cancers

Review
The TGF-β Pathway: A Pharmacological Target in
Hepatocellular Carcinoma?
Ester Gonzalez-Sanchez 1,2,3, *, Javier Vaquero 1,2 , Maite G. Férnandez-Barrena 1,4,5 , Juan José Lasarte 5,6 ,
Matías A. Avila 1,4,5 , Pablo Sarobe 1,5,6 , María Reig 1,7 , Mariona Calvo 2,8 and Isabel Fabregat 1,2,3, *

1 Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD),

National Biomedical Research Institute on Liver and Gastrointestinal Diseases, Instituto de Salud Carlos III,
28029 Madrid, Spain; jvaquero@idibell.cat (J.V.); magarfer@unav.es (M.G.F.-B.); maavila@unav.es (M.A.A.);
psarobe@unav.es (P.S.); mreig1@clinic.cat (M.R.)
2 Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, 08908 Barcelona, Spain;
mcalvo@iconcologia.net
3 Department of Physiological Sciences, Faculty of Medicine and Health Sciences, University of Barcelona,
L’Hospitalet de Llobregat, 08907 Barcelona, Spain
4 Hepatology Programme, CIMA-University of Navarra, 31008 Pamplona, Spain
5 Instituto de Investigaciones Sanitarias de Navarra IdiSNA, 31008 Pamplona, Spain; jjlasarte@unav.es
6 Immunology and Immunotherapy Programme, CIMA-University of Navarra, 31008 Pamplona, Spain
7 Barcelona Clinic Liver Cancer (BCLC) Group, Liver Unit, Hospital Clinic Barcelona, August Pi i Sunyer
Biomedical Research Institute (IDIBAPS), University of Barcelona, 08036 Barcelona, Spain
8 Oncología Médica, Institut Català d’Oncologia, L’Hospitalet del Llobregat, 08908 Barcelona, Spain



* Correspondence: m.gonzalezsanchez@idibell.cat (E.G.-S.); ifabregat@idibell.cat (I.F.);

 Tel.: +34-932607429 (E.G.-S.); +34-932607828 (I.F.)
Citation: Gonzalez-Sanchez, E.;
Vaquero, J.; Férnandez-Barrena, M.G.; Simple Summary: Transforming Growth Factor-beta (TGF-β) signaling is crucial to maintain tissue
Lasarte, J.J.; Avila, M.A.; Sarobe, P.; homeostasis. Alterations in TGF-β signaling impact tissue functions and favor the development of
Reig, M.; Calvo, M.; Fabregat, I. The diseases, including cancer. In hepatocellular carcinoma (HCC), the most frequent liver tumor, TGF-β
TGF-β Pathway: A Pharmacological plays a dual role, acting as a tumor-suppressor at early stages but contributing to tumor progression
Target in Hepatocellular Carcinoma? at late stages. TGF-β can also act on the stroma, favoring progression and driving immune evasion
Cancers 2021, 13, 3248. of cancer cells. Therefore, inhibiting the TGF-β pathway may constitute an effective option for HCC
https://doi.org/10.3390/ treatment. However, its inhibition in the wrong patients could have negative effects. To overcome
cancers13133248
this obstacle, it is mandatory to identify relevant biomarkers of the status of TGF-β signaling in HCC.
In this review we summarize the functions of TGF-β in HCC and the available strategies for targeting
Academic Editor:
TGF-β signaling. We also present the clinical results of the use of TGF-β inhibitors and their future
Hendrik Ungefroren
in HCC.
Received: 3 June 2021
Accepted: 24 June 2021
Abstract: Transforming Growth Factor-beta (TGF-β) superfamily members are essential for tissue
Published: 29 June 2021 homeostasis and consequently, dysregulation of their signaling pathways contributes to the devel-
opment of human diseases. In the liver, TGF-β signaling participates in all the stages of disease
Publisher’s Note: MDPI stays neutral progression from initial liver injury to hepatocellular carcinoma (HCC). During liver carcinogenesis,
with regard to jurisdictional claims in TGF-β plays a dual role on the malignant cell, behaving as a suppressor factor at early stages, but
published maps and institutional affil- contributing to later tumor progression once cells escape from its cytostatic effects. Moreover, TGF-β
iations. can modulate the response of the cells forming the tumor microenvironment that may also contribute
to HCC progression, and drive immune evasion of cancer cells. Thus, targeting the TGF-β pathway
may constitute an effective therapeutic option for HCC treatment. However, it is crucial to identify
biomarkers that allow to predict the response of the tumors and appropriately select the patients
Copyright: © 2021 by the authors. that could benefit from TGF-β inhibitory therapies. Here we review the functions of TGF-β on HCC
Licensee MDPI, Basel, Switzerland. malignant and tumor microenvironment cells, and the current strategies targeting TGF-β signaling
This article is an open access article for cancer therapy. We also summarize the clinical impact of TGF-β inhibitors in HCC patients and
distributed under the terms and provide a perspective on its future use alone or in combinatorial strategies for HCC treatment.
conditions of the Creative Commons
Attribution (CC BY) license (https://
Keywords: TGF-beta; TGF-beta inhibitors; HCC; HCC immunotherapy; HCC targeted therapy
creativecommons.org/licenses/by/
4.0/).

Cancers 2021, 13, 3248. https://doi.org/10.3390/cancers13133248 https://www.mdpi.com/journal/cancers

Cancers 2021, 13, 3248 2 of 24

1. Introduction
Hepatocellular carcinoma (HCC) is the most frequent form of primary liver cancer,
the sixth most diagnosed cancer and the fourth leading cause of death by cancer. Despite
its death burden, there are limited efficient therapeutic options against it. Surgery (liver
transplantation or tumor resection) may be the major curative option but is only available
for patients with early-stage cancer. Substantial advances have been made in the last years
in understanding the central events that drive malignant transformation and progression,
as well as in the systemic therapies approved for the treatment of unresectable HCC [1–3].
Recent emerging data of clinical studies showed manageable toxicity and safety for im-
munotherapeutic approaches [4], although limited therapeutic benefit. One urgent issue
is how to convert liver cancer from cold to hot and responsive. One ongoing approach is
to design combinatorial treatment of different immune checkpoint inhibitors with other
reagents and modalities, as recently approved for HCC [5]. Pathways involved both in
immunosuppression and cancer development and progression represent interesting thera-
peutic targets for liver cancer. In this regard, the Transforming Growth Factor-beta (TGF-β)
pathway [6] represents an excellent candidate. However, its role in hepatocarcinogenesis
is complex. This review will focus on the role of TGF-β in liver cancer, with the main
aim of better understanding whether, or not, TGF-β inhibitors may represent a promising
combinatorial therapy in HCC.

