Introduction Overview of Cancer

One of the global challenges we face today is cancer. It is because cancer is causing suffering on a global scale by affecting people of all ages. Despite the advances in science and technology, the projections of cancer cases are still increasing. According to an estimate made by the International Agency for Research on Cancer (IARC), there are expected to be around 26 million new cancer cases and 17 million deaths due to cancer globally by 2030.1 In cancer, cell growth is uncontrolled and it spreads through different routes throughout the entire body with a process referred to as metastasis.2 The basic hallmarks of cancer are progression through activation of invasion and metastasis processes, sustained proliferative signaling, stimulation of angiogenesis, evasion of growth suppressors, permitting replicative immorality, and resistance to cell death.3 Genetic and environmental factors cause cancer. Genetic factors involve a spectrum of somatic and germline mutations affecting different aspects of cell survival and viability processes. In contrast, environmental factors trigger these mutations by various chemical or physical agents.4 In addition to that, aging is a vital and critical risk factor in identifying and managing cancer during its initial stages.5 Due to aging, there is a decline in tissue health and intracellular communication by which various signals are ignored. In this way, tumors are formed because the cells ignore the signal transduction process. Among the myriad of molecular mechanisms inducing cancer progression, the Transforming Growth Factor-beta (TGF-β) signaling pathway stands out as a significant player, exhibiting dual roles in both tumor suppression and promotion of cancer, depending on the type and stage of the disease.6 The TGF-β conflicting effects based on the tumor stage continue to cast doubt on its involvement in cancer. In this review article, we intend to explore the signaling pathways involved in cancer and specifically examine TGF-β role in cancer treatment, highlighting various clinical trials and combination therapies.

TGF-β in Normal Physiology

Biological signals in multicellular organisms serve as crucial coordination and communication pathways, essential for these organisms’ proper functioning, growth, and development.7 These signals are essential for the proper regulation and homeostasis of living organisms. TGF-β family comprises pleiotropic cytokines and signaling molecules with diverse roles across the animal kingdom. While cytokines typically mediate cell communication and immune responses, TGF-β carries out a diverse range of biological processes in both embryonic and adult stages of life. This includes differentiation, wound healing, proliferation, and regulation of cell and tissue-specific motility.8

TGF-β consists of a family of ligands including TGF-βI, TGF-βII, and TGF-βIII.9 These ligands are TGF-β isoforms and are closely related to bone morphogenic proteins (BMPs), activins and various growth and differentiation factors (GDFS). These factors are soluble and have tissue-specific effects. They interact with cell membrane receptor complexes when they are activated, which results in activating cellular responses. The fundamental role of TGF-β lies in maintaining cell and tissue homeostasis through multiple levels of regulated signal transduction. Examples of this regulation include extracellular antagonists, co-receptor molecules, and intracellular regulators.10 These regulators have garnered significant interest from cancer biologists due to their pivotal roles in critical biological and cellular processes such as embryonic development, cytoskeletal organization, cellular homeostasis and tissue regeneration.11 Disruptions in the TGF-β signaling pathway result in a wide array of pathological issues including cancer, fibrosis and immune diseases.12 The intricacy of TGF-β signaling is underscored by its activation through multiple mechanisms. Integrins can mediate TGF-β activation by interacting with latent TGF-β complexes, facilitating their conversion to the active form. Acids and bases can alter the local microenvironment, promoting conformational shifts and releasing active TGF-β. Reactive oxygen species (ROS) can cause oxidative modifications, impacting the availability and activity of TGF-β. Thrombospondin-1 (TSP-1) which acts as a significant modulator by binding to latent TGF-β and activating it. Proteases, such as matrix metalloproteinases, can cleave latent TGF-β complexes, releasing the active cytokine.8 TGF-β signaling pathway plays an intrinsic role in physiological processes. These processes are as follows:

Cell Growth and Proliferation

The contribution of TGF-β signaling in cell growth and proliferation is multifaceted, while also having significant impacts on cell cycle. TGF-β induces cytostasis, which either upregulates or downregulates cell proliferation in accordance with the cellular context, ultimately causing cell cycle arrest.13 This arrest is mediated through two primary mechanisms: (i) regulation of cell cycle inhibitors and (ii) downregulation of c-Myc protein. First, the expression of cell cycle inhibitors, such as p15, p21, and p27, is upregulated by TGF-β signaling.8 These inhibitors bind to cyclin-dependent kinases (CDKs) and inactivate them. These enzymes are crucial and drive the progression of the cell cycle from the G1 phase to the S phase. The CDK inhibition ultimately interrupts the cell cycle at the G1 phase, preventing further division of cells. Secondly, c-Myc protein expression is decreased by TGF-β signaling. c-Myc is a potent transcription factor promoting cell cycle progression by driving the gene expression of those required for DNA synthesis and cell division.14 By downregulating c-Myc, TGF-β ensures that cells do not proliferate unchecked. Additionally, the TGF-β pathway also plays diverse roles in various cell types. For instance, studies have demonstrated its association with β cell proliferation and development, highlighting the pathway’s crucial function in maintaining cellular homeostasis and hindering uncontrolled cell growth, which is characteristic of cancerous tissues. The dual regulatory nature of TGF-β of promoting cytostasis and controlling cell cycle progression underscores its importance in both normal physiological processes and the pathogenesis of diseases, ie, cancer.15

Differentiation

TGF-β signaling plays a crucial part in cell differentiation and specialization by shaping the growth and development of numerous cells through intricate molecular mechanisms. One of its significant functions is the conversion of mesenchymal cell differentiation into myofibroblasts. This process involves the activation of SMAD-dependent (canonical) and SMAD-independent (non-canonical) signaling pathways, which regulate gene expression associated with myofibroblast markers, ie, α-smooth muscle actin (α-SMA).16,17 Myofibroblasts are essential for tissue healing and fibrosis, contributing to wound contraction and extracellular matrix deposition. Additionally, TGF-β signaling influences the differentiation of precursor cells into chondrocytes and osteoblasts, which are cartilage and bone-forming cells, respectively. In osteoblast differentiation, TGF-β activates the SMAD pathway, leading to the transcription of Runx2 which acts as a critical transcription factor for osteoblastogenesis.18 Concurrently, TGF-β regulates Sox9 expression, another transcription factor vital for chondrocyte differentiation and cartilage formation. These pathways ensure the proper growth and maintenance of skeletal tissues.

