TGFB1/INHBA Homodimer/Nodal-SMAD2/3 Signaling Network: A Pivotal Molecular Target in PDAC Treatment

Mai Abdel Mouti; Siim Pauklin

doi:10.1016/j.ymthe.2021.01.002

. 2021 Jan 9;29(3):920–936. doi: 10.1016/j.ymthe.2021.01.002

TGFB1/INHBA Homodimer/Nodal-SMAD2/3 Signaling Network: A Pivotal Molecular Target in PDAC Treatment

Mai Abdel Mouti ¹, Siim Pauklin ^1,^∗

PMCID: PMC7934636 PMID: 33429081

Abstract

Pancreatic cancer remains a grueling disease that is projected to become the second-deadliest cancer in the next decade. Standard treatment of pancreatic cancer is chemotherapy, which mainly targets the differentiated population of tumor cells; however, it paradoxically sets the roots of tumor relapse by the selective enrichment of intrinsically chemoresistant pancreatic cancer stem cells that are equipped with an indefinite capacity for self-renewal and differentiation, resulting in tumor regeneration and an overall anemic response to chemotherapy. Crosstalk between pancreatic tumor cells and the surrounding stromal microenvironment is also involved in the development of chemoresistance by creating a supportive niche, which enhances the stemness features and tumorigenicity of pancreatic cancer cells. In addition, the desmoplastic nature of the tumor-associated stroma acts as a physical barrier, which limits the intratumoral delivery of chemotherapeutics. In this review, we mainly focus on the transforming growth factor beta 1 (TGFB1)/inhibin subunit beta A (INHBA) homodimer/Nodal-SMAD2/3 signaling network in pancreatic cancer as a pivotal central node that regulates multiple key mechanisms involved in the development of chemoresistance, including enhancement of the stem cell-like properties and tumorigenicity of pancreatic cancer cells, mediating cooperative interactions between pancreatic cancer cells and the surrounding stroma, as well as regulating the deposition of extracellular matrix proteins within the tumor microenvironment.

Keywords: pancreatic cancer, chemoresistant, pancreatic cancer stem cells, TGFB1, INHBA, nodal

Graphical Abstract

TGFB1/INHBA homodimer/Nodal signaling pathways regulate several key mechanisms involved in the resistance of pancreatic adenocarcinomas to chemotherapy. Development of effective TGFB1/INHBA homodimer/Nodal-targeted therapies is highly prospective of breaching the functionally entangled territory of pancreatic cancer and blocking mechanisms of chemoresistance and tumor recurrence.

Main Text

Pancreatic ductal adenocarcinoma (PDAC), which accounts for more than 90% of all pancreatic cancers, represents a highly intractable type of human malignancy.¹ It currently ranks as the fourth leading cause of cancer-related death worldwide, with a less than 8% 5-year survival rate.² PDAC-related mortality rates are slowly increasing at an alarming rate, and are projected to overtake other types of top killer cancers (breast, prostate, and colorectal) to become the second leading cause of cancer-related death, following lung cancer, in the United States by 2030.³ In addition, PDAC is typically diagnosed at a late stage, mainly due to the lack of specific biomarkers and absence of distinctive signs and symptoms at the early stages of the disease.⁴ To date, systemic chemotherapy represents the mainstay of treatment for PDAC patients presenting with non-surgically resectable, locally advanced tumors or distant metastases.⁵^,⁶ In 1997, gemcitabine was first evaluated versus other chemotherapies such as 5-fluorouracil (5-FU) through a phase III clinical study and was found to improve the overall survival by 1 month in advanced pancreatic cancer patients as compared with 5-FU monotherapy.⁷ Significant improvements of overall survival and progression-free survival were then reported using a combination therapy of erlotinib, an epidermal growth factor receptor (EGFR) inhibitor, and gemcitabine;⁸ however, the toxicity of this combination regimen terminated its widespread adoption. FOLFIRINOX, a combination regimen of 5-FU, leucovorin, irinotecan, and oxaliplatin, significantly improved survival as compared with gemcitabine monotherapy, with a median increase in survival of 4.3 months; however, only a few patients with good performance status are eligible for FOLFIRINOX, as it is a highly toxic combination of chemotherapeutics with potential serious adverse effects.⁹ A less toxic, but also a less effective, alternative to FOLFIRINOX is a combination regimen of gemcitabine and nanoparticle albumin-bound (nab) paclitaxel which showed an improvement of overall survival, progression-free survival, and response rates as compared with gemcitabine alone; however, adverse effects such as myelosuppression and peripheral neuropathy were reported.¹⁰ To conclude, despite multiple chemotherapy-based treatments for pancreatic cancer, only a marginal improvement of overall survival is achieved, which is often associated with a high risk of developing serious adverse effects. With PDAC-related death rates still on the rise, there is an urgent need for developing treatment paradigms that are capable of optimizing the response to chemotherapy and achieving significant overall survival benefits for pancreatic cancer patients.

Stemness of Pancreatic Cancer Cells

The cancer stem cell (CSC) hypothesis proposes that a subpopulation of cells possessing distinctive stemness features and functions, known as CSCs, are located at the apex of a hierarchical organization of cells within a tumor and are likely to be responsible for driving tumorigenesis and metastasis.¹¹ PDACs are characterized by high degrees of intra- and inter-tumoral heterogeneity,¹² which meets the growing consensus that the CSC model is relevant to pancreatic cancer and that pancreatic cancer stem cells (PCSCs) play a central role in driving chemoresistance and tumor relapse. PCSCs were first identified as a subpopulation of highly tumorigenic pancreatic cancer cells expressing the cell surface markers cluster of differentiation (CD) 44, CD24, and epithelial cell adhesion molecule (EPCAM) and showing stem cell-like properties of self-renewal and differentiation.¹³ Multiple studies then followed, identifying highly tumorigenic PCSCs displaying stem cell-like properties but expressing different cell surface markers. PCSCs expressing the cell surface marker prominin 1 (PROM1) demonstrated a tumor-initiating capacity in immunodeficient mice as compared with PROM1-deficient cells and were also resistant to the apoptotic effects of gemcitabine.¹⁴ CD44⁺PROM1⁺EPCAM⁺ PCSCs displayed an increased capacity for cell growth, migration, self-renewal, and resistance to apoptosis compared with the triple-marker-negative cells (CD44⁻PROM1⁻EPCAM⁻) and also had a 100-fold higher potential of inducing tumor formation and growth in immunodeficient mice as compared with the parental cells.¹⁵ Similarly, MET^highCD44⁺ PCSCs displayed a self-renewal capacity as well as a high tumorigenic potential in immunocompromised mice, which was reduced upon treatment with MET inhibitors alone or in combination with gemcitabine.¹⁶ The origin of PCSCs still remains unknown; however, there are two potential origins: either from normal somatic stem cells that have undergone transformative genetic alterations or mutated differentiated cells that have reprogrammed to acquire stem cell-like properties.¹⁷ Thus, according to the CSC model, which is supported by in vitro cell-based assays and in vivo tumorigenicity studies, chemotherapy alone is unlikely to lead to complete tumor elimination, as the residual population of intrinsically chemoresistant CSCs are able to self-renew and differentiate indefinitely, leading to tumor regeneration (Figure 1). This review article discusses targeting PCSCs as one of the major roots of tumor relapse that regulate the development of resistance to chemotherapy through multiple mechanisms. We are mainly focusing here on the SMAD2/3-dependent transforming growth factor beta 1 (TGFB1)/inhibin subunit beta A (INHBA) homodimer (Activin A)/Nodal signaling pathways, which are crucial for sustaining the stem cell-like characteristics of PCSCs, mediating bidirectional crosstalk between pancreatic cancer cells and components of the surrounding tumor microenvironment (TME), and regulating the properties of the PDAC-associated stroma.

