Transforming growth factor‐β signalling in tumour resistance to the anti‐PD‐(L)1 therapy: Updated

Abstract Low frequency of durable responses in patients treated with immune checkpoint inhibitors (ICIs) demands for taking complementary strategies in order to boost immune responses against cancer. Transforming growth factor‐β (TGF‐β) is a multi‐tasking cytokine that is frequently expressed in tumours and acts as a critical promoter of tumour hallmarks. TGF‐β promotes an immunosuppressive tumour microenvironment (TME) and defines a bypass mechanism to the ICI therapy. A number of cells within the stroma of tumour are influenced from TGF‐β activity. There is also evidence of a relation between TGF‐β with programmed death‐ligand 1 (PD‐L1) expression within TME, and it influences the efficacy of anti‐programmed death‐1 receptor (PD‐1) or anti‐PD‐L1 therapy. Combination of TGF‐β inhibitors with anti‐PD(L)1 has come to the promising outcomes, and clinical trials are under way in order to use agents with bifunctional capacity and fusion proteins for bonding TGF‐β traps with anti‐PD‐L1 antibodies aiming at reinvigorating immune responses and promoting persistent responses against advanced stage cancers, especially tumours with immunologically cold ecosystem.


| INTRODUC TI ON
Immune checkpoint inhibitor (ICI) therapy using monoclonal antibodies against various checkpoints including programmed death-1 receptor (PD-1), programmed death-ligand 1 (PD-L1) or cytotoxic T lymphocyte associated antigen-4 (CTLA-4) is on the eye of current investigations. The strategy of application of ICIs has revolutionized the field of oncology, but clinical trials attested durable benefits just in a number of patients. Tumour cells take immune escape mechanisms in order to reduce the durability of response to immunotherapy. 1 Biomarker stratification is a useful tool for assessing ICI efficacy. Tumour mutational burden (TMB) and T-cell inflamed gene expression profile distinctly capture neoantigenicity and T-cell activation profile of cancer, so they have low correlation but representing joint predictive utility for identification of responders to immunotherapy. 2 Adenomas progressed towards colorectal cancer (CRC) are characterized by infiltration of immunosuppressive cells and their co-localization with tumour cells. 3 In fact, upregulation of immune checkpoints is presumably co-occurring with dysregulated cytokine profile in order to promote immunosuppression and tumour metastasis. 4 Transforming growth factorβ (TGFβ) release and PD-L1 upregulation are the two key contributing factors responsible for immune evasion and tumour aggression. TGFβ is a multi-tasking, 5 an anti-inflammatory 6 and a pro-fibrotic 7 cytokine that is frequently expressed in tumours 8 and acts as a key promoter of tumour resistance and metastasis. 9 High levels of TGFβ are characteristic of immunosuppressive tumour microenvironment (TME), 10 particularly in cancers like glioblastoma (GBM), 11 and are primary mechanism of immune escape in solid tumours. 12 TGFβ is, in fact, a tumour-intrinsic mechanism for evaluation of the functional tumour-immune state 13 and investigating responses to ICI therapy. Immune exclusion, noninflamed or cold immunity are terms referred to the activity of TGFβ. 14 A positive correlation is identified between high stromal TGFβ with weak prognosis and ICI resistance in lung cancer. 15 TGFβ activation, T-cell exclusion and low TMB are critical hallmarks of microsatellite stability CRC and are indicative of limited response to the ICI therapy. 12 In gastric cancer, a subtype with overexpression of genes related to the TGFβ/SMAD pathway also represents lower responses to the anti-PD-1 therapy. 16 Due to the importance of TGFβ in promoting key tumorigenic events, and its critical contribution to the tumour progression, therapy resistance and metastasis, we aimed to discuss about the impact of this cytokine in regulation of immune checkpoints with a particular focus over PD-(L)1 checkpoints, which is a hot topic of the current years in the area of cancer therapy. Mechanisms related to the TGFβ-mediated bypass of ICI therapy, and the current view over strategies to rescue tumour immunity from the impact of this cytokine in order to improve the efficacy of anti-PD-(L)1 are described in this paper.