2. TGF-β Signaling
TGF-β superfamily consists of 33 multifunctional cytokines, including TGF-βs, ac-
tivins, inhibins, bone morphogenetic proteins (BMPs) and growth and differentiation
factors (GDFs). TGF-β, the prototypical member of the family, presents three isoforms in
mammals (TGF-β1, TGF-β2 and TGF-β3), among which TGF-β1 is the most abundant
and well-studied. TGF-β signaling plays key roles in the regulation of different cellular
processes, including proliferation, differentiation, migration or cell death, which are es-
sential for tissue homeostasis. Consequently, dysregulation of its pathways contributes to
human disease.
Nearly all cells, including those of the liver [7], produce and secrete TGF-β (Figure 1A).
First, TGF-β is synthesized in the rough endoplasmic reticulum as a pro-peptide precursor
consisting of a large N-terminal pro-segment named Latency Associated Peptide (LAP)
and a C-terminal mature polypeptide (mature TGF-β). Next, pro-peptide precursor’s
dimers are processed in the Golgi network by the furin convertase to form a small latent
complex (SLC) in which the LAP portions shield the mature TGF-β, preventing it from
binding to its receptors. Finally, SLC are secreted and deposited in the extracellular matrix
(ECM) by bonding with the latent TGF-β binding proteins (LTBPs) or anchored to the
cell surface by glycoprotein-A repetition predominant protein (GARP) [8] (Figure 1A).
Although different mechanisms may account for the activation of mature TGF-β, the
integrins αv-mediated process, leading to the release of the LAP by contractile forces,
appears to have a predominant role [8,9].
Once released, mature TGF-β binds to TGF-β type I and type II serine/threonine
kinase membrane receptors (i.e., TβRI and TβRII) triggering the formation of an heterote-
trameric complex in which the constitutively activated type II receptor phosphorylates
and activates the type I receptor. After the extracellular signal is transduced across the
membrane, activated TβRI initiates the canonical TGF-β signaling pathway through the
phosphorylation of the Receptor-regulated (R)-SMADs 2 and 3 in their C-terminal serine
residues. Thereafter, phosphorylated SMAD2 and 3 form a trimeric complex with a com-
mon mediator (Co)-SMAD4 and translocate into the nucleus where they need to interact
with other transcription factors to activate or repress the transcription of target genes [10]
(Figure 1A). Besides transcriptional regulation, interaction of SMADs with specific tran-
scription factors and co-regulators enables them to regulate gene expression by alternative
mechanisms including epigenetic remodeling, RNA splicing and miRNA processing [10].
Cancers 2021, 13, 3248 3 of 24
Cancers 2021, 13, x 3 of 25

Figure1.1. TGF-β-mediated
Figure TGF-β-mediated signaling
signaling in
in liver
liver cells.
cells. (A)
(A)Canonical
Canonical(SMAD-dependent)
(SMAD-dependent)andandnon-ca-
non-
nonical (non-SMAD) signaling pathways. (B) TGF-β dual role controlling tumor suppressor and
canonical (non-SMAD) signaling pathways. (B) TGF-β dual role controlling tumor suppressor and
protumorigenic responses in hepatocellular carcinoma. Cav-1: Caveolin, ECM: extracellular matrix,
protumorigenic responses in hepatocellular carcinoma. Cav-1: Caveolin, ECM: extracellular matrix,
GF: Growth Factor, LTBP: latent TGF-β binding proteins, SLC small latent complex, LAP: Latency
GF: Growthpeptide.
associated Factor, LTBP:
Figurelatent TGF-β binding
was created proteins, SLC small latent complex, LAP: Latency
with BioRender.com.
associated peptide. Figure was created with BioRender.com.
Once released, mature TGF-β binds to TGF-β type I and type II serine/threonine ki-
In addition to the SMAD pathway, TGF-β can also initiate multiple non-SMAD or non-
nase membrane receptors (i.e., TβRI and TβRII) triggering the formation of an hetero-
canonical signaling pathways (Figure 1A) [10,11]. For example, due to its weak tyrosine
tetrameric complex in which the constitutively activated type II receptor phosphorylates
kinase activity, TβRI can induce the phosphorylation of Src homology domain 2-containing
and activates the type I receptor. After the extracellular signal is transduced across the
protein (Shc) and subsequently activate the ERK MAP kinase pathway. Besides, TβRI can
membrane, activated TβRI initiates the canonical TGF-β signaling pathway through the
also recruit TGFβ-activated kinase 1 (TAK1), through tumor necrosis factor-associated
phosphorylation of the Receptor-regulated (R)-SMADs 2 and 3 in their C-terminal serine
factor (TRAF) 4 or 6 to stimulate JNK, p38 MAPK and NF-κB pathways, or activate RHO
residues. Thereafter, phosphorylated SMAD2 and 3 form a trimeric complex with a com-
small GTPases leading to actin cytoskeleton reorganization, whereas TβRII can directly
mon mediator (Co)-SMAD4 and translocate into the nucleus where they need to interact
phosphorylate the cell polarity regulator PAR6. AKT signaling can also be activated by
with other
TGF-β transcription
in a PI3K dependent factors toMoreover,
matter. activate orthese
repress the transcription
signaling of target
pathways may genes [10]
also cross-talk
(Figure
with 1A). Besides
the TGF-β transcriptional
canonical regulation,
signaling through interaction ofcontrol
post-translational SMADs with specific
of SMAD tran-
activation
scription factors
and functions [10]. and co-regulators enables them to regulate gene expression by alternative
mechanisms including epigenetic remodeling, RNA splicing and miRNA processing [10].
Cancers 2021, 13, 3248 4 of 24

Due to its pleiotropic effects, TGF-β’s activity needs to be carefully controlled to

maintain tissue homeostasis. Multiple mechanisms may account for this strict regulation
including: the actions of (i) sequestering proteins that prevent TGF-β binding to its recep-
tors; (ii) accessory coreceptors such as TβRIII (betaglycan), which regulate the presentation
of ligands to the TβRII/TβRI receptor complexes; or (iii) inhibitory proteins as SMAD7,
which antagonize the activation of SMADs 2 and 3; (iv) the control of TGF-β response by
cell surface distribution of TβR receptors and (v) post-translational modifications of TβR
and SMADs, such as phosphorylation, ubiquitination and sumoylation [9].
In the liver, TGF-β plays a major role in physiological and pathological conditions.
Expression of TGF-β ligands is increased in chronic liver diseases and TGF-β signaling
participates in all the stages of disease progression from initial liver injury to HCC [12–15].
In cancer settings, alterations in TGF-β signaling both in the malignant cells and the tumor
microenvironment (i.e., cancer associated myofibroblasts (CAF), endothelial cells and
immune cells) may contribute to the progression of HCC as thoroughly described below.

3. Role of TGF-β in HCC Cells

Expression of TGF-β ligands is increased in liver chronic diseases and TGF-β signal-
ing participates in all stages of disease progression [12]. In liver carcinogenesis, TGF-β
plays a dual role, behaving as a suppressor factor at early stages, but contributing to
later tumor progression once cells escape from its cytostatic effects. In non-transformed
hepatocytes, TGF-β inhibits proliferation [16] and induces apoptosis [17,18]. Activation of
TGF-β signal induces antiproliferative signals in epithelial cells through SMAD-dependent
transcriptional regulation of genes that codify for proteins involved in cell cycle, such
as Retinoblastoma, Cyclin- Dependent Kinase (CDK) inhibitors, or c-Myc, among oth-
ers [19–21]. Apoptosis induced by TGF-β also requires SMAD-dependent transcription,
although other non-canonical signals have been proposed to be involved. In hepatocytes
and liver tumor cells, TGF-β-induced apoptosis requires up-regulation of the NADPH oxi-
dase NOX4 (Figure 1B) that mediates reactive oxygen (ROS) production, which is required
for regulating mitochondrial-dependent cell death [22–24].
Taking all this under consideration, it should be expected that the TGF-β pathway
plays a tumor suppressor role in liver cancer. However, malignant cells surpass the
suppressive effects of TGF-β either by inactivation of key components of the pathway or
through the overactivation of parallel pathways that counteract its suppressive effects, such
as production of autocrine factors, included EGFR ligands or PDGF and their receptors
(Figure 1B) [25,26]. The autocrine loop of EGFR activated by TGF-β in HCC cells requires
activation of the metalloprotease TACE/ADAM17 located in caveolin compartments in
the cell membrane [27]. Moreover, clathrin (CTLC) has been recently identified as a key
regulator of TGF-β-mediated EGFR transactivation in this context [28]. Thus, up-regulation
of EGFR ligands and activation of EGFR signaling enhances the capacity of the cells to
overcome the pro-apoptotic effects of TGF-β. In late stages, tumor cells that have acquired
resistance to TGF-β suppressor functions respond to it acquiring capabilities that contribute
to tumor progression. Indeed, HCC cells respond to TGF-β by inducing phenotypic changes
related to a full or a partial Epithelial-Mesenchymal Transition (EMT), that contribute to
increase the tumor cell migratory and invasive capacities and confer them properties of a
migratory tumor initiating cell [29,30]. Taking together these and many other studies, it is
very clear that TGF-β plays a dual role in the progression of HCC.
An elegant study from Coulouarn and col. proposed different liver TGF-β signatures
in HCC cell lines and patients defining a cohort of genes related to its tumor suppressor
capacities, which they designed as the “early signature” and another cohort of genes
related to its tumor promoting effects, the “late signature” [31]. The early signature pattern
correlated with longer and the late signature response pattern with shorter survival in
HCC patients. In addition, tumors expressing the late gene signature displayed invasive
phenotype, increased tumor recurrence and accurately predicted liver metastasis. Increased
TGF-β levels and mutations in the key molecules involved in the TGF-β pathway have
Cancers 2021, 13, 3248 5 of 24