TGF-β also promotes the development and differentiation of immune cells (T and B cells). In the context of T cells, TGF-β signaling is pivotal in inducing regulatory T-cells (Tregs). It does so by upregulating Foxp3 expression which is a transcriptional factor necessary for the formation and proper functioning of Treg.19 Tregs are involved in maintaining immunological tolerance and preventing autoimmune responses. Similarly, TGF-β influences B cell differentiation into regulatory B cells (Bregs) and certain plasma cell types.20 Bregs, characterized by the expression of IL-10, contribute to immune homeostasis by suppressing inflammatory responses.21 Furthermore, TGF-β signaling is involved in mucosal immunity, where it supports the development of IgA-secreting plasma cells, essential for mucosal defense.22 The precise regulation of these differentiation processes by TGF-β ensures a balanced immune response and maintenance of tissue integrity, highlighting its multifaceted role in cellular differentiation and specialization.

Immune Regulation

TGF-β also has an impact on the regulation of the immune system through its potent immunosuppressive effects, mediated by intricate molecular mechanisms. As a central immunosuppressive pathway, TGF-β signaling modulates the activity of various immune cells, ie, T and B-lymphocytes and natural killer (NK) cells. It also regulates T-lymphocytes by promoting the differentiation of Tregs, essential for maintaining immunological tolerance and preventing autoimmune responses. This process involves the activation of SMAD2/3 signaling pathway, which facilitates the transcription of Foxp3 gene referred to as a master regulator of Treg development and function.19 TGF-β also inhibits the rapid division and effector functions of conventional T-helper cells and cytotoxic T-lymphocytes by interrupting the pro-inflammatory cytokines (IL-2 and IFN-γ) expression, thus curbing excessive immune activation.21

In B-lymphocytes, TGF-β influences differentiation into regulatory B-cells (Bregs), producing anti-inflammatory cytokines, ie, IL-10 and TGF-β itself, contributing to the suppression of inflammatory responses and promoting immune tolerance.21 TGF-β’s impact on NK cells, crucial elements of innate immune system, involves downregulating their cytotoxic activity and cytokine production. NK cells are vital for identifying and destroying virally infected and malignant cells; however, TGF-β signaling diminishes their effectiveness by altering their receptor expression and reducing their cytotoxic potential.23 Moreover, TGF-β signaling decreases the pro-inflammatory cytokines (TNF-α and IL-6) production, thereby attenuating overall immune responses and promoting tolerance to self-antigens. Through these multifaceted actions, TGF-β maintains immune homeostasis, preventing autoimmunity and ensuring appropriate immune responses while facilitating tissue repair and regeneration.

Extracellular Matrix Production and Remodelling

TGF-β signaling is a major regulatory pathway responsible for the production and remodeling of extracellular matrix (ECM), playing a fundamental role in tissue and cell homeostasis and repair. This pathway influences the synthesis and organization of ECM’s different components, ie, collagen, fibulins, fibronectin and proteoglycans.24 TGF-β imposes its effects through both canonical and non-canonical signaling pathways.

Canonical pathway involves TGF-β binding to TGF-β type II receptor (TGF-βRII), which cause activation and phosphorylation of TGF-β type I receptor (TGF-βRI). When TGF-βRI activates, it phosphorylates receptor-regulated SMADs (R-SMADs), primarily SMAD2 and SMAD3. These phosphorylated SMADSs form a complex by binding with the common mediator SMAD4, which migrates to the nucleus where it regulates the transcription of the target genes. The promoter regions of genes encoding ECM proteins are directly binded with this complex, ie, collagen types I and III, proteoglycans and fibronectin, enhancing their transcription and subsequent protein production.25

TGF-β in the non-canonical pathways activates other signaling cascades such as MAPK (ERK, JNK, p38), Rho-like GTPase and PI3K/AKT pathways. These pathways further contribute to ECM regulation by modulating cellular responses, ie, migration, differentiation and proliferation, which are crucial for ECM remodeling. For instance, ERK pathway activation can lead to the phosphorylation of transcription factors like AP-1, which as a result enhances ECM genes expression.26 Moreover, TGF-β signaling modulates the activity of various transcriptional co-regulators. For instance, TGF-β-induced SMAD complexes can form interaction with co-activators such as CBP/p300, which possess histone acetyltransferase (HAT) activity, facilitating chromatin remodeling and transcriptional activation of ECM-related genes.27 On the other hand, TGF-β also activates co-repressors, ie, SnoN and Ski, which inhibit SMAD-mediated transcription, providing a fine-tuned regulatory mechanism for ECM gene expression.28

TGF-β also promotes the production of matrix metalloproteinases (MMPs) enzymes responsible for ECM degradation and tissue inhibitors of metalloproteinases (TIMPs) which inhibit MMP activity. The balance between MMPs and TIMPs is crucial for controlled ECM remodeling, allowing for tissue repair and maintenance without excessive fibrosis.29 TGF-β-induced expression of MMPs involves SMAD3-mediated transcriptional activation and interaction with transcription factors such as SP1. Conversely, TGF-β upregulates TIMPs through SMAD signaling pathways, ensuring a controlled environment for ECM turnover.

Another critical aspect of TGF-β-mediated ECM regulation is its influence on the integrin signaling. Transmembrane receptors like integrins also facilitate cell-ECM interactions and transmit signals from the ECM to the cell interior. TGF-β can enhance certain integrins expression, promoting cell adhesion and migration to ECM.30 This interaction is considered essential for ECM assembly and remodeling. Additionally, integrin-mediated activation of focal adhesion kinase (FAK) can synergize with TGF-β signaling to further regulate ECM gene expression and cellular responses. TGF-β also regulates the synthesis of connective tissue growth factor (CTGF), a downstream mediator that amplifies the effects of TGF-β on ECM production. CTGF expression is induced by SMAD-dependent pathways and plays a role in enhancing the deposition of ECM components and promoting fibroblast proliferation and differentiation.31

The enhanced expression of genes encoding ECM proteins leads to increased deposition and structural organization of the ECM, contributing to tissue strength and integrity. However, dysregulation in TGF-β signaling may result in pathological conditions characterized by excessive ECM deposition, such as fibrosis, or insufficient ECM production, such as in certain degenerative diseases. Therefore, TGF-β signaling is pivotal in maintaining dynamic balance of ECM production and remodeling, essential for normal tissue function and response to injury.