| PROG R AMMED DE ATH RECEP TOR / LI G AND IN C AN CER IMMUNIT Y AND IMMUNOTHER APY
Checkpoint is regulator of immune responses. Allison and Honjo began ground-breaking investigations over cancer immunity and immunotherapy in the year 1992, and their discovery was honoured by the 2018 Nobel Prize in physiology and medicine. 17 Studies further expanded in the area to target a number of advanced stage solid cancers, and the primary results were promising. However, therapy bypass has been a considerable issue of the current years, and many researchers are trying to find ways to hamper this bypassing system and extending the durability of therapy, especially in tumours with cold immunity. Tumoral expression of PD-L1 is a biomarker associated with therapy response to the ICI therapy. 18 Single-cell transcriptome profiling of oesophageal squamous cell carcinoma (ESCC) showed the highest expression of PD-L1 on tumour dendritic cells (DCs), which is contributed to the T-cell anergy. 19 Anti-PD(L)1 is viewed as an immune normalization approach with higher responseto-toxicity profile. Immune normalization vs. immune enhancers is like a big pipeline. Enhancers increase the pressure within the pipeline in order to overcome deficiencies in drainage, but it has a risk of pipeline breakdown when the increased pressure rate is too high.
By contrast, the normalizer approach is aimed at restoring the normal flow within the pipeline without risking pipeline breakdown. In the TME, overexpression of PD-L1 causes overregulation of T cells.
Thus, identification of a particular defect, and developing strategies for selective repair of this deficiency and restoring immune competence without causing general immune activation can be a desired anti-tumour strategy. 20

| Transforming growth factorβ regulation
TGFβ has the high affinity to bind with the TGFβ receptor II (TGF-βRII), which further recruits TGF-βRI into a heterotetrameric complex. The complex initiates SMAD-related transcriptional repression or activation of genes contributed to the differentiation, growth and migration. 8 SMAD4 is the key mediator in the TGFβ pathway; upon activation of TGFβ, complexes of SMAD are formed through bondage of SMAD2 and SMAD3 with SMAD4. The TGFβ/SMAD network exerts complex biological activities. 21 TGFβ signalling is negatively regulated by SMAD7. In fact, SMAD7 restricts PD-1-induced regulatory T-cell (Treg) differentiation and limits responses from T cells to TGFβ, which further result in increased intestinal inflammation and progression of colitis. PDL1/2 + DCs are more developed when they are SMAD7 deficient, such cells induce CD4 + T cell-to-Treg differentiation and reduce the severity of colitis in mice. 22 In cancer, a diverse path is taken in which increased activity of TGFβ and dysregulation in the TGFβ/SMAD signalling is contributed to tumour initiation and progression in several human cancers. 23 Tregs suppress T-cell effector function and dampen anti-tumour immunity, which can be abrogated after blockade of surface TGFβ on Tregs. 24 Beside promotion of canonical SMAD pathway, TGFβ signalling can take tumour-promoting activities also through non-canonical pathways, mediated via interaction with intracellular pathways, such as phosphatidylinositol 3′-kinase (PI3K)/protein kinase B (AKT) and mitogen-activated protein kinase (MAPK). 25 TGFβ is released in an inactive (or latent) form into the TME.
Virtually, latent TGFβ is secreted from all immune cells, but only some cells are involved in the activation of this cytokine. 26 Bondage between TGFβ and latency-associated peptide (LAP) is dissociated upon interaction with integrin αvβ6 subunit expressed on cancer cells, which results in TGFβ activation for further reshaping cells within the stroma of tumour. 27 Targeting LAP stimulates anti-tumour immunity. 28 Expression of TGFβ-related LAP on surface of myeloid- against LAP in such cells slows tumour progression. 29 In DCs, the integrin αvβ8 subunit is contributed to the activation of latent TGFβ. TGF-β1 is also presented on surface of Tregs in an inactive form.
Here, TGF-β1 binds to the GARP. It is found that the membrane protein GARP is contributed to the generation of active TGF-β1 from Tregs, and antibodies to suppress GARP can block immunosuppressive capacity of Tregs and boost immune responses against infection or cancer. Combination of anti-GARP antibodies with anti-PD-(L)1 improves the efficacy of immunotherapy 26 (Figure 1).