been found in a relevant percentage of HCC patients. In this sense, somatic mutations in at
least 1 gene whose product is a member of the TGF-β pathway have been found in 38% of
HCC samples [32]. Some of them correlated with loss of TGF-β tumor suppressor activity,
but other ones were related to overactivation of the TGF-β pathway, which contribute to
amplify its pro-tumorigenic, pro-inflammatory and pro-fibrotic actions. Molecular gene
signatures reflecting the TGF-β oncogenic arm have also been identified in tumors across
the different HCC molecular classification [33].
Indeed, targeting the TGF-β pathway may be a promising therapeutic option in
HCC, but it is necessary the identification of biomarkers that help to identify which is
the response of the tumor cells in patients. In this sense, in vitro and in vivo studies and
analyses in patients have identified CXCR4, CD44, SMAD7 or CLTC as genes that are
upregulated by TGF-β in the HCC tumor cell, correlating with its pro-tumorigenic arm.
The high expression of these genes, together with high expression of TGF-β1, may help to
identify patients with a “late TGF-β signature” that would benefit from TGF-β targeting
drugs [28,30,34–38]. Table 1 compiles the information regarding the expression of the
above-mentioned biomarkers in the human HCC cells lines for which more published
information can be found, according to their TGF-β signature. Recent pharmacological
studies with Galunisertib, a TβRI kinase inhibitor, are also allowing the identification
of biomarkers that may help to calculate the benefit and/or to follow up the potential
efficiency of TGF-β blockers in the progression of HCC [39].

Table 1. TGF-β signature and biomarkers in human HCC cell lines.

TGF-β Biomarkers (Expression Levels)

Cell Line Tumor Type Phenotype
Signature CDH1 VIM CD44 CXCR4 SMAD7 CLTC
HepG2 Human caucasian HCC Epithelial Early High Absent Absent Low Low No data
PLC/PRF/5 Human liver hepatoma Epithelial Early High Very Low Low Low Low Low
Huh-7 Human asian HCC Mixed Early Medium Low Low Low High Low
Hep3B Human black HCC Mixed Early Medium Low Low High High Low
SNU-449 Human asian HCC Mesenchymal Late Absent High High High No data High
HLE Human HCC Mesenchymal Late Absent High High High High high
HLF Human HCC Mesenchymal Late Absent High High High High High
CDH1: E-Cadherin, CLTC: Clathrin, Vim: Vimentin.

4. TGFβ-Related Functions in HCC Tumor Microenvironment (TME)

The tumor microenvironment (TME) consists of a variety of resident and infiltrating
host cells, secreted growth factors and cytokines, and ECM proteins that provide a scaffold
for the infiltration and migration of the different cellular components inside the tumor,
including tumor cells. Through reciprocal interactions with malignant cells, stromal cells
(CAF, endothelial cells and immune cells) contribute to the accumulation of ECM, angio-
genesis, inflammation, metastasis, and the suppression of the anti-tumorigenic adaptive
immune cell response. During hepatocarcinogenesis, TGF-β is produced by most cell types
and takes part in the dialogue between tumor cells and host stroma, placing it as a key
player in the regulation of these important hallmarks of cancer progression as detailed
hereafter (Figure 2).
 Cancers 2021, 13, x 6 of 25

Cancers 2021, 13, 3248 6 of 24

as a key player in the regulation of these important hallmarks of cancer progression as
detailed hereafter (Figure 2).

Figure 2. Schematic representation of the effects of TGF-β on the stromal cell types of hepatocellular
Figure 2. Schematic representation of the effects of TGF-β on the stromal cell types of hepatocellular
carcinoma. (A) Effects on CAF: TGF-β stimulates the activation of HSC and the maintenance of the
carcinoma. (A) Effects on CAF: TGF-β stimulates the activation of HSC and the maintenance of the
myofibroblastic phenotype, which, upon malignant transformation of hepatocytes into HCC cells,
myofibroblastic
become CAF. CAF phenotype,
producewhich, upon
(i) TGF-β, malignant
that transformation
acts on HCC cells inducingof hepatocytes
EMT, and enhance into HCC cells,
vascular
become CAF. CAF produce (i) TGF-β, that acts on HCC cells inducing EMT,
mimicry formation in HCC cells together with SDF1, (ii) other chemokines (CCL2, CCL5, CCL7 and and enhance vascular
mimicry
CXCL16) formation in HCC
that enhance TGF-βcellsactivity
together onwith
HCCSDF1, (ii) othertochemokines
cells leading metastasis. (CCL2, CCL5,
(B) Effects on CCL7 and
endothelial
CXCL16) that may
cells: TGF-β enhance TGF-β
promote activity on
migration andHCC cells leading
proliferation to metastasis.
of endothelial (B)by
cells Effects on endothelial
(i) directly acting on
endothelial
cells: TGF-β maycells promote
or (ii) inducing
migrationVEGF andsecretion by HCC
proliferation cells. (C) Effects
of endothelial cells by on(i)the immune
directly system:
acting on
TGF-β maycells
endothelial increase
or (ii)the levels of
inducing soluble
VEGF MICA by
secretion andHCC
weaken
cells.the
(C)action
Effects ofonnatural killer (NK)
the immune cells
system:
through
TGF-β may itsincrease
binding theto NKG2D. TGF-β can
levels of soluble MICAalsoanddownregulate
weaken the NKG2D
action of expression
natural killer or (NK)
repress the
cells
mTOR pathway impairing tumor recognition. TGF-β can also act on tumor associated macrophages
through its binding to NKG2D. TGF-β can also downregulate NKG2D expression or repress the
(TAM) that secrete growth factors and promote migration of endothelial cells and angiogenesis.
mTOR pathway impairing tumor recognition. TGF-β can also act on tumor associated macrophages
TGF-β can favor the generation of tolerogenic dendritic cells (DC) and impair the activation of ef-
(TAM)
fector that secrete growth
T lymphocytes factors
and their and promote
cytotoxic activity.migration of endothelial
TGF-β promotes cells andofangiogenesis.
the conversion conventional
TGF-β can favor the generation of tolerogenic dendritic cells
CD4 or CD8 T cells into immunosuppressive Treg cells that can hinder the antitumor(DC) and impair the activation
activity of ofthe
effector
adaptive T lymphocytes
immune response. and theirCAF: cytotoxic activity. TGF-β
cancer associated promotes
fibroblasts, ECM:theextracellular
conversion of conventional
matrix, EMT Ep-
ithelial-Mesenchymal
CD4 or CD8 T cells intoTransition, HSC: hepatic
immunosuppressive Treg stellate cells,
cells that can MF: myofibroblasts,
hinder the antitumor Teff: effector
activity of theT
cells, Treg:
adaptive T regulatory
immune response.cells.CAF: cancer associated fibroblasts, ECM: extracellular matrix, EMT
Epithelial-Mesenchymal Transition, HSC: hepatic stellate cells, MF: myofibroblasts, Teff: effector
T cells, Treg: T regulatory cells.
Cancers 2021, 13, 3248 7 of 24