PCSCs Drive Tumor Recurrence and Chemotherapy Failure

(A) Only non-stem cancer cells, which constitute the majority of the total tumor cell population, respond to the cytotoxic effects of chemotherapy, leading to partial tumor elimination and initial regression of the tumor size. (B) Chemoresistant PCSCs self-renew and differentiate through symmetric and asymmetric divisions, respectively, rendering them capable of recapitulating the cellular heterogeneity of the primary tumor, which is associated with increased tumor size and relapse.

Activation of TGFB1/INHBA Homodimer/Nodal Pathways

TGFB1, INHBA homodimer, and Nodal belong to the transforming growth factor superfamily of cytokines, which is subdivided into two main functional groups: the transforming growth factor beta-like group, including different isoforms of transforming growth factor beta (TGFB1, TGFB2, and TGFB3), INHBA, inhibin subunit beta B (INHBB), inhibin subunit beta C (INHBC), inhibin subunit beta E (INHBE), inhibin subunit alpha (INHA), Nodal, and some growth differentiation factors; and the bone morphogenetic proteins-like group that includes bone morphogenetic proteins, anti-mullerian hormone (AMH), and most of the growth differentiation factors. Different ligands of the transforming growth factor beta superfamily regulate various developmental processes and physiological functions, however, in a developmental context-dependent and cell type-specific manner.¹⁸ In addition, different ligands, namely TGFB1, INHBA homodimer, and BMP4, transduce signaling cascades with distinct dynamics that are largely dependent on the effect of each ligand on the overall trafficking of their respective cell surface receptors, specifically determined by the rates of their internalization from the cell surface and degradation as well as rates of renewal by recycling and/or synthesis.¹⁹

Transforming growth factor beta occurs in three isomeric forms in humans (TGFB1, 2, and 3), which are encoded by genes located on the long arms of different chromosomes: 19q13.1 (TGFB1), 1q41 (TGFB2), and 14q24 (TGFB3).20, 21, 22 They share highly conserved regions but also diverge in several amino acid sequences. Functionally, they mediate their cellular effects through the same receptors.²³^,²⁴ Of the three isoforms, TGFB1 is predominantly expressed in human tissues; synthesized and secreted by almost all cell types including fibroblasts, platelets, lymphocytes, macrophages/monocytes, epithelial cells, dendritic cells, and Treg cells; and also the most frequently upregulated in tumor cells that produce and secrete autocrine TGFB1 into the TME.25, 26, 27 Active TGFB1 occurs as a homodimer (molecular weight of 25 kDa) composed of two polypeptide chains, each containing 112 amino acid residues that are linked by a single disulphide bond stabilized by hydrophobic interactions.²⁸ This active form of TGFB1 is synthesized from latent TGFB1, a large inactive precursor molecule. Latent TGFB1 is composed of a TGFB1 dimer in a non-covalent complex with the TGFB1 propeptide known as latency-associated protein, which is linked to the latent transforming growth factor beta binding protein 1 (LTBP1) by a disulphide bond.²⁹ In the extracellular matrix (ECM), this precursor molecule acts as a reservoir for TGFB1 and is activated through different mechanisms in response to multiple molecules that liberate TGFB1 from latent complexes.³⁰

Transcripts of INHBA and INHBB are expressed in nearly all tissues; however, INHBC and INHBE transcripts are predominantly expressed in the liver. Disulphide-linked homo- or heterodimers of INHBA and INHBB, homodimers of INHBA (Activin A), homodimers of INHBB (Activin B), and heterodimers of INHBA and INHBB (Activin AB), are the currently known bioactive proteins that exist in various tissues,31, 32, 33, 34, 35, 36, 37 which are formed upon the dimerization of precursor polypeptides, followed by proteolytic cleavage.³⁸^,³⁹ Of all mature dimeric polypeptides, INHBA homodimers are predominantly expressed in metastatic prostate cancer,⁴⁰ stage 4 colorectal cancer,⁴¹ lung cancer,⁴² hepatocellular carcinomas,⁴³ pancreatic cancer,⁴⁴ and breast cancer.⁴⁵

Nodal is an embryonic morphogen⁴⁶ whose expression is mostly restricted in embryonic tissues and reproductive cell types, while being undetectable in most normal adult tissues.⁴⁷^,⁴⁸ However, recent research studies demonstrate the re-expression of Nodal during cancer progression, including malignant thyroid tumors,⁴⁹ hepatocellular carcinomas,⁵⁰ pancreatic cancer,⁵¹ melanoma,⁵² and carcinomas of the endometrium⁵³ and prostate.⁵⁴ Nodal is synthesized as an inactive precursor form consisting of a single peptide, prodomain, and mature domain. In transfection studies using exogenous mouse Nodal, extracellular proprotein convertases such as furin and proprotein convertase subtilisin/kexin type 6 (PCSK6) cleave the pro- and mature domains of Nodal into a much less stable but highly active mature form. In addition, teratocarcinoma-derived growth factor 1 (TDGF1), a crucial extracellular protein for Nodal signaling, contributes to Nodal activation by anchoring the proform of Nodal and one of the proprotein convertases in a complex at the plasma membrane.⁵⁵

Initiation of Signal Transduction by Activated Receptors

A TGFB1-mediated signaling is initiated when TGFB1 interacts with the extracellular domain of a type II transmembrane receptor, which has a cytoplasmic serine/threonine kinase domain. TGFB1 directly binds to transforming growth factor beta receptor 2 (TGFBR2), a type 2 transmembrane receptor, which in turn recruits TGFBR1, a type 1 transmembrane receptor, to the TGFB1-TGFBR2 complex, leading to receptor activation through the phosphorylation of multiple serine/threonine residues within its GS box, a conserved glycine and serine-rich region just preceding the receptor kinase domain.⁵⁶ INHBA homodimers interact with activin A receptor type 2A (ACVR2A) or (ACVR2B), which activates and phosphorylates activin A receptor type 1B (ACVR1B) within the GS domain.57, 58, 59, 60, 61 Nodal also signals through ACVR2A/ACVR2B and ACVR1B; however, it requires TDGF1 for the assembly of the type 1 and type 2 receptor complex.⁶²

SMAD-Dependent Signal Transduction

Mechanism of Signal Transduction

The canonical pathway for TGFB1/INHBA homodimer/Nodal signaling is SMAD dependent (Figure 2), where the expression of target genes is regulated through the activation of SMAD transcription factors. SMAD proteins consist of two globular domains, Mad homology (MH) 1 and MH2, which are connected by a linker region that acts as a hub for positive and negative regulatory inputs. The amino-terminal MH1 domain is a sequence-specific DNA-binding domain,63, 64, 65, 66, 67 whereas the carboxy-terminal MH2 domain mediates the interaction of SMADs with receptors,68, 69, 70 transcription cofactors,71, 72, 73 and other SMAD proteins.⁷⁴^,⁷⁵