| Transforming growth factorβ cross-talking within tumour microenvironment
TGFβ is secreted from tumour-associated macrophages (TAMs) 30 and is activated in tumour endothelial cells (ECs). TGFβ pathway is pivotal for transition of fibroblasts into myofibroblasts. 19 Cancer-associated fibroblasts (CAFs) are one of the critical components of TME that take diverse roles in tumour progression. 31 Co-option between CAFs with TAMs causes high secretion of TGFβ for hindering the effector activity of T cells and inducing generation of Tregs. 32 High TGFβ expression from pro-tumour type 2 macrophages (M2) also inhibits natural killer (NK) cell activity. 33 Tregs are the cell type abundantly presented in TME of solid tumours. 31 Induction of Tregs and inhibition of CD8 + T and Th1 cells by TGFβ are contributed to the immune dysfunction within TME. 8 Myeloid cells activate TGFβ to promote tumour metastasis. 34 TGFβ is placed upstream to the regulation of mammalian target of rapamycin (mTOR) complex 2 (mTORC2). The inducible effect of TGFβ on mTORC2 promotes invasion of bladder cancer cells 35 ; in NK cells, TGFβ represses mTOR pathway, which results in the suppression of NK cell activation and function 36 (Figure 2).

| CELLUL AR MED IATOR S OF RE S IS TAN CE REL ATED TO THE IMPAC T OF TR AN S FORMING G ROW TH FAC TOR-Β O N A N T I -P D -( L ) 1
TGFβ promotes resistance to the anti-PD-(L)1 through several mechanisms, a summary of which is presented in the Figure 3.

| CD8 + T-cell exclusion
Immunosuppressive signals, such as TGFβ hamper persistence of tumour-reactive T cells in solid tumours. 37 Anti-PD-(L)1 therapy is effective only in a number of patients with metastatic urothelial cancer. Resistance to the PD-L1 inhibitor atezolizumab is related to the CD8 + T-cell exclusion from tumour interior, instead shifting the cells towards tumour stroma. This is mediated through the inducible effect of TGFβ on CAFs for constructing a collagenous-rich stroma. 38 TGFβ is among cytokines contributed to the promotion of interactions between SPP1 + macrophages with FAP + fibroblasts. This interaction stimulates formation of desmoplastic structures that are immune excluded and restrict the infiltration of T cells. High expression of SPP1 or FAP in CRC patients is contributed to the low efficacy of anti-PD-L1 therapy. Conversely, disruption of interactions between the two cell types improve immunotherapy outcomes. 39 Pancreatic ductal adenocarcinoma (PDAC) displays high PD-L1 expression, major histocompatibility complex class I (MHC-I) sequestration, insufficient antigenicity, DC exclusion, and Treg and MDSC attraction, all of which are favouring highly efficient immune evasion profile of such cancer type. 40 PDAC is the best example of a desmoplastic tumour that secrets high amount of TGFβ. 41 In such cancer type, presence of CD8 + T cells near to the PDAC cells is indicative of higher overall survival. 42 Increasing intra-tumoral infiltration of cytotoxic or effector T cells sensitizes basal-like mesenchymal PDAC to the anti-PD-L1 therapy. 43 CD8 + T-cell exclusion is promoted by integrin αv-mediated TGFβ activation within TME. Targeting αv integrin on lung tumour cells increases the density of CD8 + T cells and improves the efficacy of anti-PD-1 therapy. 27 Tumoral infiltration of CD8 + T cells is affected from the activity of sympathetic nervous system (SNS), which has harmful and advantageous effects on tumour stroma. In liver cancer model, placing mice in an enrichment environment characterized by social interactions for making the animals happier resulted in a dramatic fall in the expression of cytokines, such as TGFβ. The environment enrichment also helped F I G U R E 1 Transforming growth factorβ (TGFβ) activation in cancer. TGFβ is released into the tumour microenvironment (TME) in an inactive form through binding to latencyassociated peptide (LAP). Interaction between the latent TGFβ with integrin αvβ6 (on cancer cells) or αvβ8 (on dendritic cells/DCs) is contributed to the activation of this cytokine, which acts on its receptor on TME cells, such as cancer-associated fibroblasts (CAFs). In regulatory T cells (Tregs), bondage between latent TGFβ with GRAP generates active TGFβ.
to overcome anti-PD-1 resistance. This is mediated through involvement of SNS/β-adrenergic receptors (β-ARs) signalling for silencing CCL2 and further promoting the infiltration of CD8 + T cells. 44