4.1. HSC and CAF

HCC usually develops from a background of chronic liver disease that in most cases
curses through premalignant states of fibrosis and subsequent cirrhosis, which provide the
proper environment for hepatocyte malignant transformation [40]. One of the hallmarks
of liver fibrosis is the activation of hepatic stellate cells (HSC) to myofibroblasts, which
in turn triggers the transformation of the microenvironment by producing ECM deposits
and releasing pro-fibrotic and pro-inflammatory factors that contribute to chronic liver
disease progression [40]. In fact, most studies agree that, in the liver, the major sources
of myofibroblasts in experimental models of fibrosis are HSC [41–45]. Eventually these
activated myofibroblasts will evolve to CAF during hepatocarcinogenesis, although other
sources, such as the differentiation of recruited bone marrow derived mesenchymal cells
or the transformation of epithelial cells through EMT, that have been described in other
cancers [46], cannot be entirely ruled out.
The master role of TGF-β in the activation of HSC to myofibroblasts during liver
fibrosis has been profoundly studied (detailed revisions on this subject can be found [47]).
Briefly, TGF-β stimulates the activation of HSC and the maintenance of the myofibrob-
lastic phenotype [42,48,49]. This effect is mediated through the activation of canonical
(SMAD3) [12,50–52] and non-canonical (ERK, JNK, p38, and STAT3) [53,54] intracellular
signaling pathways that induce the expression of pro-fibrogenic genes, including COL1A1
(encoding collagen 1) and CCN2 (encoding CTGF). In turn, CTGF stimulates the production
of ECM components. In addition, NOX4, by the production of ROS, has been described as
a central mediator of the TGF-β-induced activation of HSC [49,55].
As in the fibrotic tissue, in the TME the activation of CAF supports tumor progression
by producing ECM and cytokines, stimulating immune evasion, and promoting angio-
genesis [46]. However, despite the extensive information on the role of TGF-β in HSC
activation in the fibrotic liver or CAF generation in other cancers [56], the studies related to
TGF-β and CAF in HCC are scarce. The best-known effects of TGF-β on CAF consist of
the induction of a pro-fibrotic phenotype, which is negatively regulated by LXRα through
the interaction of this nuclear receptor with SMAD3 at the ACTA2 promoter sites [57].
Furthermore, LXRα antagonistic effects were able to impair the pro-tumorigenic effects
of CAF on malignant HCC cells in a 3D co-culture model [57]. But CAF not only respond
to TGF-β, they also produce and secrete this cytokine affecting the crosstalk between
tumor and stromal cells (Figure 2A). In this sense, CAF-secreted TGF-β in conjunction with
SDF1 has been shown to enhance the expression of VE-cadherin, MMP2 and laminin5γ2,
leading to vascular mimicry formation in HCC cells in a mechanism negatively regulated
by miR-101 [58]. Moreover, CAF secrete multiple chemokines (CCL2, CCL5, CCL7 and
CXCL16) that promote HCC cell migration and invasion through enhancing TGF-β activity
in HCC cells, leading to HCC metastasis [59]. Indeed, a study modelling tumor-stroma
interaction revealed that TGF-β secreted by HSC and myofibroblasts can mediate EMT in
HCC cells [60].
Despite these advances, much is still to elucidate about the role of TGF-β in HCC
CAF. For instance, CAF autocrine TGF-β has been associated in other cancers to the
recruitment of more CAF and the subsequent deposition of high amounts of ECM. These
deposits of ECM increase tumor stiffness reducing the density of blood vessels, thus
forming a barrier that can impair the access of anti-cancer drugs and immune cells to the
tumor [61–64]. In the same direction, recent advances in single cell sequencing techniques
have allowed the characterization of distinct CAF subpopulations with different specific
functions, such as CAF subtypes with high α-SMA expression, that demonstrate a strong
TGF-β responsiveness [65–68]. Further characterization of HCC CAF subpopulations
could shed light on the role of TGF-β in CAF and other cell types in the TME, providing
opportunities for their specific targeting.
Cancers 2021, 13, 3248 8 of 24

4.2. Endothelial Cells

Angiogenesis is a hallmark of cancer progression as it facilitates tumor growth and
metastasis. Endothelial cells lining newly formed blood vessels nourish tumors and medi-
ate the entry and exit of immune cells and other substances. TGF-β has been demonstrated
as a pro-angiogenic factor in different tumors. In HCC, TGF-β secretion by mesenchymal
stem cells was related to an increased angiogenesis in mouse models [69]. Another study
linked the angiogenic effects of TGF-β secreted by HCC cells to the signaling mediated
by its coreceptor CD105 [70]. Indeed, endothelial cells isolated from HCC showed higher
expression of CD105 and enhanced capacity to migrate in response to TGF-β than normal
endothelial cells [70] (Figure 2B). In HCC patients, the worst malignant features were
correlated with the highest expression of TGF-β, CD105 and angiogenic markers [70]. Thus,
inhibition of TGF-β signaling using LY2109761 was able to reduce vessel formation in
tumors but showed no effects on physiological angiogenetic development [70]. On the
other side of this crosstalk, exposure to TGF-β induces production of VEGF in HCC cells
in a mechanism mediated by TGF-β/SMAD3/NF-κB signaling cascade [71] (Figure 2B).
Consequently, VEGF actions on endothelial cells ultimately led to promotion of angiogene-
sis. The same study showed that treatment with arsenic trioxide impaired this effect by
upregulating miR-491, which directly targets the expression of SMAD3.

4.3. Immune System

The liver is the largest peripheral immunomodulatory organ and is filled with a
multitude of innate and adaptive immune cells, including Kupffer cells, natural killer
(NK) cells, NK T (NKT) cells, and liver-transiting and/or -resident CD8+ T cells and
CD4+ T cells [72]. TGF-β critically regulates immune cells in the liver to maintain a
balance between immune tolerance and activation. This immune homeostasis is required
to properly control inflammatory processes and prevent autoimmune alterations. But this
equilibrium can be altered by TGF-β released within the TME, which may promote cancer
progression through differential effects on multiple key cell types that orchestrate innate
and adaptive immunity.
As it is well known, immune TME is heterogeneous. The HCC immune landscape
shows that approximately 25% of cases have a high degree of immune infiltration (so-
called ‘immune class’ of HCC), with high expression levels of PD-1/PD-L1 [73–75] and
likely well suited for PD-1-blocking immunotherapy. Notably, intratumoral immune cell
densities of CD3+ and CD8+ cells associate significantly with recurrence and relapse free
survival [76,77]. The HCC immune class was further subdivided into an active immune
response subtype (65% of immune class samples), characterized by overexpression of
adaptive immune response genes, and an immune exhaustion subtype (35%), characterized
by the presence of immunosuppressive signals and cells (TGF-β and M2 macrophages).
Importantly, patients in the active immune response cluster showed improved survival
and lower rates of tumor recurrence compared with patients in the exhausted immune
response cluster. This study allows the development of classification tools for HCC patients
to predict their response to immunotherapy or to design new therapeutic strategies [75]. A
strong association between the TGF-β signature and the exhausted immune signature in
HCC was identified, suggesting that the TGF-β pathway is an important immune regulator
and biomarker for HCC [32,74,75]. In fact, several types of innate and adaptive immune
cells respond to TGF-β released by cancer cells, stromal cells and immune cells themselves,
resulting in an immunosuppressive TME (Figure 2C).
TGF-β in innate immune cells in HCC: Natural killer cells (NK) are part of the first
line of immunological defence against cancer development. Defects in NK cell numbers
and functions are recognized as important mechanisms for immune evasion of tumor cells
in HCC [78]. NK cell function appears to be attenuated in HCC and various mechanisms
seem to be involved in their malfunction. On the one hand, TGF-β directly inhibits
the activation and functions of NK cells by repressing the mTOR pathway [79]. On the
other hand, TGF-β, directly or through post-transcriptional mechanisms, controls the
Cancers 2021, 13, 3248 9 of 24