Chemoresistant PCSCs Enhance Tumor Regeneration through the Activation of TGFB1/INHBA Homodimer/Nodal Signaling

Binding of TGFB1, INHBA homodimer, and Nodal to the extracellular domain of their respective transmembrane type II receptors, TGFBR2 for TGFB1 and ACVR2A/ACVR2B for INHBA homodimer/Nodal, recruits type I transmembrane receptor, TGFBR1 for TGFB1 and ACVR1B for INHBA homodimer/Nodal, to the ligand-receptor complex, leading to receptor activation through the phosphorylation of multiple serine and threonine residues within its GS domain. ZFYVE9 regulates the access of SMAD2/3 to the activated receptor complex, where the interaction between SMAD2/3 and the activated receptor is mediated through the phosphorylated GS domain and L45 loop of the activated type I receptor and L3 loop of SMAD2/3, leading to SMAD2/3 phosphorylation followed by subsequent dissociation from ZFYVE9 and binding to SMAD4. The active SMAD2/3/4 transcription complex then translocates into the nucleus, where binding to a specific SBE sequence occurs in association with DNA-binding transcription factors and coregulators to control the expression of target genes. TF, transcription factor.

In response to receptor activation, zinc double finger FYVE-type containing 9 protein (ZFYVE9) mediates the binding of receptor-regulated SMADs to the activated receptor complex.⁷⁰ SMAD2 and SMAD3, two types of receptor-regulated SMADs, mediate signaling initiated by ligands of the transforming growth factor beta-like group.⁷³^,76, 77, 78, 79, 80, 81, 82, 83 SMAD2/3 bind to activated receptors through the phosphorylated GS domain and the L45 loop of the receptor kinase domain of type I receptors that is adjacent to the GS region, with the L3 loop of SMAD2/3 serving as the corresponding specificity determinant.⁶⁸ SMAD2/3 are phosphorylated by the activated type I receptors on the last two serine residues at the carboxy-terminal end within a highly conserved Ser-Ser-X-Ser (SSXS) motif, in which X is either methionine (SMAD2) or valine (SMAD3).⁷³^,⁷⁸^,80, 81, 82 This decreases the binding affinity of ZFYVE9 to SMAD2/3, leading to their subsequent dissociation⁷⁰ and binding to SMAD4 in the cytoplasm,⁷⁴^,⁸⁰ which is stabilized by the carboxy-terminal phosphoserine residues on SMAD2/3.⁸⁴^,⁸⁵ SMAD complexes then translocate into the nucleus, where they positively and negatively regulate the transcription of target genes.

Shuttling of SMADs

SMADs shuttle continuously between the cytoplasm and nucleus, also referred to as the “basal shuttling equilibrium,” in the absence of signaling.86, 87, 88 Under these conditions, the distribution of SMADs between the cytoplasm and the nucleus is mainly dependent on the relative strength of their nuclear import-to-nuclear export signals.⁸⁹ The nuclear export of SMAD2/3 occurs at a faster rate than their nuclear import, and they are thus predominantly cytoplasmic,⁹⁰ whereas SMAD4 is more or less equally distributed between the two compartments.⁸⁷ In the presence of a signal leading to SMAD complex formation followed by nuclear translocation, export becomes limited,⁹¹ as the structural SMAD motifs mediating their nuclear export are masked in the complex.⁹² In addition, binding of SMADs to DNA and DNA-binding factors is involved in retaining their presence in the nucleus.⁸⁹ Upon the dissociation of the SMAD complex as a result of constitutive SMAD2/3 dephosphorylation, monomeric SMADs are released into the cytoplasm, where they re-enter their basal shuttling equilibrium.⁹³

More than one SMAD2/3 phosphatase has been proposed to dephosphorylate SMAD2/3, which impairs their transcriptional activity. SMAD2/3-tail phosphatases, which are able to dephosphorylate SMAD2/3-tail SXS motif, include protein phosphatase Mg2+/Mn2+-dependent 1A (PPM1A);⁹⁴ protein phosphatase 2 phosphatase activator (PTPA), which regulates the dephosphorylation of SMAD3, but not SMAD2, only under hypoxic conditions;⁹⁵ and myotubularin-related protein 4 (MTMR4), which has been recently proposed to dephosphorylate SMAD2/3.⁹⁶ SMAD2/3 linker phosphatases include CTD small phosphatase 1 (CTDSP1) and CTD small phosphatase 2 (CTDSP2), which have been reported to efficiently dephosphorylate the SMAD2 linker sites at Ser245, Ser250, Ser255, and analogous SMAD3 sites.⁹⁷^,⁹⁸ However, the efficiency of these phosphatases to dephosphorylate SMAD2/3 has not yet been confirmed in vivo, and, hence, there is still a controversy regarding the true SMAD2/3 phosphatases and the mechanisms that regulate their binding specificity to the substrate and phosphatase activities.

Nucleocytoplasmic Trafficking of SMADs

The mechanisms by which SMADs translocate through the nuclear pore are controversial. The transport of SMADs through a karyopherin-independent transport has been suggested,⁸⁸^,⁹⁹ but a karyopherin-dependent mechanism of transport has also been reported.⁹³ SMADs are translocated into the nucleus through the nuclear pore by several karyopherins, such as karyopherin subunit beta 1 (KPNB1), which directly binds to SMAD3 through the nuclear localization signal (NLS) located within its MH1 domain¹⁰⁰^,¹⁰¹ as well as SMAD1 through a corresponding motif.¹⁰² Karyopherin subunit alpha 2 (KPNA2) binds to SMAD4 and facilitates its nuclear import through an extended, bipartite NLS.¹⁰³ An alternative mechanism for the transport of SMAD2, SMAD3, and SMAD4 through direct binding to the nuclear pore complex (NPC) has also been suggested.⁸⁸^,⁹⁹ However, these results are mainly based on in vitro studies using isolated SMAD domains, which might provide an explanation for this controversy regarding the nuclear import of SMADs.

The nuclear export of SMAD4 from the nucleus is mainly mediated by exportin 1 (XPO1) through a nuclear export signal (NES) located within the SMAD4 linker region.⁸⁷^,¹⁰⁴ Exportin 4 (XPO4) regulates the nuclear export of SMAD3,¹⁰⁵ whereas a karyopherin-independent mechanism has been suggested for the nuclear export of SMAD2.⁸⁸

Interactions of SMADs in the Nucleus

Through the amino-terminal MH1 domain, SMAD3 and SMAD4 bind to a specific DNA sequence (CAGA), known as the SMAD-binding element (SBE).⁶⁷ However, as a result of the relatively weak binding affinity of SMAD proteins to DNA (K_d ≈1 × 10⁻⁷ M),⁶⁵ excluding SMAD2, which lacks any DNA-binding affinity due to a 30 amino acid insertion within its MH1 domain,¹⁰⁶ additional DNA-binding factors are required for high-affinity and high-specificity recruitment and binding of SMADs to DNA (Table 1). In addition, SBE concatemers are seldom present in SMAD target promoters, and even SMAD target genes containing up to 4 SBEs still require DNA-binding factors.¹⁰⁷ In addition to mediating DNA binding, transcription factors bound to nuclear SMAD complex are also involved in regulating various cellular responses to TGFB1/INHBA homodimer/Nodal stimulation. SMAD proteins also bind to transcription coactivators¹⁰⁸ or corepressors,¹⁰⁹ which regulate the transcription of TGFB1/INHBA homodimer/Nodal-responsive genes (Table 2). Some of these transcription coactivators, with intrinsic histone acetyltransferase (HAT) activity, acetylate histones and open up the chromatin structure, which increases the accessibility of active transcription complexes to DNA-binding sites, resulting in transcriptional activation. Other coactivators, lacking intrinsic HAT activity, stimulate gene transcription by mediating an interaction between SMAD complexes and transcription coactivators possessing HAT activity. On the other hand, some transcription corepressors function by recruiting histone deacetylases (HDACs) to SMAD complexes and/or interact with SMAD proteins to block their interaction with transcription coactivators, leading to transcriptional repression.