| Th17 cell stimulation
PD-1 + Th17 cells are the population of CD4 + T cells that highly express TGFβ. Co-culture of PD-1 + Th17 cells with fibroblasts induce production of collagen-1 and pulmonary fibrosis. 45 TGFβ is an inducer of Th17 lineage development. 46

| Myeloid-derived suppressor cell accumulation
Presence of myeloid cells is required for activation of PD-(L)1 and establishing an immunosuppressive TME in pancreatic cancer. 50 F I G U R E 2 Transforming growth factorβ (TGFβ)-mediated cross-talking within tumour microenvironment (TME). The activity of TGFβ is contributed to the progression of tumour through increasing the activity of pro-tumour cells and dampening the activity of anti-tumour cells within tumour ecosystem. M2, type 2 macrophage; Treg, regulatory T cell; CAF, cancer-associated fibroblast; MDSC, myeloid-derived suppressor cell; NK, natural killer; DC, dendritic cell; and Th1, T helper1.
MDSCs are poorly mature cells that are belonged to the innate immunity, and their accumulation in tumour area confers resistance to the ICI therapy. TGFβ is released from MDSCs to stimulate the activity of Tregs. 51 Single-cell RNA sequencing has shown the impact of S100A9 as a mediator for promoting cross-talk between tumour and stroma in metastatic tumours. 52 TGFβ activity is contributed to the cell surface translocation of S100A9 and its further secretion. S100A9-C-X-C chemokine ligand 12 (CXCL12) signalling in response to the breast cancer-associated gene 1 (BRCA1) deficiency induces MDSC accumulation in tumour area and promotes anti-PD-1 insensitivity. 53 Targeting CXCL12 can be used as an approach for rendering synergistic effects with anti-PD-L1 in pancreatic cancer. 54