expression of the activation receptor NKG2D in NK cells [80]. Intra-tumoral NK cells have
NKG2D downregulation in comparison to NK cells in non-tumor liver [81]. Furthermore,
some tumor cells also downregulate NKG2D ligands, such as MICA, on the tumor cell
membrane. The increase of soluble MICA by the action of MICA-shedding proteases has
been reported in several patients with HCC, impairing the action of NK cells and leading
to the defective recognition of the tumor [82], and TGF-β may be playing a role in this
process [83]. NK cell function is also regulated by the crosstalk between immune cells in
the TME. Multiple immune cell subpopulations, such as the myeloid derived suppressor
cells (MDSC), T regulatory cells (Tregs), macrophages polarized to the immunoregulatory
phenotype (M2), and immature Dendritic cells (DC) facilitate NK cell disfunction. MDSC
and Treg may act on NK cells via membrane-bound TGF-β [84,85]. In addition to their
direct effect on NK cells, myeloid cells [86] as well as on Tregs [87], TGF-β signaling has
been demonstrated as a critical mediator in tumor invasion and metastasis through the
action of tumor associated macrophages (TAM) that secrete growth factors, including
TGF-β, which promote migration of endothelial cells and angiogenesis [88,89].
TGF-β in adaptive immune cells in cancer: The effect of TGF-β in the adaptive immune
response against HCC is very broad and affects to all T lymphocyte subpopulations.
TGF-β produced by cancer cells can induce an immature differentiation state of DC,
converting them into tolerogenic DC [90] with a downregulated expression of MHC class-II
molecules [91], impaired cross-presenting capacities and downregulated costimulatory
molecule expression [92]. These TGF-β-induced immature DC facilitate tumor tolerance
by inducing antigen-specific CD8+ Tregs, suppressing the function of other effector T
cells [92,93]. TGF-β can directly inhibit the cytotoxic functions of CD8 T cells [94]. On CD4
T cells, TGF-β affects the differentiation of both Th1 and Th2 subsets by downregulation
of their key transcription factors [95–97] and ultimately favouring a shift of Th1 towards
Th2 cell differentiation [98,99]. TGF-β affects T cell proliferation and effector functions
by impairing IL-2 production during T cell activation [100], inducing cell cycle arrest and
favouring apoptosis of T cells [101,102].
TGF-β activates SMAD2/3 and, in cooperation with IL-21 and IL-23, promotes the
generation of Th17 cells contributing to NAFLD-associated liver inflammation and HCC
development [103–105]. In CD8+ cytotoxic T cells, TGF-β cooperates with the transcription
factor ATF1 to suppress the expression of IFN-γ to inhibit its antitumor activity [98].
Intratumoral TGF-β suppresses NKT cells, a population responsible for recruiting effector
immune cells to the tumor through the production of large amounts of IFN-γ. Notably, the
development of Invariant NKT (iNKT) cells, which represent a subclass of NKT cells with
regulatory functions, is orchestrated by TGF-β [106], and plays an important role switching
from inflammation to resolution of liver injury [107], but also may affect the outcome and
overall survival in HCC [108,109].
The production of TGF-β, mainly by liver sinusoidal endothelial cells (LSEC) and
HSC contribute to hepatic regulatory T (Treg) cell induction. There is a correlation between
TGF-β and Treg cells in HCC patients. Moreover, the Treg-associated expression of both
TGF-β and IL-10 was shown to be associated with HCC progression [110]. Both CD4+
Foxp3+ and CD4+ Foxp3- suppressor cells induced by TGF-β are increased in HCC patients
and correlate with poor overall survival [111–113]. Notably, TGF-β can also elicit the
production of other factors such as amphiregulin [114], that has a promoting role on the
immunosuppressive activity of Treg cells [115,116]. The presence of Treg cells may play an
important homeostatic role in tissue repair after injury. However, its immunosuppressive
role may affect dramatically the antitumor activity of other immune cells.
On the other hand, TGF-β1 enhances antigen-induced PD-1 expression through
SMAD3-dependent transcriptional activation in antigen-specific T cells, suggesting that
the TGF-β pathway directly participates in immune-checkpoint regulation [117].
All these data point to TGF-β as one of the master immunosuppressive molecules in
HCC and imply that targeting the TGF-β pathway might enhance antitumor immunity in
HCC patients.
Cancers 2021, 13, x 10 of 25

All these data point to TGF-β as one of the master immunosuppressive molecules in
Cancers 2021, 13, 3248 10 of 24
HCC and imply that targeting the TGF-β pathway might enhance antitumor immunity in
HCC patients.

5. TGF-β
5. TGF-β Inhibitors
Inhibitors
The tumorigenic role
The tumorigenic roleofofTGF-β
TGF-βininlate-stage
late-stage solid
solidmalignancies
malignancies hashas
spurred
spurredthe the
de-
velopment of a variety of anti-TGF-β drugs [11,118]. All the evidence
development of a variety of anti-TGF-β drugs [11,118]. All the evidence discussed in discussed in previ-
ous sections
previous indicates
sections that targeting
indicates the TGF-β
that targeting pathway
the TGF-β may also
pathway mayconstitute an effective
also constitute an ef-
strategystrategy
fective for HCCfor treatment in appropriately
HCC treatment selected patients.
in appropriately AvailableAvailable
selected patients. TGF-β inhibitors
TGF-β
are of different
inhibitors are of chemical nature and
different chemical present
nature diverse diverse
and present mechanisms of action
mechanisms including:
of action (i)
includ-
the suppression
ing: of the production
(i) the suppression of TGF-β;
of the production (ii) the inhibition
of TGF-β; of TGF-β
(ii) the inhibition of activity; (iii) the
TGF-β activity;
blockage
(iii) of the interaction
the blockage of TGF-βofwith
of the interaction its receptors;
TGF-β and (iv) the
with its receptors; andinhibition of the kinase
(iv) the inhibition of
activity
the kinaseof activity
the TGF-β receptor.
of the TGF-βThese pharmacological
receptor. effects mayeffects
These pharmacological be achieved
may bewith anti-
achieved
senseantisense
with oligonucleotides, neutralizing
oligonucleotides, antibodies,
neutralizing ligand traps
antibodies, ligandand small
traps andmolecule inhibi-
small molecule
inhibitors
tors (Figure (Figure 3). Many
3). Many of these
of these inhibitors
inhibitors havehaveshownshown promising
promising anti-tumoral
anti-tumoral activity
activity in
in preclinical
preclinical models,
models, andand ananincreasing
increasingnumber
numberofofthem themhavehavebeen,
been, or
or are
are currently being
being
tested in clinical trials of almost all types of solid solid tumors,
tumors, alone
alone and
and in
in combination
combination with with
other agents [11,118–120].

Schematicrepresentation
Figure 3. Schematic representationofofthe thedifferent
differentstrategies
strategiestargeting
targetingTGF-β
TGF-β signaling
signaling forfor can-
cancer
therapy. According to their mechanism of action, directed against TGF-β expression,
cer therapy. According to their mechanism of action, directed against TGF-β expression, cytokinecytokine block-
ade or signaling
blockade inhibition,
or signaling thesethese
inhibition, strategies fall under
strategies different
fall under categories:
different (i) Antisense
categories: oligonucle-
(i) Antisense oligonu-
otides; (ii) Neutralizing antibodies; (iii) Ligand traps; (iv) Small molecule inhibitors (kinase
cleotides; (ii) Neutralizing antibodies; (iii) Ligand traps; (iv) Small molecule inhibitors (kinase inhibi-
tors). LAP: Latency Associated Peptide, LTBP: latent TGF-β binding proteins. Figure
inhibitors). LAP: Latency Associated Peptide, LTBP: latent TGF-β binding proteins. Figure was was created
with BioRender.com.
created with BioRender.com.