Wound Healing

In the case of wound healing process and various cellular activities, the TGF-β pathway also plays an integral role. TGF-β is released by platelets at initial stages of wound healing, which leads to the development of inflammatory cells at the wound site.32 It promotes cells, ie, fibroblasts and keratinocytes movement and proliferation. These cells are essential for the tissue repair process. It also enhances the production of granulation and angiogenesis of tissues which increases the ECM production and further helps in wound healing activity. This can help in the development of advanced therapeutic methods for wound healing.

Angiogenesis

Angiogenesis, a process involving the formation of new blood vessels from pre-existing ones, is critical in development, wound healing and disease. TGF-β exerts a significant part in regulating angiogenesis through its complex signaling pathways modulating the behavior of endothelial cells. Depending on the context and interacting molecules, TGF-β can either inhibit or promote angiogenesis, making its role highly versatile and context dependent.

In the context of promoting angiogenesis, TGF-β signaling induces pro-angiogenic factor expression, most notably vascular endothelial growth factor (VEGF). VEGF is a powerful stimulator in the endothelial cell proliferation and migration which are essential steps in new blood vessel formation. This can be achieved through SMAD-dependent and SMAD-independent pathway activation where it activates transcription of VEGF and other angiogenic genes in the nuclease.33 This non-canonical signaling pathway is involved in the fine-tuning of angiogenic responses by modulating the stability and translation of VEGF mRNA. Additionally, TGF-β signaling can stimulate the production of other angiogenic factors, ie, fibroblast growth factor (FGF) and platelet-derived growth factor (PDGF), which synergistically induce endothelial cell proliferation and new vessel formation.34 TGF-β also contributes to the maturation and stabilization of newly formed blood vessels. It regulates the integrins and other adhesion molecule expression on endothelial cells, facilitating their interaction with ECM and pericytes.35

This interaction is crucial for structural integrity and functional maturation of blood vessels. For instance, TGF-β induces integrin αvβ3 expression, which further enhances endothelial cell adhesion and migration of ECM components, thereby supporting the formation of stable vascular structures.36

Interestingly, TGF-β’s role in angiogenesis is context-dependent and can also be inhibitory. In certain settings, particularly in the case of high levels of TGF-β or in conjunction with other signaling molecules, TGF-β can induce anti-angiogenic responses. This involves the upregulation of angiogenesis inhibitors, ie, thrombospondin-1 (TSP-1) and the suppression of VEGF signaling.37 The TGF-β inhibitory effects are mediated via SMAD-independent pathways, such as activation of the p38 MAPK pathway, which can enhance the expression of anti-angiogenic genes and suppression of endothelial cell growth, proliferation and migration.

Furthermore, TGF-β signaling influences the recruitment and differentiation of mesenchymal stem cells (MSCs) and pericytes, which are essential for vessel stabilization and the prevention of excessive angiogenesis. By promoting MSCs differentiation into pericytes and smooth muscle cells, TGF-β aids in the structural support and functional regulation of newly formed vessels, ensuring proper vascular remodeling and homeostasis.38 In the case of pathological conditions for instance cancer, the dual role of TGF-β in angiogenesis becomes particularly evident. Tumors exploit the pro-angiogenic properties of TGF-β in promoting vascularization and sustaining their growth and metastasis. On the other hand, therapeutic strategies targeting TGF-β signaling aim to disrupt its pro-angiogenic effects and inhibit tumor angiogenesis.

Development and Differentiation in Embryogenesis

Another crucial function of TGF-β signaling pathway is its role in growth, development and differentiation during embryogenesis. Embryogenesis, the process beginning with the fertilization of an egg with sperm cell, involves a series of highly regulated events leading to the formation of new organs and tissues. TGF-β signaling has been instrumental in orchestrating these events, ensuring the proper organization, differentiation, and maintenance of cells in the developing embryo.8

During early embryogenesis, TGF-β signaling holds a significant role in the formation of the 3 primary germ layers: (i) ectoderm (ii) endoderm (iii) mesoderm. These layers form all tissues and organs in the body. TGF-β signaling is pivotal in inducing mesoderm formation, a process facilitated by Nodal activation, a member of TGF-β superfamily.39 Nodal signaling, in conjunction with SMAD2/3, regulates mesodermal markers, ie, goosecoid and brachyury expression, driving the differentiation of mesodermal progenitors. In addition to mesoderm induction, TGF-β signaling is essential in neural differentiation from ectoderm. The balance between TGF-β and BMPs is crucial for neural development. BMP signaling promotes epidermal fate, while TGF-β signaling via BMP pathway inhibition, favors neural differentiation by inducing neural-specific transcription factors, ie, Sox2 and Neurogenin.40 TGF-β signaling also provides a significant role in endodermal differentiation, influencing the development of internal organs such as liver, pancreas and lungs. By regulating transcription factor expression like Sox17 and Foxa2, TGF-β signaling ensures proper formation and patterning of endodermal tissues.41 Furthermore, TGF-β signaling regulates epithelial-to-mesenchymal transition (EMT) process critical for the formation of various tissues and organs. EMT is regulated by TGF-β-induced transcription factors, ie, Snail, Slug and Twist, which repress epithelial markers and activate mesenchymal markers, facilitating tissue remodeling and organ development.42

TGF-β Signaling Pathways Synthesis of TGF-β

TGF-β is as a large, complex and inactive precursor protein synthesized in a rough endoplasmic reticulum (RER). It consists of a signal peptide which includes; (i) large N-terminal pro-domain referred as latency-associated peptide (LAP) preventing TGF-β activation,43 (ii) short mature peptide C-terminal domain.44,45 TGF-β and other members of this superfamily are synthesized in dimer form. The pro-domain then assembles into a homodimer via two disulphide bonds linking LAP portions, while the mature TGF-β moieties interact by single disulphide bond46 and form a small latent complex (SLC) by binding non-covalently with LAP. A proteolytic cleavage side is located between pro and mature domains. The bond between pro-domain LAP and short domain is cleaved with the help of convertase furin located in trans-Golgi.47,48 The LAP proteins then enfold mature domain which form small latent complex (SLC) by non-covalent bonds and protect the binding of mature TGF-β with its receptors. SLC forms a large, inactive complex as it interacts by with latent TGF-β binding molecule (LTBP) by disulphide bond which is a glycoprotein that acts as TGF-β chaperone and mediates its folding and secretes it into extracellular matrix (ECM).