| TR AN S FORMING G ROW TH FAC TOR-Β IN CELLUL AR IMMATURIT Y AND ANTI -PD (L)1 RE S P ON S E S
TGFβ and PD-L1 are mediators of immaturity in tumour ecosystem.
TGFβ stimulates inhibitor of differentiation 1 (Id1) that its activity further blocks DC maturation. 55 Under the influence of TGFβ, LAP is expressed on immature DCs and is contributed to the suppression of T-cell activation. 56  Epithelial-mesenchymal transition (EMT) is a required process during embryo development and at the time of tissue repair, but it confers as a feature of malignant profile 57 and a promoter of tumour aggression in cancer. 58 EMT is required for invasion of tumour cells, 59 but is repressed in the metastatic colonization step, indicated by the reduced EMT inducer Prrx1. 60 Zeb, 61 Snail and Slug (Snail2) are transcription factors related to the EMT profile in tumour cells. 62 The activity of TGFβ/SMAD is critical for promotion towards acquisition of EMT and metastasis. 21,63 TGFβ is released from TAMs, 30 and there is a positive cross-talk between TAMs with mesenchymallike tumour cells for promotion of metastasis of breast 64 and CRC cells. 65 Chronic exposure to the TGFβ promotes a stable EMT state in mammary carcinoma cells accompanied by stable generation of cancer stem cells (CSCs) and drug resistance that cannot be reversed after withdrawal of TGFβ, but it is responsive to the mTOR inhibition. 66 Beside tumour cells, CAFs also show a signature of EMT profile. 67 Metastatic renal cancer samples are enriched in TGFβ and EMT signalling. 59 ESCC is equipped with CST1 + myofibroblasts that are characterized by high EMT profile and TGFβ pathway. In such tumour type, interaction score for the CTLA4-CD80/86 and PD1-PDL1 (CD274) is higher in tumour samples compared with normal counterpart. 68 Zeb1 is required for expression of PD-L1 on invading lung cancer cells, 69 but there are tumours that show high EMT but low PD-L1 expression profile. Enriched TGFβ signalling and EMT is a characteristic of mesenchymal and basal-like 2 (BL2) subtypes of triple-negative breast cancer (TNBC). The mesenchymal subtype shows low expression of PD-L1 along with suppression of MHC-I and displays resistance to many compounds, but represents a level of sensitivity to the TGF-βRI suppressor SB-505124. 70 Dual targeting of TGFβ/PD-L1 is presumed to hamper EMT/stemness, tumour aggression and resistance. 71 Whether it is also applicable in tumours under chronic stimulation with TGFβ is an open question, requiring further illustrations.

| TR AN S FORMING G ROW TH FAC TOR-Β IN TUMOUR ME TABOLIS M , HYP OXIA AND CHECK P OINT REG U L ATI ON
Metabolic reprogramming is a characteristic hallmark of solid tumours. 72  Hypoxia is a state of O 2 low condition developed as an adaptive response within the TME 74 and acts as a promoter of immunosuppression. 75 Hampering hypoxia sensitizes tumour to therapy. 76 The activity of TGFβ is induced under hypoxia, 77 and that suppression of TGFβ attenuates the level of hypoxia in tumour area. 41 A point here is that, TGFβ signalling stimulates angiogenesis in tumours, mediated through increased expression of vascular endothelial growth factor (VEGF), 5 and an increase in the tumour vasculature will finally boost hypoxia due to being aberrant and leaky. 75 These are indicative of a bi-directional association between TGFβ with increased hypoxia in tumour ecosystem. A point of notice here is that shortterm and long-term hypoxia promote reversible and perpetual EMT, respectively, with the latter depended on the activity of HIF-1. 78 Besides, chronic TGFβ exposure is contributed to the chronic hypoxia. 5 Thus, relation between hypoxia with increased TGFβ signalling and EMT state of tumour is predictable, and it seems that such interrelations finally promote PD-L1 upregulation in tumour area, as it also attested a positive correlation between HIF-1 activity with increased PD-L1 expression on tumour cells. 73 Glycogen synthase kinase 3β (GSK3β) is a metabolic enzyme and a tumour suppressor that its activity is hampered under hypoxic conditions of TME. 79

| Targeting lysine specific demethylase 1
Growth and differentiation factor 1 (GDF1) is the member of TGFβ superfamily that shows high expression profile in weakly differentiated high-grade hepatocellular carcinoma (HCC). GDF1 induces a dedifferentiation program in HCC cells and increases tumour cell dissemination and metastasis. GDF1 acts through ALK7-SMAD2/3 and suppresses lysine specific demethylase 1 (LSD1) to reactivate cancer testis antigens (CTAs). CTAs stimulate HCC immunogenicity. LSD1 ablation may sensitize GDF1 + HCC patients to the anti-PD-1 therapy. 88 In tumours with cold immunity, ablation of LSD1 enhances T-cell infiltration and tumour immunogenicity and increases responses to anti-PD-1 in ICI refractory tumours. 89