5.1. Antisense Oligonucleotides

oligonucleotides (AON) are designed to specifically bind target
Antisense oligonucleotides target mRNAs
mRNAs
and induce their degradation. AP12009
AP12009 (Trabedersen)
(Trabedersen) is an 18-mer AON targeting TGF-
β2 mRNA
mRNA thatthathas
hasbeen
beenevaluated
evaluatedininclinical trials
clinical forfor
trials several advanced
several advancedsolid tumors,
solid in-
tumors,
cluding pancreatic
including and
pancreatic colorectal
and carcinoma,
colorectal carcinoma,melanoma
melanoma andandglioma with
glioma encouraging
with encouragingre-
sults and
results anda agood
goodsafety
safetyprofile
profile[11,120].
[11,120].Downregulation
Downregulation of of TGF-β2
TGF-β2 expression
expression not only
results in the inhibition of cancer cell growth,
growth, itit may
may also
also enhance
enhance immunity
immunity against
against the
the
tumor [121].
[121].This
This notion
notion ledled to the
to the development
development of tumor
of tumor cell vaccines
cell vaccines like Lucanix,
like Lucanix, which
which expresses TGF-β2 AON, or Vigil that harbors a short hairpin RNAi targeting furin
convertase, involved in TGF-β1 and TGF-β2 precursors processing [120]. These vaccines
have been tested with promising results in patients with different solid tumors, such as
lung, ovarian and metastatic Ewing’s sarcoma, but still not in HCC patients [120].
Cancers 2021, 13, 3248 11 of 24

5.2. Neutralizing Antibodies

Several neutralizing monoclonal antibodies targeting binding of TGF-β to receptors
have been developed and tested at different levels. After demonstrating the efficacy of the
murine anti-TGF-β 1D11 antibody in preclinical in vivo models [122–124], the humanized
version (Fresolimumab; GC-1008) was developed. It is a pan-neutralizing IgG4 antibody
that binds to all three TGF-β isoforms and has been tested in the clinic in several neo-
plastic and non-neoplastic indications, as reviewed in [125], but not in HCC. In patients
with melanoma or renal cell carcinoma [126] or with relapsed malignant pleural mesothe-
lioma [127] therapy was well tolerated and a patient with complete response and several
with stable disease were observed. Development of this antibody was discontinued and an
improved version, SAR439459, has shown important immunomodulatory effects in preclin-
ical in vitro and in vivo models [128]. SAR439459 has entered clinical trials as monotherapy
in several solid tumors including melanoma, colorectal adenocarcinoma, urothelial can-
cer, HCC and non-small cell lung cancer (NCT03192345). In addition to their effects as
monotherapy, this antibody improved immunogenicity and antitumor efficacy triggered by
PD-1 blockade, suggesting its potential as partner in combinatorial immunotherapies [128].
Anti-PD-1-based therapies may enhance the Treg/CD4+ Th ratio and increase pSMAD3 ex-
pression in tumor cells. Since anti-TGF-β antibody administration attenuates these effects,
the combined blockade of both molecules results in a synergistic effect, where additional
immune-independent mechanisms are also targeted by this combination [129].
NIS793 is another anti-TGF-β antibody that is being tested in a phase I/Ib clinical trial
in patients with different solid tumors (breast, lung, HCC, colorectal, pancreatic and renal)
NCT02947165. In this case, as for other antibodies, it is being administered in combination
with PD-1 blockade.
Besides cytokine blockade, antibodies targeting the receptor have also been developed.
LY3022859 is an IgG1 antibody that, upon binding to TβRII, prevents the formation of the
ligand-receptor complex and thus inhibits the ensuing signaling activation. Preclinical data
with this antibody showed antitumor effects mediated through several mechanisms [130].
However, results obtained in a clinical trial in patients with advanced solid tumors raised
safety concerns related to uncontrolled cytokine release [131].
Other antibody-based strategies targeting additional TGF-β-related elements have
been tested. The role played by some integrins in the activation process of surface
TGF-β [132] suggested that anti-integrin antibodies would inhibit TGF-β activation, by-
passing the effect of systemic TGF-β inhibition [133,134]. Indeed, expression of β8 integrin
by cells that rely on TGF-β for their immunosuppressive effects, such as tumor cells [134] or
Treg cells [135], controls some TGF-β-associated events, and blockade of this integrin results
in antitumor activity in several preclinical murine models. In this regard, a clinical trial
based on the administration of the αvβ8-blocking antibody PF-06940434 (NCT04152018)
is testing this drug in several advanced and metastatic solid tumors (not including HCC).
Interestingly, as for other inhibitory antibodies, some trial arms include a combination with
anti-PD-1 antibodies. Finally, targeting of latent TGF-β1 and inhibition of its activation has
been achieved with SRK-181 antibody. By considering the lower sequence similarity in
the sequences of TGF-β1, 2, and 3 prodomains, this antibody was selected to specifically
bind and block TGF-β1 activation [136]. Although monotherapy with this antibody did not
provide any beneficial effect on tumor growth, combination with PD-1 blockade overcome
resistance to this immunotherapy, associated to a profound remodeling of the tumor im-
mune profile. These results have prompted a new clinical trial (NCT04291079) in patients
with locally advanced or metastatic solid tumors, where SRK-181 is also considered in
combination with PD-(L)1 blocking antibodies.

5.3. Ligand Traps

The basic rationale behind the use of ligand traps is to sequester the cytokine and avoid
thus its binding to the receptors [137]. In the case of TGF-β, several compounds in this field
have been developed [138–141] based on the use of soluble TβRII or III or receptors fused
Cancers 2021, 13, 3248 12 of 24

to immunoglobulins. AVID200 is a TGF-β1 and 3 inhibitor of this class [142,143] that has
entered clinical trials (NCT03834662) in patients with advanced and metastatic malignancies.
In addition to molecules with TGF-β-binding domains, more complex molecules
have been designed to enrich their number of functions, tackling thus different effector
and/or immunosuppressive mechanisms. FIST15 is a molecule containing the IL15Rα-
sushi domain bound to IL15 as well as the TβRII ectodomain [144]. FIST15 enhanced
functional activity of CD8 T cells and NK cells, resulting in a higher antitumor effect in
preclinical murine models. Resistance to checkpoint inhibitors has been associated with
TGF-β expression [145,146]. Based on this, TGF-β ligand traps have been conjugated
to PD-1/PD-L1 blocking molecules or to antiCTLA-4 [147,148]. These dual molecules
have demonstrated superior preclinical antitumor efficacy compared with monotherapies
blocking either TGF-β or the corresponding checkpoints, due to their capacity to activate
innate and adaptive immunity. M7824 (Bintrafusp alfa) is being tested in many clinical
trials in patients with a variety of solid tumors [149–151], including HCC [152]. In this
last case, the phase I study showed a manageable safety profile and preliminary efficacy,
warranting further assays in larger patient groups.

5.4. Small Molecule Inhibitors

The most explored strategy for TGF-β inhibition in cancer is based on small molecules
that interfere with intracellular signaling from TGF-β receptors, suppressing canonical
and non-canonical pathways. Preclinical studies with the TβRI kinase inhibitor SB-431542
demonstrated antitumoral activity in different tumors such as renal carcinoma and malig-
nant glioma [120], and also in HCC [153,154]. Vactosertib (TEW-7197) is an orally available
more potent and specific TβRI kinase inhibitor that has also shown efficacy in preclinical
models of myeloma, breast and pancreatic carcinomas [119]. Vactosertib is currently un-
dergoing clinical studies in patients with different solid tumors, with a favorable safety
profile and its efficacy is being evaluated [119,120]. In vitro studies also showed inhibitory
activity of Vactosertib in HCC cells growth [155]. Galunisertib (LY2157299) is another orally
available drug and the most extensively studied of all small molecule inhibitors of TβRI ki-
nase [156]. Galunisertib showed excellent antitumor activity in preclinical models of breast
and colon cancer, among other solid tumors [120], and also demonstrated efficacy in HCC
cell lines and experimental models [38,157,158]. Interestingly, Galunisertib treatment also
increased the efficacy of Sorafenib on HCC cells growth inhibition and apoptosis [158,159].
Extensive preclinical and phase I studies led to the clinical development of Galunisertib,
including the establishment of dosing strategies and its therapeutic window, and providing
evidence of good tolerability [160]. The clinical efficacy of Galunisertib has been recently
assessed in patients with advanced HCC who progressed on or were ineligible to receive
Sorafenib [161]. This study showed that Galunisertib had a manageable safety profile, and
that those patients in which circulating alpha fetoprotein (AFP) and TGF- β1 levels were re-
duced upon Galunisertib administration had longer survival (NCT01246986). A follow-up
study from the same team confirmed the usefulness of plasma TGF-β1 levels as a biomarker
to assess the clinical activity of this TβRI inhibitor [162]. Subsequent phase Ib and phase II
studies demonstrated that the combination of Galunisertib and Sorafenib in patients with
advanced HCC was well tolerated and prolonged overall survival [15,163,164]. Together,
these studies underscore the potential of Galunisertib for advanced HCC treatment, and
open the door for additional combinations of this drug with other antitumoral agents.