Activation of TGF-β

For binding with the receptor, the latent TGF-β needs to get activated. Different processes are observed to activate TGF-β. Latent TGF-β activation occurs when mature TGF-β portions are dissociated from the LAP portions. The cleavage may operate in both in vitro and in vivo. The in vitro cleavage includes heating of TGF-β with mild acid lowering the pH to 4.549–51 or by oxidative modification where reactive oxygen species (ROS) cause loss of ability in LAP to bind with mature TGF-β.52–54 The in vivo cleavage includes the proteolytic cleavage of LAP via various ECM serine proteases, ie, plasmin, Leucine-rich repeat consisting protein 33 (LRRC33), matrix metalloproteinases (MMPs), ie, MMP9 and MMP14, Cathepsin D and thrombospondin-1 (TSP-1) release the active TGF-β.49,55–58 Additionally, LTBP can be associated with LAP by covalent bonding forming a large latent complex (LLC) and deposits SLC in the ECM.48,59–61 LLC then covalently binds with a particular ECM protein (fibrillin and fibronectin) through LTBP in a large N-terminal domain.59,62,63 Furthermore, LTBP is associated with glycoprotein A repetition predominant protein (GARP), a transmembrane protein of various cells, ie, regulatory T (Treg), endothelial and platelets which activate latent TGF-β.64,65 Epithelial restricted integrins, which are cell adhesion receptor proteins, play a part in invasion, proliferation and survival migration of cells while also activating latent TGF-β.66–68 Integrins comprises heterodimeric α, β subunits (αvβ6, αvβ8) which are known as transmembrane receptors type I and found in a variety of different cells.66 It has been observed that some integrins also bind to Arg-Gly-Asp (RGD) which is a motif of LAP and generates a mechanical force that deforms the structure of LLP and undergoes cellular contractions that releases active TGF-β.47,69–72 The active TGF-β half-life is faster than that of latent TGF-β and if its receptor is absent, then it can be cleared rapidly from the ECM.73 Once latent TGF-β is activated, it controls the timing and location of TGF-β signaling.

Canonical and Non-Canonical TGF-β Signaling Pathways

TGF-β is a versatile, pleiotropic, multifunctional cytokine belonging to a superfamily having ubiquitous cell growth factors such as activins, Bone Morphogenic Proteins (BMPs), inhibitions and anti-Mullerian hormone,51 expressed in mammals in three isoforms: TGF-βI, II, and III. TGF-βI is considered to be the most abundant and ubiquitously expressed in humans among all, and all three isoforms show 75% of homology via the same receptor complex. TGF-β undergoes transmission of signals through canonical or non-canonical pathways as demonstrated in Figure 1.74

Figure 1 This figure illustrates Canonical (SMAD dependent) and Non-canonical (SMAD independent) TGF-β signaling pathways.11,75

Abbreviations: P13K, Phosphoinositide 3-kinase; mTOR, Mammalian target of rapamycin; S6K, ribosomal protein S6 kinase; PaR6, Partitioning-defective protein 6; TAK1, Transforming growth factor β-activated kinase 1; P, Phosphorus; P38MAPK, Mitogen-activated protein kinases; JNK, C-Jun N-terminal kinase; RAS, Rat sarcoma; RAF, Rapidly accelerated fibrosarcoma; RHO, Rho-associated coiled-coil forming kinase; C-MYC, Myelocytomatosis oncogene cellular homolog; ERK1/2, Extracellular-signal-regulated kinase; LIMK, LIM kinases; EMT, Epithelial–mesenchymal transition; SMAD-R, Receptor-regulated SMADs.

TGF-β receptor complex, a tetramer that is comprised of two paired transmembrane serine/threonine protein kinases; 2 TβRIs (ALK 1) and 2 TβRIIs.76,77 Betaglycan is the third type of TGF-β receptor (TβRIII) which is a low affinity, non-signaling, co-receptor abundant on different cell surfaces binding TGF-β ligands to high-affinity TGF-β receptor complex.78,79 In the case of canonical TGF-β signaling pathways, initially active TGF-β ligands bind with TGF-β receptor type II (TβRII).79 It can cause phosphorylation and recruitment in TGF-β receptor type I (TβRI). According to a recent whole exome sequencing (WES) study, TβRII has been observed in 16 most commonly mutated genes in the case of pancreatic cancer.80 The TGF-β ligand binding and recruitment trigger TβRII that results in the kinase activation that trans-phosphorylates specific serine/threonine residues of TβRI located in GS domain and intracellular juxta-membrane region consisting of serine and glycine residues.81 Active TβRI undergoes intracellular signaling with the help of SMADs, proteins transferring signals from TGF-β receptors present on the cell membrane to the nucleus. SMADs have been classified into 3 categories; (i) receptor regulated R-SMADs, (ii) common SMADs, and (iii) inhibitory SMADs. The activated TβRI or activing type I receptors phosphorylates R-SMADs family member 2 (SMAD 2) or 3 (SMAD3) at their two carboxyl-terminal serine residues.82 BMP type I receptors, on the other hand, phosphorylate SMAD 1, 5 and 8.83 After phosphorylation, SMAD2/3 dissociates from TβRI and undergo oligomerization of SMAD2 or SMAD3 with SMAD family member 4 (SMAD4), the only known common partner SMAD forming a complex. The heteromeric complex SMAD2/3-SMAD4 results in nuclear translocation84 where it is associated with different transcriptional factors regulating the transcriptional repression or activation of target genes highlighted in Table 1.85–89