| Targeting hedgehog signalling
Presence of cancer-associated mesenchymal stem cells (CA-MSCs) in a tumour is closely associated with CD8 + T-cell exclusion in both human and mice. In mouse model of ovarian cancer with hot immunity, CA-MSCs are negatively correlated with the number of intratumoral CD8 + T cells and responses to the anti-PD-L1 therapy, and this is mediated through secretion of chemokines and cytokines, such as TGFβ. Such effects were found to be counteracted after treatment with hedgehog inhibitors, so targeting this signalling in CA-MSCs can reduce the expression of TGFβ, turn the tumourimmune ecosystem into hot, and restore responses to the anti-PD-L1 therapy. 33 Sonic hedgehog (SHH) is a hedgehog ligand that its secretion from tumour cells is contributed to the polarization of macrophages towards pro-tumour TGFβ high M2 phenotype, which further act for exclusion of CD8 + T cells. Combination of the hedgehog inhibitor vismodegib with anti-PD-1 synergistically reduced tumour growth in mice model of hepatoma and lung carcinoma. 90

| Combined TGFβ/PD-L1 blockade therapy
Targeting TGFβ pathway is a promising approach in cancer immunotherapy. 91 Targeting TGFβ in mice with liver metastasis rendered tumour sensitivity to the PD-(L)1 inhibitor therapy. 12 Antibodies against TGFβ and PD-L1 co-administered in immuneexcluded mammary mouse model enhanced T-cell penetration towards tumour interior and reduced tumour burden. 38 Y-traps are antibodyligand traps that contain an antibody against PD-L1 or CTLA-4 fused to an ectodomain sequence of TGF-βRII, which suppresses both autocrine and paracrine TGFβ signalling in TME. The efficacy of anti-CTLA-4 TGF-βRIIecd in reducing Treg activity in TME and hampering tumour progression is superior to that for the CTLA-4 inhibitor ipilimumab.
Similarly, the efficacy of anti-PD-L1 TGF-βRIIecd is higher compared to that for the PD-L1 inhibitors avelumab or atezolizumab. 8  therapy. Thus, reinstating the activity of anti-tumour immunity using TGFβ inhibitors can be an effective strategy for improving the efficacy and durability of ICI therapy. A suggested strategy is to use fusion proteins or peptides for targeting both TGFβ signalling and PD-L1, which is the current focus in cancer immunotherapy. PD-L1 expression, however, shows considerable heterogeneity among tumours, which indicates the essence of the utility of indication-specific strategies to formulate treatment approaches. Besides, the upregulation of alternative checkpoints 104 and implication of non-immune pathways to the ICI outcomes also require attention. 105 Another point to consider is that although TGFβ is a critical driver of immunosuppression in TME, it is not the solo factor in this context. There are other cytokines and chemokine that implement such activity either in relation with or independent on TGFβ activity, and their effects are different from one tumour to another. This indicates that the inspection towards the microenvironment of tumour must be tumour-type specific, and therapeutic regimen must be designed based on considering dominant drivers of immune evasion in the TME of each tumour type. Gathering more information about interactions between cells and driver signalling pathways in tumour stroma will be without a doubt outstanding in order to choose the best therapy or combinatory agent and to improve the idea of personalized therapy.

| FUTURE DIREC TIONS
Understanding more about epigenetic regulators of TGFβ pathway in tumours and developing strategies to interfere with their expression profile can be effective for improving responses to ICI therapy. Histone demethylases can be a focus in this context. 106 Beside the applicability in ICI therapy, targeting TGFβ can also be effective in adoptive T-cell therapy, which is the current focus in cancer immunotherapy. Chimeric antigen receptor (CAR) T cells armoured against TGFβ can be developed as a feasible and safe strategy for this purpose. 10 Another point is that due to the effect of psychological stress on TGFβ activity and responses to the anti-PD-(L)1 44 and limited studies about possible contribution of stressors in different types of solid cancers, it is suggested to have more focus in ongoing research in this area.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Not applicable.