Table 1.

Interaction of SMADs with DNA-Binding Transcription Factors

Cofactor	Family	Interacting SMADs	Function	References
Enhancers

Forkhead box H1 (FOXH1)	forkhead boxes (FOX)	SMAD2 SMAD3	activate gene expression	⁷¹^,¹¹⁰
Nanog homeobox (NANOG)	NKL subclass homeoboxes and pseudogenes	SMAD2 SMAD3	recruit DPY30-COMPASS histone modifiers to regulate transcription of developmental genes	¹¹¹
Eomesodermin (EOMES)	T-box transcription factors	SMAD2 SMAD3	activate the transcription network required for endoderm formation	¹¹²
Ras responsive element binding protein 1 (RREB1)	zinc fingers C2H2-type	SMAD2 SMAD3	regulate the expression of mesendoderm genes in pluripotent stem cells and fibrogenic factors in pancreatic cancer cells	¹¹³
Jun proto-oncogene, Fos proto-oncogene	basic leucine zipper proteins	SMAD3	induce transcription of TGFB1-responsive genes	¹¹⁴
Mix paired-like homeobox (MIXL1)	PRD class homeoboxes and pseudogenes	SMAD2	activate TGFB1/INHBA homodimer-mediated transcription	¹¹⁵
POU class 5 homeobox 1 (POU5F1)	POU class homeoboxes and pseudogenes	SMAD3	induce the expression of snail family transcriptional repressor 1 (SNAI1), snail family transcriptional repressor 2 (SNAI2), and C-X-C motif chemokine ligand 13 (CXCL13)	¹¹⁶
POU class 5 homeobox 1 (POU5F1)	POU class homeoboxes and pseudogenes	SMAD2 SMAD3	cooperate with transcription effectors of the Hippo pathway and SMAD2/3 to induce the expression of pluripotency genes in response to INHBA homodimer/Nodal signaling in ESCs	¹¹⁷
Activating transcription factor 2 (ATF2)	basic leucine zipper proteins	SMAD3	activate TGFB1-mediated transcription	¹¹⁸
Core-binding factor subunit beta (CBFB)	beta subunit of a heterodimeric core-binding transcription factor belonging to the CBFB transcription factor family	SMAD3	stimulate transcription of the germline immunoglobulin (Ig) Calpha promoter	¹¹⁹
Vitamin D receptor (VDR)	nuclear hormone receptors, protein phosphatase 1 regulatory subunits (PPP1R)	SMAD3	SMAD3 forms a complex with VDR and acts as its coactivator	¹²⁰
Tumor protein p53 (TP53)	member of the p53 gene family of transcription factors	SMAD2 SMAD3	activate the transcription of SERPINE1 by recruiting acetyltransferase histone CREB-binding protein (CREBBP)	¹²¹
Forkhead box O1 (FOXO1), Forkhead box O3 (FOXO3), Forkhead box O4 (FOXO4)	forkhead boxes	SMAD3	increase expression of CDKN1A	¹⁰⁷

Repressors

Zinc finger E-Box binding homeobox 2 (ZEB2)	homeoboxes, zinc fingers	SMAD3	repress transcription through the recruitment of c-terminal binding protein 1 (CTBP1) corepressor	¹²²
Nuclear receptor subfamily 3 group C member 1 (NR3C1)	nuclear hormone receptors	SMAD3	repress TGFB1-mediated transcriptional activation of SERPINE1	¹²³
TGFB induced factor homeobox 1 (TGIF1)	TALE class homeoboxes and pseudogenes	SMAD2	repress TGFB1-mediated transcription which acts in part by recruiting HDACs	¹²⁴^,¹²⁵

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Table 2.

Interaction of SMADs with Transcription Coactivators and Corepressors

Cofactor	Family	Interacting SMADs	Function	References
Coactivators

E1A binding protein p300 (EP300)/CREBBP	chromatin-modifying enzymes	SMAD2, SMAD3, SMAD4	remodel/open up chromatin by histone acetylation	126, 127, 128
Lysine acetyltransferase 2B (KAT2B)	chromatin-modifying enzymes	SMAD3	potentiate TGFB1/SMAD3-mediated transcriptional responses independently or in cooperation with p300/phosphoprotein membrane anchor with glycosphingolipid (PAG1)	¹²⁹
Lysine acetyltransferase 2A (KAT2A)	chromatin-modifying enzymes	SMAD2 SMAD3	remodel chromatin and enhance transcriptional activity in response to TGFB1 and BMP stimulation	¹³⁰
CREBBP/p300 interacting transactivator with Glu/Asp rich carboxy-terminal domain 1 (CITED1)	member of CITED family of proteins	SMAD4	promote CREBBP/p300-SMAD4 interaction and enhance SMAD4-mediated transcriptional activation	¹³¹^,¹³²

Corepressors

Histone deacetylase 1 (HDAC1)	member of HDAC superfamily	SMAD 2	interact with SMAD2 via TGIF1 for the repression of TGFB1-responsive genes	¹²⁴^,¹²⁵
SKI proto-oncogene (SKI)	SKI transcriptional corepressors	SMAD2, SMAD3, SMAD4	block the interaction of SMAD2 with binding cofactors	¹³³^,¹³⁴
SKI like proto-oncogene (SKIL)	SKI transcriptional corepressors	SMAD2, SMAD4	recruit nuclear receptor corepressor 1 (NCoR1) and participate in the negative feedback regulation of TGFB1 signaling	¹³⁵
E1A binding protein p300 (EP300)	chromatin-modifying enzymes	SMAD3	inhibit the interaction between SMAD3 and CREBBP/p300 coactivators	¹³⁶
MDS1 and EVI1 complex locus (MECOM)	zinc fingers CH2H2-type	SMAD3	repress TGFB1 signaling by inhibiting the transcriptional activity of SMAD3	¹³⁷^,¹³⁸
SMAD nuclear interacting protein 1 (SNIP1)	spliceosomal P complex	SMAD4	bind to SMAD4 and p300 which inhibits the formation of functional SMAD4/p300 complex	¹³⁹

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SMAD-Independent Signal Transduction