Table 1 The Table Illustrates the TGF-β Pathway Target Genes for Tumour Promotion and Suppression

Canonical signaling carries out the modulation by various mechanism feedback. For instance, TGF-β induces SMAD6 and SMAD7 expression for a negative regulator in TGF-β/SMAD signaling pathway. SMAD7 protein inhibits TGF-β signaling by undergoing various mechanisms, ie, interacting with TβRI and blocking the interactions and phosphorylation between SMAD2/3 and activated TGF-β receptors.111 Moreover, SMAD7 also inhibits SMAD2-SMAD4 complex formation and its nuclear translocation112,113 along with the interruption in SMAD-DNA complex formation inhibiting TGF-β signalling.114 SMAD ubiquitination regulatory factor 1 (Smurf-1) and E3 ubiquitin ligases also aids in TGF-β signaling regulation due to proteasomal degradation of TβRI.116 Adaptor protein, ie, SMAD anchor for receptor activation (SARA), microtubules and embryonic liver fodrin (ELF) also mediate SMAD’s interaction with TβRI necessary for signaling.117

Apart from canonical pathways, TGF-β also activates different intracellular non-canonical (SMAD-independent) signalling pathways in certain type of cells by TGF-β receptor activation.118 In non-canonical signalling pathways, the regulation of actin cytoskeleton changes leading to cell motility, adhesion and growth takes place via Rhodopsin (Rho) like GTPase pathway,119 cell migration and tight junctions via PAR6 regulators,120 cell proliferation, survival and metastasis via Extracellular Signal-Regulated Kinases (ERK)/Mitogen Activated Protein Kinases (MAPK) and Phosphatidylinositol-3 Kinase (PI3K)/Akt signaling,121–123 cell migration via the RHO/ROCK pathway124 and immune evasion, cell survival and inflammation via NF-Κb pathway. These pathways can directly affect the R-SMADs activity. For example, in the ERK signaling pathway, SMAD2/3 is activated via phosphorylation, whereas SMAD3 is sequestered in the cytoplasm for its regulation in the case of the AKT pathway. TGF-β signaling can be activated in many known human cancer types; hence, it is considered an active research topic.

Mucin-1 (MUC1) known as a Type 1 transmembrane glycoprotein, is an oncogene that plays a fundamental role in the modulation of TGF-β signaling in cancer progression and metastasis.125–127 MUC1 in normal conditions is restricted to the apical surface of epithelial cells where it serves as a protective barrier.128 Meanwhile, in the case of malignant cells, MUC1 does not remain localized to the apical surface; instead, its glycosylation reduces and becomes hypo-glycosylated and causes overexpression of proteins across the cell surface interacting with various growth factor receptors including TGF-β receptors.127

In different cancers, tumor-associated MUC1 is overexpressed to enhance EMT, a critical process for cancer metastasis resulting in enhanced drug resistance, metastasis and invasiveness, particularly of EMT-inducing genes.125,129–131 As TGF-β induces EMT, MUC1 interacts with TGF-β signaling pathways to regulate its function. Unlike the MUC1 extracellular domain, which acts as a ligand for different receptors, ie, cell adhesion receptors, cytoplasmic tail of MUC1 (MUC1-CT) causes oncogenic signal transduction by undergoing phosphorylation, which serves in cell invasiveness and metastasis.132–134 Once phosphorylated, it gets released from MUC1 N-terminus and binds with β-catenin along with other transcription factors, resulting in the translocation towards nucleus where it undergoes downstream signaling pathways, ie, PI3K/AKT and MAPK pathways.135 MUC1-CT is 72 amino acid long, highly conserved domain with seven tyrosine residues phosphorylated by intracellular tyrosine kinases, ie, c-Src, a proto-oncogene molecule having a role in cancer progression.125,136,137

TGF-β/AP-1 Signaling Axis in Cancer Progression

At the core of the shift between TGF-β dual roles, is its interaction with the Activator Protein-1 (AP-1) transcription factor complex, which includes key proteins like c-Fos and c-Jun. This partnership between TGF-β and AP-1 orchestrates numerous downstream effects, driving processes like cell survival, proliferation, invasion, and metastasis. The multifaceted influence of TGF-β and AP-1 on gene expression is critical to the cellular and micro-environmental transformations that define aggressive cancer phenotypes.

TGF-β signals through both canonical (SMAD-dependent) and non-canonical (SMAD-independent) pathways. In the canonical pathway, the translocation of SMAD4 to the nucleus collaborates with AP-1 components like c-Fos and c-Jun, allowing it to regulate genes involved in cellular functions such as growth and differentiation.138 This TGF-β/AP-1 collaboration influences genes that modulate ECM production and degradation, promoting invasive behaviors that facilitate cancer cell migration through tissue barriers. In the non-canonical pathway, TGF-β activates AP-1 through other signaling cascades, such as the MAPK, JNK, and PI3K/AKT pathways. For instance, the JNK pathway enhances AP-1 activity by directly phosphorylating c-Jun, which supports gene expression related to stress responses, cellular motility, and invasion.139 Meanwhile, the PI3K/AKT pathway promotes apoptosis resistance by stabilizing anti-apoptotic proteins. Through these pathways, AP-1 contributes to cancer cell survival and adaptation, enhancing their resilience to treatments that typically induce cell death.140

One of the critical roles of TGF-β/AP-1 signaling in cancer is its promotion of epithelial-to-mesenchymal transition (EMT), a process that enables cancer cells to become more migratory and invasive.141 During EMT, cells lose epithelial characteristics like cell–cell adhesion and gain mesenchymal traits such as motility, which are essential for metastatic spread. TGF-β signaling upregulates EMT-related transcription factors (eg, Snail, Slug, and Twist), often in coordination with AP-1.142,143 This combined effect represses epithelial markers like E-cadherin and enhances mesenchymal markers such as N-cadherin and vimentin, which reduce cellular adhesion and support migration. Additionally, non-canonical signaling via the JNK pathway enhances AP-1’s ability to orchestrate cytoskeletal changes and ECM degradation, facilitating the structural alterations necessary for cancer cell dissemination.144 In addition, the non-canonical pathways of TGF-β/AP-1 signaling axis are also instrumental in fostering drug resistance. AP-1 can upregulate genes related to drug efflux, DNA repair, and stress response, enabling cancer cells to resist chemotherapy and other targeted treatments.145

