying biomarkers for the prediction of tumor grade as well as become a target for novel therapies (32–35). CSCs are hypoth- prognostic value has led to interest in Nodal as a potential marker. esized to contribute their tumorigenic and metastatic potential via To evaluate this, Hendrix and colleagues, in a recent study, their capacity for regeneration and differentiation (32–35). Most obtained tissue specimens from 431 treatment-na€ve patients importantly, CSCs have been postulated to be a cause of resistance diagnosed with both benign breast disease and breast cancer and to conventional cytotoxic chemotherapy and radiation (32, 35). blindly investigated Nodal expression (8). Nodal expression In normal adult pancreatic tissue, the Nodal/Activin pathway is was determined by IHC, with staining scores ranging from 0 to inactive (36), and expression of mRNA for the Nodal genes is 3(0¼ no staining; 1 10%; 2 ¼ 10%–50%, and 3 50%). The undetectable (7). Flow cytometry and IHC have validated that degree of staining was correlated with clinical data. The results there is upregulation of Nodal and its associated genes (Cripto-1, suggested that Nodal index score was highest in malignant, FoxH1, Smad2, Smad4, Alk4, and Activin) in pancreatic cancer undifferentiated, invasive, and advanced stage breast cancer com- development and tumor progression (7). Lentivirus models of pared with benign breast disease or early-stage cancer (P < 0.0001; pancreatic stem cells demonstrated that knockdown of Nodal ref. 8). Nodal expression also correlated with breast cancer dif- resulted in significantly lower in vivo tumorigenic effects (7). ferentiation as determined by the Nottingham grade score, with Lonardo and colleagues demonstrated in in vitro studies that the higher Nodal scoring seen in poorly differentiated cancer tissues pancreatic CSCs are inherently resistant to chemotherapy and compared with well-differentiated tissues. Correlation with have an increased expression of Nodal signaling genes. As such, tumor stage and lymph node involvement status illustrated that when CD133 expression is used as a marker for CSC content, advanced T stage or lymph node–positive disease had higher antagonizing the Nodal/Activin pathway leads to complete elim- Nodal scoring (T stage P ¼ 0.0003 and N stage P ¼ 0.009; ref. 8). ination of CSCs. The effect is further augmented in the presence of When Nodal was targeted with an anti-Nodal antibody in human gemcitabine (7). Pancreatic cancer cell lines were exposed to metastatic breast cancer cell lines (MDA-MB-468 and MDA-MB- gemcitabine alone, an Alk4/7 receptor inhibitor (inhibitor of the 231), the dose–response curve demonstrated a significant reduc- Nodal pathway) or a combination of both agents. These cells were tion in the expression of Nodal protein and Smad2 phosphory- then implanted into the pancreas of immunocompromised mice. lated levels with increasing concentrations of anti-Nodal antibody Tumor size evaluation, as determined by PET scan, demonstrated administration (P < 0.05; ref. 8). These findings cautiously suggest that combination treatment with gemcitabine and an Alk4/7 the potential role of Nodal as a prognostic biomarker and perhaps inhibitor eliminated in vivo tumors entirely. Furthermore, there þ a therapeutic target in breast cancer. was a 3-fold increase in the apoptotic CD133 cells with the Recent understanding of Cerberus as a natural inhibitor of combination of gemcitabine and Alk4/7 inhibitor use. The use of Nodal has led to numerous studies evaluating its role on breast single-agent gemcitabine or Alk4/7 inhibitor individually did not þ cancer cell lines. Five human breast cancer cell lines were evalu- irreversibly eliminate CD133 cells (a marker for CSCs; ref. 7). ated using Cerberus-Fc, including high Nodal expression (MDA- Therapeutically, these findings indicate that an Alk4/7 inhibitor in MB-231, BT-549), weak metastatic potential with low Nodal combination with gemcitabine is potentially capable of overcom- expression (Hs578t), and noninvasive breast cancer (MCF-7, ing chemoresistance of the pancreatic CSCs. These promising T47D; ref. 31). The amount of viable cells after treatment with results led to the evaluation of whether inhibition of the Nod- human Cerberus-Fc was compared with cells that were untreated. al/Activin signal would translate to meaningful progression-free Cerberus-Fc inhibited the proliferation of breast cancer cell lines survival (PFS) in xenograft models. As expected, in xenograft with Nodal expression compared with control (untreated). Inhi- models developed after orthotopic implantation of pancreatic bition was greatest in the cell lines with higher Nodal expression cancer cells, treatment with a combination of gemcitabine and (31). In addition, cell migration in Nodal-expressing breast cancer Alk4/7 inhibitor showed no detectable tumor by days 40 and 100 cell lines was also restricted when treated with Cerberus-Fc, and almost 100% survival in the combination arm (7). However, lending merit to the theory that inhibition of this pathway can when this same concept was tested in human pancreatic cancer potentially decrease or halt invasion and metastases in breast tissue xenografts, the combination treatment did not improve PFS cancer. or decelerate tumor growth (7). The RAS–ERK signaling pathway is active in several human As previously described in this review, the tumor microen- malignancies. Upon activation, RAS-ERK contributes to the vironment is a crucial component of Nodal signaling. Lonardo aggressive nature of triple-negative breast cancer by promoting and colleagues further explored the role of a Nodal inhibitor. resistance to therapy, self-renewal, and plasticity. Nodal signaling They hypothesized that the disappointing results of combina- activates the ERK pathway and contributes to its associated tion therapy in human pancreatic cancer tumor xenografts were downstream effects (described above). When Nodal signal is due to poor drug delivery in stromal-rich pancreatic tumors. absent, these breast cancer cell lines submit to apoptosis, cell- Rather surprisingly, the addition of Smoothened inhibitor cycle arrest, and reduced invasiveness (2). These same features are (Smo), which targets the Hedgehog pathway in stromal cells, observed when ERK signal is inhibited. These findings support the to gemcitabine and an Alk4/7 inhibitor caused almost com- premise that Nodal can be considered a therapeutic target to limit plete resolution of the tumors in the xenografts with improved plasticity and self-renewal of breast cancer cells. PFS and OS in these models (7). Taken together, these data suggest that triple therapy could overcome pancreatic CSCs and offer a potential therapeutic target. These results suggest that Nodal in Pancreatic Cancer tumor-associated stroma offers an additional source of Nodal Pancreatic adenocarcinomas are increasing in incidence, and and modulates tumor vascularity, thereby interfering with drug therapy options remain limited with dismal survival rates. In vitro delivery. The stromal expression of Nodal has been explored in