Although TGFB1/INHBA homodimer/Nodal mainly signals through a SMAD-dependent mechanism, increasing evidence suggests that a more complex network that incorporates non-SMAD proteins, acting independently or crosstalking with the SMAD-dependent branch, regulates the biological activities of those ligands. In a SMAD-independent manner, binding of TGFB1/INHBA homodimer/Nodal to its cognate receptors activates various SMAD-independent signaling pathways directly or indirectly via adaptor proteins, including mitogen-activated protein kinase 1/2 (MAPK1/2),¹⁴⁰^,¹⁴¹ mitogen-activated protein kinase kinase kinase 7 (MAP3K7), an upstream signaling molecule that regulates TGFB1-mediated activation of the mitogen-activated protein kinase 8 (MAPK8)¹⁴² and mitogen-activated protein kinase 14 (MAPK14)¹⁴³ pathways, phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)/AKT serine/threonine kinase 1 (AKT1),144, 145, 146, 147 ras homolog family member A (RhoA),¹⁴⁸^,¹⁴⁹ and PTPA.¹⁵⁰

The regulation of both branches, SMAD dependent and SMAD independent, to one another is extensive, but also diverse, involving direct and indirect mechanisms of regulation, and is dependent on the physiological context. For instance,the SMAD-independent PIK3CA/AKT1 pathway could be directly stimulated by TGFB1 through AKT1 phosphorylation, which in turn mediates TGFB1-induced epithelial-mesenchymal transition (EMT) through the stimulation of AKT1 downstream effectors such as mechanistic target of rapamycin kinase (MTOR), eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1), and ribosomal protein S6 kinase B1 (RPS6KB1), leading to an increase in cell size and invasion.¹⁵¹ Moreover, TGFB1 promotes prostate cancer cell migration by indirectly activating PIK3CA/AKT1 signaling through the ubiquitylation of phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) by the tumor necrosis factor receptor associated factor 6 (TRAF6).¹⁵² On the other hand, TGFB1 mediates its apoptotic effects in hematopoietic cells through the downregulation of PIK3CA/AKT1 signaling through SMAD-induced transcription of inositol polyphosphate-5-phosphatase D (INPP5D), leading to the inhibition of AKT1 phosphorylation.¹⁵³

Non-SMAD proteins could also modulate the transcriptional activity of SMAD proteins, most often negatively, in a cell type-dependent context. Multiple sources of evidence show that the activation of MAPK1/2,154, 155, 156, 157 MAPK14,158, 159, 160 and MAPK8¹⁶¹^,¹⁶² inhibit the nuclear translocation of pSMAD2/3 through the phosphorylation of serine and threonine residues within the linker region, which impairs SMADs-mediated signal transduction. TGFB1-mediated cytostatic and proapoptotic effects are antagonized by the PIK3CA/AKT1 pathway, where the transcriptional activity of SMAD3 is inhibited upon its interaction with AKT1.¹⁶³^,¹⁶⁴ Furthermore, AKT1 phosphorylates the forkhead box O1 (FOXO1) transcription factor, which prevents the formation of SMAD-FOXO1 transcriptional complex, inducer of cyclin-dependent kinase inhibitor 2B (CDKN2B), and cyclin-dependent kinase inhibitor 1A (CDKN1A) expression in response to TGFB1 stimulation,¹⁰⁷ impairing the growth inhibitory effects of TGFB1.¹⁶⁵

TGFB1/INHBA Homodimer/Nodal Signaling in PCSCs

While mounting evidence demonstrates the important role of TGFB1/INHBA homodimer/Nodal signaling pathways in the maintenance of pluripotency of human embryonic stem cells (hESCs), mainly by regulating the expression of pluripotency transcription factors such as Nanog,¹⁶⁶^,¹⁶⁷ the mechanism(s) by which TGFB1/INHBA homodimer/Nodal signaling maintains the stem cell-like characteristics of PCSCs are not fully understood.

INHBA homodimer/Nodal signaling is inactive in adult tissue cells⁴⁷^,¹⁶⁸ but functionally operating in PCSCs expressing pluripotency-associated genes such as NANOG, POU5F1, SOX2, STAT3, and KLF4. In addition, mRNA expression levels of components of INHBA homodimer/Nodal signaling are significantly higher in PCSCs as compared with non-stem cancer cells (NSCCs). In PROM1⁺ PCSCs, NODAL, INHBA, TDGF1, TDGF1P3, and ACVR1B were found to be overexpressed as compared with PROM1⁻ cells. Functionally, recombinant Nodal and to a lesser extent recombinant INHBA homodimer (in short-term experiments) promoted the self-renewal capacity of A6L and 185-derived PCSCs, which was impaired by recombinant left-right determination factor 1 (LEFTY1; a specific inhibitor of Nodal signaling), and SB431542 (inhibitor of TGFBR1, ACVR1B, and ACVR1C), but neither TGFBR2 neutralizing antibody nor LY2157299, a TGFBR1 inhibitor, affected the sphere-forming ability of PCSCs.⁴⁴ However, other studies showed that TGFB1 increased the proportion of ATP binding cassette subfamily G member 2 (ABCG2)-expressing MIA PaCa-2-derived PCSCs and enhanced their sphere-forming capacity,¹⁶⁹ and also increased the percentage of PANC1-derived CD44⁺CD24⁺ PCSCs,¹⁷⁰ suggesting that the self-renewal promoting effects of TGFB1 are cell type-dependent. While the mechanisms underlying the biological effects of TGFB1/INHBA homodimer/Nodal in PCSCs remain largely unknown, one study reported that the self-renewal effects of INHBA homodimer are mediated by the T-box transcription factor 3 (TBX3), a downstream effector of INHBA homodimer signaling, through an autocrine Nodal/INHBA homodimer-TBX3-Nodal/INHBA homodimer feedback loop.¹⁷¹

In addition to the maintenance of the stemness features, the invasiveness of PCSCs is largely dependent on the activity of TGFB1, INHBA homodimer, and Nodal; suppressing the activities of INHBA homodimer and Nodal by blocking ACVR1B and that of TGFB1 by blocking TGFBR1 impairs the invasive potential of PCSCs.⁴⁴ In side population (SP) cells isolated from pancreatic cancer cell lines, TGFB1 induces an EMT phenotype, which enhances the invasiveness and in vivo metastatic activity of cells as compared with the main population (MP) cells. Interestingly, the cells reacquire their epithelial phenotype through a mesenchymal-epithelial transition following the removal of TGFB1 stimulus, suggesting that the plasticity of PCSCs to switch from an epithelial to a mesenchymal phenotype and vice versa is largely TGFB1 dependent. In addition, TGFB1 alters the mRNA expression levels of epithelial and mesenchymal markers, significantly reducing the expression of CDH1 and inducing the expression of SNAI1 and MMP2.¹⁷²

The inherent chemoresistant profile of PCSCs is one of the key drivers of tumor relapse post-chemotherapy and treatment failure,¹³^,¹⁴ where the acquisition of a stem cell-like phenotype and development of chemoresistance are closely associated.¹⁷³ PCSCs are also enriched by chemotherapy, which only induces the death of NSCCs.¹⁷⁴ Several studies demonstrated the involvement of TGFB1/INHBA homodimer/Nodal signaling in regulating the properties of chemoresistant PCSCs. PROM1⁺ PCSCs, in which TGFB1/INHBA homodimer/Nodal pathways are functionally active, are resistant to the apoptotic effects of gemcitabine.¹⁴^,⁴⁴ Interestingly, cell death was observed upon the addition of SB431542, suggesting that by blocking the activity of TGFB1/INHBA homodimer/Nodal pathways, the self-renewal ability of PCSCs is impaired, reversibly driving the cells to a differentiated phenotype that is sensitive to the cytotoxic effects of chemotherapy.⁴⁴