Beyond acting on cancer cells directly, TGF-β and AP-1 modify the tumor microenvironment to favor malignancy. AP-1 regulates ECM-remodeling enzymes, such as matrix metalloproteinases (MMPs), which facilitate tissue breakdown and invasion.142,146 This ECM remodeling, enhanced by TGF-β-driven AP-1 activity, enables cancer cells to breach physical barriers, supporting their spread to distant organs. Additionally, AP-1 mediates the expression of pro-inflammatory cytokines like IL-6 and TNF-α, promoting a chronic inflammatory environment that nurtures cancer progression.147 TGF-β’s activation of AP-1 in this context supports angiogenesis, immune evasion, and additional ECM remodeling, creating a microenvironment conducive to cancer cell survival and adaptation. This inflammatory milieu also supplies cancer cells with growth signals, sustaining tumor expansion and enhancing the resilience of the tumor against therapies. Thus, TGF-β enables AP-1 to coordinate gene expression patterns that promote aggressive cancer traits. This dual influence on cellular and microenvironmental factors highlights the importance of the TGF-β/AP-1 axis as a therapeutic target, especially for slowing tumor growth and enhancing cancer cell susceptibility to treatment.

Cross Talk of TGF-β Signaling Pathway with Other Pathways

TGF-β signaling pathways cross talk with various other intrinsic complex networks, which is a perennial topic in TGF-β study.148 This cross talk can enhance the understanding of TGF-β role in mediating different biological responses, its effect on cellular physiology and its role in therapeutics. The cross talk can take place at different levels including ligands, receptor, antagonists and signaling component expression level. These components associate with transcription complexes, induce chromatin modifications, change gene expression and directly interact with SMADs.149 At early developmental stage, the interactions of TGF-β with BMP, Hedgehog (Hh), Wnt/ Wg, MAPK, Notch and other pathways play a role in cell fate, organogenesis, body configuration and maintenance150,151 as highlighted in Figure 2.

Figure 2 This figure represents the TGF-β signaling pathway cross talk with other related signaling pathways.

Abbreviations: FAK, Focal Adhesion Kinase; NICD, Notch intracellular domain; SMO, Smoothened; FGFs, Fibroblast growth factors; HGF, Hepatocyte growth factor; EGF, Epidermal growth factor; FZDs, Frizzled receptors; LRP, Low-density lipoprotein receptor-related protein; APC, Adenomatous polyposis coli; mTOR, Mammalian target of rapamycin.

Cross Talk with Wnt Signaling Pathway

Wnt signaling pathway involves secreted, lipid-modified signaling molecules responsible for regulating tissue homeostasis, cell fate, migration, survival, self-renewal, and the maintenance of early progenitor and stem cells.152 Dysregulation in the Wnt pathway is implicated in different cancers, including leukemia and colorectal cancer, where it can lead to aberrant cellular processes.153

The canonical Wnt signaling pathway is initiated by Wnt ligands binding to the Frizzled (Fz) receptor along with LRP5/6 co-receptor. This interaction triggers a signaling cascade and is mediated by β-catenin which is a crucial transcriptional co-activator. Upon ligand binding, the intracellular protein Dishevelled (Dvl) becomes activated and inhibits β-catenin destruction complex, which includes Axin, APC (Adenomatous Polyposis Coli), and GSK-3 (Glycogen Synthase Kinase 3). Inhibition of GSK-3 activity prevents the phosphorylation and subsequent degradation of β-catenin, resulting in its accumulation and stabilization in the cytoplasm. Stabilized β-catenin then translocates to nucleus, where it further binds to T-cell factor (TCF) or lymphoid enhancer-binding factor (LEF) transcription factors. This complex recruits co-activators such as CREB-binding protein (CBP) and p300 to drive the expression of Wnt target genes, promoting cell proliferation, differentiation and self-renewal. Hyperactivation of this pathway is a key driver of oncogenesis in various cancers.153

The cross talk between TGF-β/BMP and Wnt signaling pathways has been extensively studied, revealing interactions at multiple levels that regulate crucial cellular processes ranging from early development to post-natal tissue homeostasis.154,155 First, TGF-β/BMP and Wnt signaling reciprocally regulate their respective ligand production. Second, these pathways interact in the nucleus, where SMAD proteins (mediators of TGF-β signaling pathway) can create complexes with β-catenin and LEF/TCF transcription factors, co-regulating a shared set of target genes and modulating transcriptional activity. Third, cytoplasmic interactions between these pathways are also significant. For example, in Xenopus, SMAD4 has been shown to associate with β-catenin in the context of Spemann’s organizer, influencing early developmental processes.156 Additionally, β-catenin signaling is activated by TGF-β via GSK-3β inactivation, further integrating these pathways.125

In the context of cancer, the interaction between TGF-β/Wnt signaling pathways has a pivotal role in promoting metastasis, particularly through their collective influence on epithelial-to-mesenchymal transition (EMT), a process in cancer progression and metastasis. This cross talk not only enhances the invasiveness of cancer cells rather it also contributes in maintaining stem cell-like properties, facilitating tumor spreading and recurrence.