6. Current Therapies in HCC and New Perspectives for TGF-β Inhibitors

The landscape of HCC has been substantially changed with the incorporation of
Atezolizumab and Bevacizumab in 2020 as first-line treatment for advanced HCC [165].
The breakthrough impacted not only on the clinical decision-making but also into the
clinical trials design. Until 2018 when the REFLECT trial (Lenvatinib vs. Sorafenib, no-
inferiority design) was positive [166], the only first-line treatment was Sorafenib and all
evidence-based data in second-line treatments were demonstrated in patients treated with
Sorafenib as first-line treatment [167–169] (Several revisions describe these clinical trials
Cancers 2021, 13, 3248 13 of 24

characteristics and outcome [1,170,171]). However, despite currently having several options
in clinical practice, the benefits of sequential HCC treatments vary across patients and
its impact is not only related to the baseline tumor burden or related symptoms. Indeed,
despite having predictors of outcome such as baseline BCLC stage, ECOG-PS, or AFP or
evolutionary-events such us early-dermatologic adverse events in patient’s treatment with
tyrosine-kinase inhibitors [172–174] that could be associated to better outcome, the rate of
tumor progression and time to progression would be defined by factors that have not been
identified yet. So, precision oncology aims to identify molecular factors that could help
clinicians in their clinical practice at the time of defining the need of changing from one
treatment-line to the other.
In this regard, strong evidences suggest that an immunosuppressive TME may con-
tribute to therapeutic failure. Therefore, it was no wonder that drugs that block the
binding of PD-L1 to PD-1 were effective in the treatment of advanced HCC. Indeed,
Nivolumab [175] and Pembrolizumab [176] have been granted an accelerated FDA ap-
proval in second-line treatment based on radiologic tumor response. However, despite the
incredible clinical advance in HCC treatment represented by PD-1-PD-L1 interaction in-
hibitors, a recent phase III trial in second-line comparing the PD-1 inhibitor Pembrolizumab
vs. placebo failed to improve overall survival (OS) and progression-free survival (PFS) [177].
Moreover, a phase III study of Nivolumab vs. Sorafenib in first-line treatment (Checkmate
459, NCT02576509) [178] showed that HCC patients treated with Nivolumab presented
better median OS (16.4 months compared to 14.7 in the patients who received Sorafenib
[HR 0.85 (95% CI; 0.72–1.02; 0.075)] although the results did not reach statistical signif-
icance. Therefore, despite the clinical and quality of life benefits of Nivolumab, this is
another negative study. This deceiving failure of immunotherapy in HCC exposes the
specific molecular characteristics of this cancer and the need to unravel the mechanisms
of such resistance to treatment. Subsequently highlighting the urgent need for specific
biomarkers which allow to identify patients that would benefit from therapy with immune
checkpoint inhibitors.
The rise of new drugs available in HCC prompts an attractive attempt to combine
different approaches, such as the immune checkpoint inhibitors and targeted therapy
with the aim of improving response and survival. Indeed, there are currently 4 phase
III studies recruiting or pending to report results of combination treatments: LEAP-002
(Lenvatinib + Pembrolizumab vs. Lenvatinib) (NCT03713593), COSMIC-312 (Cabozantinib +
Atezolizumab vs. Sorafenib) (NCT0329851), HIMALAYA (Duravalumab ± Tremelilumab
vs. Sorafenib) (NCT03298451) and CheckMate 9DW (Nivolumab + Ipililumab vs. Sorafenib
or Lenvatinib).
In this context, it is essential to keep in mind that the rationale for combinations relies
not only on the additive therapeutic effect, but also on the potential immunomodulation
property of target agents and their impact on the immunosuppressive TME. In this sense,
targeting TGF-β1 is of particular interest due to its potent immunosuppressive effects thor-
oughly described above. However, none of the current phase I-II trials in advanced HCC
(Table 2) or the ongoing phase III trials focus on TGF-β signaling, despite data from prior
studies showing promising results of TGF-β modulation in HCC as previously mentioned.

Table 2. Current phase I-II trials in intermediated/advanced HCC.

No. Clinical Trial Population Arms N Aim Status

NCT01988493 Advanced Tepotinib vs. Sorafenib 117 Safety and TTP Active, no Recruiting
Tivozanib + Durvalumab
NCT03970616 Advanced 42 Safety Recruiting
(1st Line)
Donafenib Tosilate +
NCT04503902 Advanced 46 Safety and ORR Not yet Recruiting
Toripalimab
SynOV1.1 monotherapy
NCT04612504 Advanced vs. SynOV1.1 + 45 Safety Not yet Recruiting
Atezolizumab
Cancers 2021, 13, 3248 14 of 24

Table 2. Cont.

No. Clinical Trial Population Arms N Aim Status

TQB2450 injection +
NCT03825705 Advanced 60 ORR Recruiting
Anlotinib
GT90001 + Nivolumab
Intermediate (not lo-
NCT03893695 (dose escalation 20 Safety Active, no Recruiting
coregional)/Advanced
and expansion)
Intermediate (not lo- Toriplimab montherapy
NCT03864211 130 PFS Recruiting
coregional)/Advanced vs. toriplimab + Ablation
Intermediate (not lo- ET140203 autologous
NCT04502082 50 Safety Recruiting
coregional)/Advanced T cell product (3rd Line)
Intermediate (not lo- ET140202 autologous
NCT03998033 50 Safety Active, no Recruiting
coregional)/Advanced T cell product
Camrelizumab +
NCT04035876 Intermediate Apatinib (Downstaging 120 ORR and RFS Recruiting
for TOH)
Toripalimab + Sorafenib Safety and 6-month
NCT04069949 Advanced 39 Not yet Recruiting
(1st Line) PFS
Intermediate (not ABX196 + Nivolumab
NCT03897543 48 Safety Recruiting
locoregional)/Advanced (2nd Line)
MGD013 monotheray vs.
Intermediate (not
NCT04212221 MGD013 + Brivanib 300 Safety Recruiting
locoregional)/Advanced
(2nd Line)
KN046 + Ningetinib
NCT04601610 Advanced 70 Safety and ORR Not yet Recruiting
(1st or 2nd Line)
Fluorouracil +
Intermediate (not Nivolumab +
NCT04380545 15 Safety Not yet Recruiting
locoregional)/Advanced Recombinant Interferon
Alpha 2b-like protein
Sitravatinib monotherapy
Intermediate (not vs. Sitravatinib +
NCT03941873 104 Safety and ORR Recruiting
locoregional)/Advanced Tislelizumab
(1st o 2nd Line)
PTX-9908 + TACE vs.
NCT03812874 Intermediate 50 Safety Recruiting
PBO + TACE
Safety and
GNOS-PV02 + INO-9012 Immunogenicity of a
Intermediate (not
NCT04251117 + Pembrolizumab 24 personalized Recruiting
locoregional)/Advanced
(2nd Line) neoantigen DNA
vaccine.
Unresectable HCC/no CAR-T/TCR-T cells
NCT03941626 50 Safety Recruiting
effective treatment immunotherapy
KY1044 monotherapy vs.
NCT03829501 Advanced 412 Safety Recruiting
KY1044 + Atezolizumab
TTP: time to progression, ORR: overall response rate, PFS: progression- free survival, RFS: recurrence free survival, TACE: transarterial
chemoembolization, PBO: placebo.