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pancreatic cancer to date and does lend itself to be further onic and oncogenic signaling pathway. Nodal morphogen is evaluated in other malignancies. expressed in several malignancies, including melanoma, breast More recently, the expression patterns of Lefty and Nodal in cancer, and pancreatic cancer. Furthermore, there is a direct pancreatic ductal adenocarcinomas (PDAC) was investigated correlation between levels of Nodal expression and higher grade using 54 PDAC specimens (37). Control specimens from normal of tumor. Antagonizing Nodal by its innate inhibitor (Lefty) or pancreatic acinar and ductal cells did not have any Nodal or Lefty an mAb has shown to reduce the tumorigenic potential of cells, expression. However, variable expression of Nodal was seen in the when using proliferation and degree of metastases as markers. PDAC specimens (37). High levels of Nodal in the stroma were Increasing evidence supports the premise that Nodal can be associated with reduced patient survival compared with low utilized as a therapeutic target in addition to being a predictive Nodal expression (overall median survival, 17.8 months vs. 33 and prognostic marker of breast cancer. Further studies are months; P ¼ 0.013; ref. 37). These results reinforce the hypothesis needed to evaluate the therapeutic aspect of Nodal in combi- that Nodal expression is associated with poor prognosis in PDAC. nation therapy in addition to the already known monotherapy effects in melanoma and breast cancer. Conclusions/Future Implications Disclosure of Potential Conflicts of Interest There is a growing body of evidence suggesting that embry- M.J.C. Hendrix holds a patent for targeting Nodal. All other authors named onic stem cells and cancer cells share several features, including have no financial conflicts. plasticity and key signaling pathways. The Nodal signaling pathway, a member of the TGFb family, is directly related to Received April 12, 2016; revised November 22, 2016; accepted December 5, tumor cell plasticity and represents a merging of both embry- 2016; published online May 3, 2017.

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Aparna Kalyan, Benedito A. Carneiro, Sunandana Chandra, et al.

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