Cross Talk with PI3K/ Akt Signaling Pathway

The PI3K/Akt signaling pathway can show a crucial role in regulating multiple cellular and physiological processes, ie, cell proliferation, invasion, growth, and survival.126 Phosphoinositide 3-kinases (PI3Ks) are among the family of lipid kinases and exist in heterodimeric forms and classified into three classes (I, II, and III) based on their (a) structure, (b) distribution, (c) phospholipid substrate specificity, (d) regulatory mechanisms.127

PI3K/Akt pathway activation is triggered by various growth factors, cytokines, and cellular stressors through G-protein-coupled receptors (GPCRs) or multiple receptor tyrosine kinases (RTKs). Once it gets activated, PI3K carries out the conversion of PIP2 (phosphatidylinositol 4.5-bisphosphate) into PIP3 (phosphatidylinositol 3,4,5-trisphosphate), a critical second messenger that recruits Akt (also referred as protein kinase B) to the plasma membrane. Akt is then phosphorylated and activated by phosphoinositide-dependent kinase 1 (PDK1) and the mTORC2 complex. Activated Akt regulates numerous downstream targets that are involved in cell survival, metabolism and growth. PI3K/Akt pathway is negatively regulated by the lipid phosphatase PTEN (phosphatase and tensin homolog) dephosphorylating PIP3 back to PIP2, thus acting as a tumor suppressor by inhibiting this pathway.128

In many cancers, hyper-activation of PI3K/Akt pathway has been noticed, often due to the mutations or loss of function in PTEN, causing continuous cell growth and survival. The interaction between TGF-β/PI3K/Akt pathway causes additional complexity to cellular regulation. PI3K can be directly or indirectly activated by TGF-β receptors, leading to the activation of PI3K/Akt pathway. This cross talk influences cell fate and self-renewal by upregulating Nanog expression, a key transcription factor responsible for maintaining stem cell pluripotency.151 Moreover, the PI3K/Akt pathway can modulate TGF-β signaling. For instance, Akt can phosphorylate and inhibit SMAD3, TGF-β pathway key mediator, thereby preventing TGF-β-induced apoptosis in hepatocytes.129 Akt can also phosphorylate transcription factor FoxO, which undergoes interaction with SMAD3 and inhibits its nuclear translocation, blocking the transcriptional expression of pro-apoptotic genes.130 This interaction between TGF-β/PI3K/Akt pathways can promote epithelial–mesenchymal transition (EMT), a critical procedure in cancer progression that enhances cell migration, metastasis and invasion.149

The cross talk between these pathways also modulates the tumor microenvironment. For example, SMAD-dependent TGF-β signaling can interact with p38 MAPK and PI3K/Akt pathways to activate PFKFB3, an enzyme that drives glycolysis, thus supporting the metabolic demands of rapidly proliferating cancer cells. Conversely, in normal murine mammary gland epithelial cells, the interaction between these pathways can lead to the activation of connexin 43 expression, which is associated with cell–cell communication and homeostasis.131,132

The intricate interaction of TGF-β with PI3K/Akt signaling pathways highlights their combined functions in regulating key cellular processes, including cell survival and differentiation along with cancer progression. This cross talk not only promotes oncogenic processes such as EMT and metastasis but also affects the metabolic adaptation of tumor cells, contributing to their growth and survival in a hostile microenvironment.

Cross Talk with NF-Kb/Rel Signaling Pathway

NF-κB/Rel signaling pathway is a crucial regulatory network in cellular processes, ie, cell adhesion, senescence, proliferation and survival. NF-κB/Rel proteins function as dimeric transcription factors and bind to specific DNA sequences present in the nucleus, including the enhancer region of the κ-light chain of the immunoglobulin family. NF-κB family is classified into two subfamilies; (i) “NF-κB” proteins (p50/NF-κB1 and p52/NF-κB2), (ii) “Rel” proteins (RelA/p65, c-Rel, and RelB).133 Dysregulation in NF-κB pathway has been linked with various diseases, including arthritis, cancer, cardiovascular diseases, chronic inflammation, asthma, and neurodegenerative disorders.134

In the non-canonical NF-κB pathway, the activation is mediated by a specific group of receptors, such as lymphotoxin-α/β or CD40L receptors. These receptors activate NF-κB-inducing kinase (NIK), which subsequently phosphorylates IKKα. Phosphorylated IKKα then phosphorylates the carboxy-terminal residues of NF-κB2 p100, leading to the activation of RelB. NF-κB2 p100/RelB complex translocates towards the nucleus regulating the expression of a distinct set of genes which is involved in immune responses and cell survival.134

The cross talk of TGF-β/NF-κB pathway is a significant area of study, as these pathways interact in various cellular contexts, particularly in cancer and immune responses. TGF-β activates NF-κB in a non-canonical manner in various cell types such as head and neck squamous cell carcinoma (HNSCC), osteoblasts, hepatocytes and murine B cells. This activation occurs through TGF-β-activated kinase 1 (TAK1), which is a crucial mediator in this cross talk. Upon TGF-β stimulation, TAK1 is activated which subsequently phosphorylates and activates IKK, resulting in the further activation of NF-κB. This interaction results in the nuclear translocation of NF-κB dimers, where they can influence gene expression related to inflammation, cell survival, and proliferation.135

In cancer, TGF-β/NF-κB pathways can have profound implications for tumor progression and metastasis. For instance, in the case of head and neck squamous cell carcinoma (HNSCC), the TGF-β-mediated activation of NF-κB adds to the aggressive behavior of these tumors by promoting cell survival and apoptosis resistance. Similarly, in the context of chronic inflammation, TGF-β/NF-κB signaling can cooperate to sustain a pro-inflammatory environment, contributing in the production and progression of cancer and other chronic diseases.

This cross talk highlights the intricate balance of TGF-β/NF-κB signaling in regulating immune responses and maintaining cellular homeostasis. Dysregulation of this interaction can lead to pathological conditions, including cancer, where the combined activity of these pathways promotes tumor growth, invasion, and therapy resistance. Understanding the molecular mechanisms which underlie this cross talk offers potential therapeutic targets for treating diseases which are relevant to aberrant TGF-β and NF-κB signaling.