TGFB1/INHBA Homodimer/Nodal Signaling in PDAC-Associated Stroma

Pancreatic Stellate Cells

Through a highly interconnected network, pancreatic cancer cells receive signals from the surrounding stromal components that support their maintenance and functions, one of which is the pancreatic stellate cells (PSCs).¹⁷⁵ PSCs represent one of the main components of the PDAC-associated stroma, which were first identified in murine pancreatic tissues as storage cells of vitamin A-containing lipid droplets, following hypervitaminosis, in the connective tissues and at the periphery of blood capillaries.¹⁷⁶ They were then observed in the periacinar regions, with long cytoplasmic projections extending toward the basolateral aspects of the acinar cells in normal human and rat pancreas,¹⁷⁷ after which they have been isolated and cultured from the rat pancreas for further characterization.¹⁷⁸

There are two known biological phenotypes of PSCs: quiescent and activated. Low numbers of quiescent PSCs are located interlobular and in the periacinar space of a normal pancreas¹⁷⁸^,¹⁷⁹ and are distinguished from normal fibroblasts by the presence of vitamin A-containing lipid droplets as well as expression of vimentin (VIM), desmin (DES), nestin (NES), synemin (SYNM), and the glial fibrillary acidic protein (GFAP).¹⁸⁰ In response to pancreatic injury or inflammation, cytokines and growth factors secreted by the injured cells transform quiescent PSCs to activated myofibroblast-like cells. Moreover, PSCs are activated in a paracrine manner by cytokines and growth factors secreted by pancreatic cancer cells,¹⁸¹^,¹⁸² pancreatic acinar cells, inflammatory cells, platelets, and endothelial cells, and by the PSCs themselves in an autocrine manner.178, 179, 180^,183, 184, 185 Activated PSCs are distinguished from their quiescent counterparts by the expression of survival of motor neuron 1, telomeric (SMN1; a selective marker for activated PSCs) and loss of vitamin A-containing lipid droplets. The typical features of activated PSCs are: high mitotic index and migration capacity, secretion of excessive amounts of ECM components such as collagen type I (composed of two pro-collagen type I alpha 1 chains encoded by COL1A1 gene and one pro-collagen type I alpha 2 chains encoded by COL1A2 gene) and fibronectin 1 (FN1) into the pancreatic stroma, as well as expression of cytokines and growth factors, including interleukin 1 beta (IL1B), IL6, tumor necrosis factor (TNF), TGFB1, and platelet-derived growth factor subunit B (PDGFB), chemokines, and cell adhesion molecules.¹⁸⁰^,¹⁸⁶^,¹⁸⁷

There is an increasing recognition of the distinct parallels between PSCs and PCSCs, one of which is the activity of TGFB1/INHBA homodimer/Nodal signaling pathways. In both cell types, TGFB1/INHBA homodimer/Nodal signaling contributes to the development of chemoresistance, although through different mechanisms. INHBA homodimer and Nodal are highly expressed in PSCs¹⁸⁸^,¹⁸⁹ and PCSCs.⁴⁴ In activated PSCs, TGFB1 propagates through SMAD3 to regulate the synthesis and deposition of collagen type I into the ECM by rescuing the proteolytic degradation of the reversion inducing cysteine-rich protein with kazal motifs (RECK), a membrane-anchored inhibitor of matrix metallopeptidase 9 (MMP9), which is post-transcriptionally processed in quiescent PSCs.¹⁹⁰ Extensive fibrosis mediated by PSCs within the TME impairs the effective delivery of therapeutic drugs to their target tumor cells191, 192, 193 and triggers intratumoral hypoxia, which results in a more chemoresistant phenotype by promoting EMT and genetic instability of cancer cells.194, 195, 196 In PCSCs, TGFB1/INHBA homodimer/Nodal signaling enhances their tumor-initiating potential and resistance to chemotherapy; however, the mechanism remains unclear and requires further investigation.⁴⁴

PSCs also create a supportive paracrine niche for PCSCs. Secretion of INHBA homodimer/Nodal by PSCs significantly enhances the self-renewal capacity and invasiveness of PCSCs in an ACVR1B-dependent manner.¹⁹⁷ PSCs also stimulate the expression of CSC-associated markers such as ABCG2, NES, and lin-28 homolog A (LIN28A) in PCSCs, enhancing their self-renewal capacity and in vivo tumorigenicity. On the other hand, PCSCs regulate the expression of INHBA homodimer/Nodal in PSCs; in vitro culturing of PSCs in a medium conditioned by PROM1⁺ PCSCs for 24 h was associated with a slight increase of Nodal expression but a significant increase of INHBA homodimer expression in PSCs.¹⁹⁸

Tumor-Associated Macrophages

Among all types of immune cells, macrophages are the most abundant immune-related stromal cells in the PDAC microenvironment and hence are commonly referred to as the tumor-associated macrophages (TAMs).¹⁹⁹ Macrophages represent an extremely plastic type of immune cells that tend to display multiple phenotypes in response to external environmental stimuli such as infection, injury, and cancer. Based on their states of polarization, macrophages are classified into classically activated (M1), which are usually activated by interferon gamma (IFNG) and ligands of the Toll like receptors (TLR), or alternatively activated (M2), which are commonly activated by IL4 and IL13.²⁰⁰^,²⁰¹ In general, TAMs share more phenotypes and features that closely resemble the M2 macrophages; however, there are several distinct populations of macrophages that share characteristics and phenotypes of both M1 and M2 subtypes.202, 203, 204, 205 Within the TME, TAMs support immunosuppressive206, 207, 208 and pro-tumorigenic functions, including cell proliferation, angiogenesis, tumor invasion, and metastasis.²⁰²

In PDAC, a high density of TAMs is frequently associated with poor prognosis and low survival rates due to their high capabilities of promoting tumor growth, invasion, metastasis, and chemoresistance.209, 210, 211 TAMs are known to secrete TGFB1 into the TME, which contributes to the resistance of PDAC cells to gemcitabine (Figure 3). Upregulation of the growth factor independent 1 transcriptional repressor (GFI1) by simvastatin reduces the secretion of TGFB1 by TAMs, which sensitizes PDAC cells to gemcitabine.²¹² Consistently, combined inhibition of TGFB1 and colony stimulating factor 2 (CSF2) in pancreatic cancer cells improves the chemotherapeutic effects of gemcitabine by blocking the activity of M2-polarized TAMs and inducing the cytotoxic effects of CD8A⁺ T cells.²¹³ TGFB1 signaling in PDAC also promotes fibrosis and immunosuppression, leading to tumor progression.²¹⁴ In addition, TAMs have been reported to drive self-renewal and tumorigenicity of CSCs in various types of cancer,²¹⁵ including pancreatic cancer.²¹⁶ A recent study by Zhang et al.²¹⁷ shows that integrin subunit alpha V (ITGAV), overexpressed in macrophages, stimulates the secretion of TGFB1 by macrophages and enhances the stemness of pancreatic cancer cells through the TGFB1-SMAD2/3 signaling axis. In addition, a study conducted by Yin et al.²¹⁸ shows that exosomal miR-501-3p derived from M2-polarized TAMs promotes PDAC progression by stimulating TGFB1 signaling. Moreover, the expression of vasohibin 1 (VASH1), an intrinsic angiogenesis inhibitor, is negatively regulated by TGFB1/BMP signaling between TAMs and pancreatic cancer cells, resulting in enhanced angiogenesis.²¹⁹