Indeed, Galunisertib has been tested as second-line monotherapy in 149 HCC patients
in a phase II study (Table 3), predefining baseline AFP values as a biomarker (greater
than 400 ng/mL) (NCT01246986) [162]. The median OS was 7.3 months for patients with
AFP > 400 and 16.8 months for patients with AFP ≤ 400 ng/mL. In this study high baseline
levels of plasma TGF-β1 and E-Cadherin have also associated with poor outcome and
patients who decrease more than 20% from baseline in AFP and TGF-β1 levels had a signif-
icantly prolonged OS (21.5 months). Results of Galunisertib in combination with Sorafenib
400 mg BID were also reported in the same phase II clinical trial (NCT01246986) [164]
(Table 3). The median TTP was 4.1 months and median OS was 18.8 months. Both were
analyzed by baseline biomarker values: AFP < 400 ng/mL and ≥400 ng/mL and TGF-β1
less than or greater than/equal to baseline median. OS was significantly longer in TGF-β1
Cancers 2021, 13, 3248 15 of 24

responders (decrease more than 20% from baseline) (22.8 vs. 12 months, p < 0.038). More-
over, Bintrafusp alfa, designed to simultaneously target TGF-β and PD-L1, has shown
antitumor activity both as monotherapy and in combination with chemotherapy in preclin-
ical studies. This drug previously designated M7824 was also evaluated in Asian patients
with advanced solid tumors, including an HCC in a phase I, open-label, dose escalation
study at different doses 3, 10, and 20 mg/kg, every 2 weeks (NCT02699515) which the
primary objective was safety and tolerability and the secondary objective was best overall
response. Authors concluded that the Bintrafusp alfa had a manageable safety profile and
showed preliminary efficacy in data [152]. However, there were only 10 patients with HCC,
1 was screening failure and there is not data available from no-Asian patients.

Table 3. Clinical trials evaluating TGF-β inhibitors in advanced HCC.

No. Clinical
Population Phase Arms N Aim Status
Trial
Association of
NCT01246986 circulating AFP
Advanced Galunisertib 149 Completed
(Parts A and B) and TGF-β1
II levels with OS
NCT01246986 Safety, TTP, OS,
Advanced Galunisertib + Sorafenib 47 Completed
(Part C) PFF and ORR
Safety,
NCT02240433 Unresectable I Galunisertib + Sorafenib 14 tolerability, PK, Completed
TTP and PFS
Active, no
NCT02906397 Advanced I Galunisertib + SBRT 15 Safety, PFS, OS
Recruiting
Safety,
75
NCT02423343 Advanced I/II Galunisertib + Nivolumab tolerability, Completed
(10 HCC)
PFS, ORR
Safety,
Bintrafusp alfa MSB0011359C Active, no
NCT02699515 Advanced I 114 tolerability
(M7824) Recruiting
and ORR
Safety,
I SAR439459 monotherapy vs.
NCT03192345 Advanced ~350 tolerability, TTP Recruiting
(Basket trial) SAR439459 + Cemiplimab
and PFS
I NI5793 monotherapy vs. Active, no
NCT02947165 Advanced 120 ORR and PFS
(Basket trial) NI5793 + PDR001 Recruiting
TTP: time to progression, ORR: overall response rate, OS: Overall Survival, PFS: progression-free survival, PK: Pharmacokinetics, SBRT:
Stereotactic Body Radiotherapy.

The promising survival observed for the combination of Galunisertib with Sorafenib
supports further exploration of its potential in other combinatorial strategies, including
combination with immune checkpoint inhibitors. In this regard, preclinical studies have
shown the synergistic activity of Galunisertib and checkpoint inhibitors in different tumor
types [179], and a clinical trial is testing the combination of Galunisertib and Nivolumab in
patients with solid tumors, including HCC (NCT02423343). In addition, Reiss et al. found
that Galunisertib combined with Stereotactic Body Radiotherapy (SBRT) is well tolerated
and associated with antitumor activity in patients with HCC [180]. Besides Galunisertib
and Bintrafusp alfa, two TGF-β neutralizing antibodies are currently under evaluation in
two independent ongoing clinical trials (NCT03192345 and NCT02947165) in combination
with immune checkpoint inhibitors in HCC (Table 3).
Further research is needed to translate the current knowledge of HCC biology to
prognostic and predictive biomarkers in order to guide clinical decision to improve patient
outcomes. In this regard, analyzing the molecular landscape of tumor samples obtained
from patients starting first line and also upon progression prior to second line or to third
line is crucial to identify resistance signatures of signaling pathways and ultimately, design
novel therapeutic strategies using a personalized decision-making pipeline.
Cancers 2021, 13, 3248 16 of 24

7. Concluding Remarks
TGF-β pathway is difficult to target, considering that its inhibition in the wrong
patients could do more harm than good. However, there is no doubt about its potential as
a therapeutic option in HCC, due to the strong pro-tumorigenic effects that TGF-β might
mediate at later stages in the tumor cell and at all the stages in the liver tumor stroma.
Furthermore, TGF-β could favor immune evasion and is an interesting target to inhibit in
case of immunotherapy approaches. Nevertheless, to efficiently target the TGF-β pathway,
it is mandatory to deepen into the molecular mechanisms through which TGF-β promotes
tumor progression as well as to identify relevant biomarkers of the TGF-β oncogenic arms.

Author Contributions: Conceptualization, writing—original draft preparation and writing—review

and editing, E.G.-S., J.V., M.G.F.-B., J.J.L., M.A.A., P.S., M.R., M.C. and I.F.; visualization and supervi-
sion, E.G.-S. and I.F.; funding acquisition, E.G.-S., J.V., M.G.F.-B., J.J.L., M.A.A., P.S., M.R., M.C. and
I.F. All authors have read and agreed to the published version of the manuscript.
Funding: This study has been funded by (i) CIBEREHD through financial support to groups
(grant numbers: CB06/04/0006, CB06/04/0005 and CB17/04/00017), and through the Emergent
Investigators’ Program grant (to E.G-S., J.V., M.G.F-B. and M.R.); (ii) Agencia Estatal de Inves-
tigación (AEI), Ministry of Science and Innovation, through the “Retos Investigación grants”,
grant numbers: SAF2017-88933-R (to M.G.F-B.), RTI2018-094079-B-100 (to I.F.), PID2019-108651RJ-
I00/DOI:10.13039/501100011033 (to J.V.), PID2019-108989RB-I00 (to J.J.L.) and PID2019-104878RB-
100/AEI/10.13039/50110001103 (to M.A.A.); (iii), Fundación Científica de la Asociación Española
contra el Cáncer AECC, call AECC LAB 2020 (to M.G.F-B.); (iv) Instituto de Salud Carlos III co-
financed by European FEDER funds grant numbers PI17/00249 (to P.S.) and FIS18/00358 (to M.R.)
and (v) “Murchante contra el cáncer” initiative (to P.S.). M.G.F-B is also a recipient of a Ramón y
Cajal Program Contract (RYC2018-024475-1). The CIBER, a National Biomedical Research Institute,
is funded by the Instituto de Salud Carlos III, Spain. We thank CERCA Programme/Generalitat de
Catalunya for institutional support.
Conflicts of Interest: E.G.-S., J.V., M.G.F.-B., J.J.L., M.A.A., P.S., M.C. and I.F. declare no conflict
of interest. M.R. declare the following conflicts of interest: consultancy fees from Bayer-Shering
Pharma, BMS, Roche, Ipsen, AstraZeneca, Lilly and BTG; lecture fees from Bayer-Shering Pharma,
BMS, Gilead, Lilly and Roche; research grants from Bayer-Shering Pharma and Ipsen. The funders
had no role in the writing of the manuscript.

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