Cross Talk with Hedgehog (Hh) Signaling Pathway

Hedgehog (Hh) signaling pathway is a highly conserved molecular mechanism having a fundamental role in numerous cellular functions, ie, embryonic development and regeneration of tissues. Aberrations occurred in Hh signaling can cause severe developmental defects and tumorigenesis, including the formation of basal cell carcinomas (BCCs) and medulloblastomas.136 In mammals, Hh pathway is mediated by three key proteins: (i) Indian Hedgehog (Ihh), (ii) Sonic Hedgehog (Shh), (iii) Desert Hedgehog (Dhh). The initiation takes place by the interaction of Hh ligands with their cell surface receptors; Smoothened (SMO) and Patched (PTCH1 or PTCH2) and controlled intracellularly by Gli (glioma-associated oncogene homolog) factors, specifically Gli 1.2 and 3.149 When Hh ligands are absent, PTCH 1 and 2 inhibit SMO activity, thereby preventing activation of Gli transcription factors. Upon binding of ligands of Hh to PTCH receptors, the inhibition is relieved, causing SMO to activate Gli, which then migrated to the nucleus regulating target gene expression involved in cell proliferation, survival and differentiation. TGF-β/Hh signaling pathways cross talk occurs on various levels, influencing various functions of cell, particularly during tumorigenesis and embryonic development.148 TGF-β signaling regulate Hh ligands expression and the activity of Gli transcription factors, thereby modulating the Hh pathway’s influence on cell cycle control and differentiation. For example, during embryogenesis, the expression of Shh and other Hh ligands is regulated by TGF-β, influencing the patterning and growth of developing tissues.137

One of the key interactions between these pathways involves SMAD3, a major mediator of TGF-β signaling, which interacts with Gli1, enhancing its transcriptional activity. This interaction promotes cell proliferation and survival, which allows to co-regulate cell cycle and differentiation by both TGF-β and Hh pathways. This co-regulation is particularly evident in developmental processes where TGF-β and Hh signaling cooperate in controlling lineage-specific differentiation and tissue patterning.

In some contexts, Hh/Gli proteins induce the expression of TGF-β signaling components, thereby establishing a feedback loop that further refines cellular responses during development and in disease states such as cancer. SMAD proteins also interact with Gli3, although the functional consequences of this interaction remain to be fully elucidated. However, it is known that in the developing cerebellum, Bone Morphogenetic Proteins (BMP-2 and BMP-4), which are part of TGF-β superfamily, can antagonize the proliferative effects of Sonic Hedgehog (Shh) by downregulating the expression of SMO and Gli1.157

Additionally, it has been seen that TGF-β inhibits Protein Kinase A (PKA) activity while constantly inducing the expression of Gli1 and Gli2, further modulating the Hh signaling pathway and its downstream effects. This intricate interplay between TGF-β and Hh signaling pathways highlights their combined roles in regulating essential developmental processes and in contributing to pathogenesis of a wide range of diseases, including cancer. Understanding the molecular mechanisms underlying this cross talk offers potential avenues for therapeutic intervention in conditions where these pathways are dysregulated.

Dysregulation of TGF-β Signaling in Cancer

TGF-β is a highly versatile signaling pathway that has a critical part in numerous biological and cellular processes, including the development and regulation of immune system and tissue homeostasis. However, any dysregulation in this pathway can lead to a variety of pathologies, particularly cancer. Different genetic and environmental factors can disrupt TGF-β signaling, leading to impaired cellular functions and contributing to tumorigenesis.158,159 One major mechanism of dysregulation in cancer involves genetic along with epigenetic alterations that affect TGF-β receptors, leading to their downregulation or loss of function.159 This disruption compromises tumor-suppressive effects in TGF-β signaling and facilitates cancer progression. For instance, in certain Mendelian diseases, mutations in TGF-β pathway components result in the impaired development and immune responses, highlighting the essential role of this pathway to maintain normal cellular functions.160 Gene defects affecting the ligands of TGF-β cytokine family often lead to specific phenotypes, while mutations in downstream signaling components can result in broader and more severe genetic defects. These defects, collectively termed as TGF-β signalopathies, are rare disorders that provide insights into the essential TGF-β signaling functions in the immune system and other biological processes. By understanding that these signalopathies have advanced the development of targeted therapies, it is aimed to correct TGF-β dysregulation with some therapies being safe and effective in clinical studies. In the context of cancer, TGF-β signaling dysregulation allows the tumor cells to evade immune detection and compromise the ability of immune system to fight against the tumor. This immune evasion occurs through two primary mechanisms: (i) immunosuppressive cells induction, ie, regulatory T cells (Tregs), (ii) myeloid-derived suppressor cells (MDSCs), and suppression of immune cell activation. Consequently, the tumor microenvironment can become immunosuppressive, facilitating tumor growth and metastasis.161

The consequences of TGF-β dysregulation in the case of cancer are profound. The pathway, which normally acts as a tumor suppressing pathway by promoting apoptosis and inhibiting cell proliferation, can become pro-tumorigenic when dysregulated. This switch occurs through several mechanisms: First, the loss of tumor-suppressive functions disrupts the ability of TGF-β to activate growth suppressors, leading to unchecked cell proliferation. This alteration often involves changes in both autocrine and paracrine signaling, which inhibit growth-inhibitory effects of TGF-β.162 Second, during cancer progression, TGF-β signaling pathway may interfere with SMAD proteins, leading to resistance to apoptosis.163 This resistance enhances the survival of cancer cell survival, contributing to tumor growth. Third, dysregulation of TGF-β signaling promotes pro-angiogenic factor production, increasing the tumor blood supply and facilitating its growth and metastasis.164 Fourth, TGF-β dysregulation enhances epithelial–mesenchymal transition (EMT), a process that increases invasiveness in cancer cells and their ability to metastasize to distant organs.42,165 Fifth, dysregulation in TGF-β signaling leads to cause defects in DNA repair mechanisms, increasing genomic instability and promoting cancer progression.166 Sixth, dysregulation in TGF-β signaling contributes in the creation of an inflammatory tumor microenvironment, further promoting tumor growth and survival.167 Lastly, TGF-β dysregulation can drive metabolic changes in cancer cells, such as altered lipid metabolism, increased glycolysis and enhanced oxidative phosphorylation.168 These metabolic shifts provide the energy and biosynthetic precursors necessary for rapid tumor growth. The dysregulation in TGF-β signaling is particularly prevalent in cancers including breast, pancreatic, and colorectal cancer, where it plays a central role in causing metastasis, tumorigenesis and resistance to therapy.169 Understanding the molecular mechanisms highlighting TGF-β signaling dysregulation in cancer is essential for developing novel therapeutic strategies that aim to restore tumor-suppressive functions of this pathway and combating cancer more effectively.

Dual Role of TGF-β in Cancer Progression

TGF-β has a dual role in cancer progression as shown in Figure 3.170 It has tumor-suppressive effects during early stages while during advanced stages, it promotes development of tumors in the cells.

Figure 3