Stromal Components of the PDAC Microenvironment Enhance the Stem Cell-Like Features, Tumorigenicty, and Chemoresistance of PCSCs through TGFB1/INHBA Homodimer/Nodal

Secretion of TGFB1 by TAMs and INHBA homodimer/Nodal by PSCs activates TGFB1/INHBA homodimer/Nodal signaling cascade in PCSCs, which maintains their stem cell-like properties and enhances their tumorigenicity and resistance to chemotherapy, leading to tumor recurrence and therapy failure. TGFB1-SMAD3 signaling in PSCs also contributes to chemoresistance by regulating the synthesis and deposition of collagen type I into the ECM, leading to extensive fibrosis, which impairs effective intratumoral delivery of therapeutic drugs. In response to activation by INHBA homodimer/Nodal secreted by PCSCs, TAMs secrete LL-37/hCAP-18 peptide, which drives self-renewal of PCSCs and promotes their tumorigenicity.

In response to activation by INHBA homodimer/Nodal secreted by PCSCs into the PDAC-associated stroma, TAMs secrete LL-37/hCAP-18, a peptide with broad antimicrobial activities, which upregulates the expression of pluripotency-associated genes, promotes the self-renewal capacity of PCSCs, and enhances their tumorigenic and invasive potentials through formyl peptide receptor 2 (FPR2)- and purinergic receptor P2X 7 (P2RX7)-dependent mechanisms (Figure 3).²²⁰ Nodal is also involved in the phenotypic transformation of macrophages, promoting the polarization of macrophages to the M2/TAM phenotype.²²¹

Therapeutics Targeting TGFB1/INHBA Homodimer/Nodal Signaling in PDAC

Several therapeutics/drug candidates targeting the TGFB1 signaling pathway in PDAC have been evaluated through preclinical studies and clinical trials. Therapeutics acting on the ligand level involve the use of antisense RNA molecules to decrease TGFB1 synthesis. Trabedersen (AP 12009), a phosphorothioate antisense oligodeoxynucleotide specific for human TGFB2 mRNA, reduced the secretion of TGFB2 in human pancreatic cancer cell lines, which was associated with a significant decrease in cell proliferation and inhibition of cell migration in vitro. In addition, trabedersen attenuated tumor growth, lymph node metastasis, and angiogenesis in an orthotopic mouse model of metastatic pancreatic cancer.²²² In a phase I/II clinical trial performed on patients with advanced pancreatic cancer, the drug was safe, well-tolerated, and showed promising survival data, leading to an active-controlled study on stage IV pancreatic cancer patients to be receiving trabedersen as a second-line treatment (Oettle, H., et al., 2012, J. Clin. Oncol., abstract).

Targeting TGFB1 signaling on the ligand-receptor level has also proved to be an efficient strategy for inhibiting signal induction. Blocking the responsiveness of the cell to TGFB1 using soluble TGFBR2 suppressed pancreatic cancer cell metastasis and also decreased the expression of serpin family E member 1 (SERPINE1) and the metastasis-associated plasminogen activator urokinase (PLAU) in an orthotopic mouse model.²²³^,²²⁴ SB431542 sensitized pancreatic cancer cells to the apoptotic effects of gemcitabine by impairing the self-renewal capacity of PCSCs and reversibly driving the cell to a differentiated phenotype that is more responsive to chemotherapy.⁴⁴ However, pancreatic tumor xenografts in immunodeficient mice were resistant to SB431542 and gemcitabine as a result of the highly desmoplastic tumor-associated stroma, which impaired intratumoral drug delivery. Targeting the PDAC stroma using a hedgehog inhibitor, CUR199691, significantly improved the delivery of both SB431542 and gemcitabine, which led to the suppression of tumor growth.¹⁷⁶ However, future clinical studies are required to validate preclinical data in mice, especially that hedgehog inhibitors have not proved to be as successful in humans. In phase IB/randomized phase II study, no significant differences were observed in median progression-free survival or overall survival in patients receiving a combined treatment of GDC-0449, a small molecule inhibitor of the hedgehog pathway, and gemcitabine versus gemcitabine monotherapy.²²⁵ In addition, efficacy of gemcitabine was not enhanced by IPI-926 in a phase II clinical study.²²⁶ To conclude, effective targeting of TGFB1/INHBA homodimer/Nodal signaling in PDAC involves depleting the PDAC-associated stroma, which requires the clinical evaluation of various stroma-targeting agents as an adjunct treatment to improve drug delivery and optimize the response of PDAC cells to TGFB1/INHBA homodimer/Nodal-targeted therapies.

Most of the drug inhibitors interfering with the intracellular signaling transduction in response to TGFB1 stimulation target the kinase domain of TGFB1 receptors. SD-208 is an ATP-competitive inhibitor of TGFBR1 kinase, which suppresses the phosphorylation of SMAD2 and expression of TGFB1-responsive genes regulating tumor growth, metastasis, and angiogenesis. In an orthotopic mouse model of PDAC, SD-208 suppressed tumor growth and metastasis²²⁷ and limited the extent of fibrosis in the surrounding stroma.²²⁸ SD-093 is also a selective inhibitor of TGFBR1 kinase, which has been shown to significantly limit the motility and invasiveness of SMAD4-deficient PDAC cells in vitro.²²⁹ In an orthotopic mouse model of metastatic pancreatic cancer and experimental model of liver metastasis, LY2109761, a dual inhibitor of TGFBR1/2 kinase, significantly inhibited tumor growth, prolonged median survival duration, and suppressed spontaneous abdominal metastases when used in combination with gemcitabine.²³⁰ However, LY2157299 (also known as galunisertib) has been the first selective inhibitor of TGFBR1 kinase to be evaluated in a clinical setting.²³¹ In patients with unresectable pancreatic tumors, galunisertib combined with gemcitabine improved overall survival compared with gemcitabine monotherapy (8.9 months for galunisertib-gemcitabine versus 7.1 months for placebo-gemcitabine; hazard ratio, 0.79; 95% confidence interval, 0.59–1.09).²³² In a phase II, double blind study, treatment of PDAC patients with a combination of galunisertib and gemcitabine showed an improved overall survival compared with gemcitabine and placebo (10.9 versus 7.2 months) (Melisi, D., et al., 2017, J. Clin. Oncol., abstract). Furthermore, in a phase I clinical study involving Japanese patients with advanced pancreatic and lung cancers, galunisertib demonstrated an acceptable tolerability and safety profile.²³³ However, another study showed that the inhibition of TGFB1 signaling by SB431542 and galunisertib was associated with a significant increase in pancreatic cancer cell invasion enhanced by hepatocyte growth factor (HGF), an invasion-promoting factor, which is secreted as a result of the loss of the HGF-suppressing activity of cancer cell-derived TGFB1,²³⁴ suggesting that the pleiotropic effects of TGFB1 signaling in various cell types could sometimes interfere with the pharmacological effects of therapeutics targeting the pathway. In a study conducted by Gore et al.²³⁵, combined targeting of TGFBR1 and EGFR/erb-b2 receptor tyrosine kinase 2 (ERBB2) using galunisertib and lapatinib, respectively, attenuated tumor growth, lymphangiogenesis, angiogenesis, and metastasis in orthotopic mouse models of pancreatic cancer. Recent preliminary data of a phase I clinical study of galunisertib combined with durvalumab, a CD274 inhibitor, demonstrated the safety and acceptable tolerability of the combination therapy in refractory cases of pancreatic cancer. Adverse effects included grade 3 elevation of hepatic enzymes and grade 3 neutropenia. One partial response (PR) and 7/32 stable disease responses (SD) were observed, giving a 25% (8/32 patients) disease control rate (Melisi et al., 2019, J. Clin. Oncol., abstract). Furthermore, naringenin (Nar, 4′, 5, 7-trihydroxy flavanone), a natural predominant flavanone, has been shown to inhibit SMAD3-induced transcription of TGFB1-responsive genes in pancreatic cancer cells, leading to the suppression of cell migration and invasion, while increasing the sensitivity of cells to gemcitabine.²³⁶ To conclude, these findings accentuate the pivotal role of TGFB1/INHBA homodimer/Nodal signaling in driving the tumorigenicity and chemoresistance of pancreatic cancer cells; however, it should be taken into account that patients with TGFB1/INHBA homodimer/Nodal-dependent pancreatic tumors are the most likely to benefit from TGFB1/INHBA homodimer/Nodal-targeted therapies.

SMAD4 Mutations and Targeting TGFB1/INHBA Homodimer/Nodal Signaling

As mentioned earlier, the canonical pathway by which TGFB1/INHBA homodimer/Nodal mediate their tumorigenic effects is mainly regulated by the transcriptional activity of SMAD2/3/4 complex. SMAD4 gene mutations represent one of the major genetic alterations in pancreatic cancer, where about 50% of pancreatic carcinomas harbor inactivating mutations or deletions of the SMAD4 gene, usually occurring at a late stage of tumor progression.²³⁷ PDAC tumors bearing inactivating SMAD4 mutations or deletions might not be good candidates for therapies targeting TGFB1/INHBA homodimer/Nodal signaling due to the loss of the transcriptional activity of SMADs. Experimentally, knockdown of SMAD4 in pancreatic cancer cells with wild-type SMAD4 inhibited the activity of INHBA homodimer/Nodal signaling and limited in vivo tumorigenicity. However, some PDAC tumors with SMAD4 mutations still demonstrated functional SMAD2/3 signaling cascade and were found to be responsive to SB431542, but the mechanisms involved remain unclear.⁴⁴ Thus, an effective clinical application of TGFB1/INHBA homodimer/Nodal-targeted therapies requires further mechanistic studies that would enable us to understand how SMAD4 is involved in regulating the activity of TGFB1/INHBA homodimer/Nodal signaling and determining treatment response.

Concluding Remarks and Future Perspectives

PDAC is one of the most lethal malignancies, with grim prognosis and alarmingly increasing mortality rates. The conventional therapeutic approaches, including radio- and chemotherapy, are challenged by a highly heterogeneous type of malignancy that is supported by the surrounding TME. On that account, an alternative therapeutic framework that targets different cellular compartments of PDAC should be considered to achieve complete tumor elimination and prevent disease recurrence. Chemotherapeutic drugs induce apoptosis of the majority of pancreatic tumor cells. This, however, leads to a selection process of the inherently chemoresistant PCSCs, which then self-renew and differentiate, giving rise to a new tumor mass that mimics the heterogeneity of the primary tumor. Multiple strands of evidence highlight the critical role of TGFB1/INHBA homodimer/Nodal in driving self-renewal of PCSCs and enhancing their tumorigenicity and resistance to chemotherapy, which render them as potential therapeutic targets in PDAC. Nevertheless, the mechanisms by which TGFB1/INHBA homodimer/Nodal signaling regulates various tumorigenic functions in PDAC remain largely unknown and require further investigation that could ultimately identify molecular targets leading to more efficacious drug treatments.

Acknowledgments

This work in the laboratory of S.P. was supported by a Cancer Research UK Career Development Fellowship, grant ID C59392/A25064.

Author Contributions

M.A.M. wrote and revised the manuscript. S.P. contributed to the revision of the manuscript and approved the final version for publication.

Declaration of Interests

The authors declare no competing interests.

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PERMALINK

TGFB1/INHBA Homodimer/Nodal-SMAD2/3 Signaling Network: A Pivotal Molecular Target in PDAC Treatment

Mai Abdel Mouti

Siim Pauklin

Abstract

Graphical Abstract

Main Text

Stemness of Pancreatic Cancer Cells

Figure 1.

Activation of TGFB1/INHBA Homodimer/Nodal Pathways

Initiation of Signal Transduction by Activated Receptors

SMAD-Dependent Signal Transduction

Mechanism of Signal Transduction

Figure 2.

Shuttling of SMADs

Nucleocytoplasmic Trafficking of SMADs

Interactions of SMADs in the Nucleus

Table 1.

Table 2.

SMAD-Independent Signal Transduction

TGFB1/INHBA Homodimer/Nodal Signaling in PCSCs

TGFB1/INHBA Homodimer/Nodal Signaling in PDAC-Associated Stroma

Pancreatic Stellate Cells

Tumor-Associated Macrophages

Figure 3.

Therapeutics Targeting TGFB1/INHBA Homodimer/Nodal Signaling in PDAC

SMAD4 Mutations and Targeting TGFB1/INHBA Homodimer/Nodal Signaling

Concluding Remarks and Future Perspectives

Acknowledgments

Author Contributions

Declaration of Interests

References

ACTIONS

PERMALINK

RESOURCES

Cite

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PERMALINK

TGFB1/INHBA Homodimer/Nodal-SMAD2/3 Signaling Network: A Pivotal Molecular Target in PDAC Treatment

Mai Abdel Mouti

Siim Pauklin

Abstract

Graphical Abstract

Main Text

Stemness of Pancreatic Cancer Cells

Figure 1.

Activation of TGFB1/INHBA Homodimer/Nodal Pathways

Initiation of Signal Transduction by Activated Receptors

SMAD-Dependent Signal Transduction

Mechanism of Signal Transduction

Figure 2.

Shuttling of SMADs

Nucleocytoplasmic Trafficking of SMADs

Interactions of SMADs in the Nucleus

Table 1.

Table 2.

SMAD-Independent Signal Transduction

TGFB1/INHBA Homodimer/Nodal Signaling in PCSCs

TGFB1/INHBA Homodimer/Nodal Signaling in PDAC-Associated Stroma

Pancreatic Stellate Cells

Tumor-Associated Macrophages

Figure 3.

Therapeutics Targeting TGFB1/INHBA Homodimer/Nodal Signaling in PDAC

SMAD4 Mutations and Targeting TGFB1/INHBA Homodimer/Nodal Signaling

Concluding Remarks and Future Perspectives

Acknowledgments

Author Contributions

Declaration of Interests

References

ACTIONS

PERMALINK

RESOURCES

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