Targeting TGF-β Signaling in Cancer
Selcuk Colak1,* and Peter ten Dijke1,2,3,*
The transforming growth factor (TGF)-β signaling pathway is deregulated in many diseases, including cancer. In healthy cells and early-stage cancer cells, this pathway has tumor-suppressor functions, including cell-cycle arrest and apoptosis. However, its activation in late-stage cancer can promote tumorigen- esis, including metastasis and chemoresistance. The dual function and pleio- tropic nature of TGF-β signaling make it a challenging target and imply the need for careful therapeutic dosing of TGF-β drugs and patient selection. We review here the rationale for targeting TGF-β signaling in cancer and summarize the clinical status of pharmacological inhibitors. We discuss the direct effects of TGF-β signaling blockade on tumor and stromal cells, as well as biomarkers that can predict the efficacy of TGF-β inhibitors in cancer patients.
TGF-β Signaling: An Emerging Player in Cancer Progression
Activation of the TGF-β signaling pathway induces potent cell-cycle arrest in healthy non- cancerous cells and in early-stage cancerous cells, suggesting that this pathway plays a prominent role in tumor suppression. However, elevated TGF-β expression and activation of TGF-β receptor-initiated intracellular signaling is observed in many cancers [1]. Interestingly, patient stratifi cation based on gene expression studies reveals high activity of the TGF-β pathway in cohorts with the worst prognosis [2,3]. Accordingly, the activation of this pathway in cancer cells can induce epithelial-to-mesenchymal transition (EMT) in which epithelial cells lose their apicobasal polarity and cell-cell adhesion, and take on characteristics of motile mesenchymal cells [4]. Recently, using high-resolution intravital imaging in an invasive ductal carcinoma mouse model, EMT was shown in vivo. A transient acquirement of a mesenchy- mal state is key for tumor cell migration, but not for metastatic outgrowth itself [5]. In addition to its importance in migration, EMT has also been linked to chemotherapy resistance [6,7]
and to tumor cell evasion of immune surveillance [8]. TGF-β is frequently produced in high amounts by tumor and stromal cells, and in turn may also stimulate tumorigenesis by promoting angiogenesis and suppressing the immune system. This suggests that cancer progression prompts the switch of TGF-β signaling from being tumor-suppressive to tumor- promoting [9–11].
Identifi cation of a tumor-promoting role for TGF-β led to clinical trials testing TGF-β inhibitors in different types of cancers [12]. To select patients that can benefit from therapy and minimize on- target side effects, it is crucial to understand the mechanisms that underlie the dichotomous role of TGF-β and the cell type-specific effects of TGF-β inhibition. We present here an overview of TGF-β targeting agents currently being tested in cancer clinical trials, and the effects of TGF-β pathway activation in tumor and stromal cells. We also discuss biomarkers for patient stratifi- cation, such as genetic profiling of tumor biopsies, circulating tumor DNA, and circulating tumor cells. Such biomarkers can be used to select patients who will benefit from TGF-β pathway inhibitors, frequently those with cancer cells expressing a mesenchymal phenotype, and to follow the responses of patients to therapy.
Trends
TGF-β has a dual action in cancer as a tumor suppressor and a tumor promoter.
As a tumor suppressor, it inhibits tumorigenesis by inducing growth arrest and apoptosis.
As a tumor promoter, it induces tumor cell migration and stimulates epithelial to mesenchymal transition.
TGF-β also promotes tumorigenesis indirectly by acting on the tumor microenvironment.
Epithelial-to-mesenchymal transition induced by TGF-β contributes to a chemoresistant phenotype.
1Department of Molecular Cell Biology, Cancer Genomics Centre
Netherlands, Leiden University Medical Center, Postbus 9600, 2300 RC Leiden, The Netherlands
2Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Uppsala, Sweden 3Department of Cancer Signaling, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan
*Correspondence: [email protected] (S. Colak) and [email protected] (P. ten Dijke).
56 Trends in Cancer, January 2017, Vol. 3, No. 1 http://dx.doi.org/10.1016/j.trecan.2016.11.008 © 2016 Elsevier Inc. All rights reserved.
Canonical TGF-β/SMAD and Non-SMAD Signaling
The TGF-β signaling pathway can be activated upon interaction of dimeric TGF-β ligand with its specific cell-surface transmembrane receptors that are endowed with intrinsic serine/threonine kinase activity [13,14]. Three TGF receptor ligands have been identified, TGF-β1, 2, and 3, which largely have similar but not identical biological activities in vitro. The individual knockout phenotypes are distinct, and this may be attributed to their differential expression patterns [15]. TGF-βs are secreted by cells and sequestered in an inactive form in the matrix, and can be activated in an integrin-dependent manner [16]. Activated TGF-β ligands interact with TGF-β type II receptors (TβRII), which recruit and phosphorylate the TGF-β type I receptors (TβRI, also termed activin receptor-like kinase ALK5) at specifi c serine and threonine residues. In turn, activated TβRI phosphorylates SMAD2 and SMAD3 at C-terminal serine residues. Phosphory- lated SMAD and SMAD3 can then assemble into heterodimeric and trimeric complexes with SMAD4, and then translocate to the nucleus where they regulate the expression of TGF-β target genes, including the induction of plasminogen activator inhibitor 1 (PAI1) expression (Figure 1). Whereas SMAD3 and SMAD4 bind directly to DNA, SMAD2 (i.e., the predominant splice variant containing exon 3 that disrupts DNA binding by the encoded protein) does not. However, the affinity of SMAD3 and SMAD4 for DNA is relatively weak and they need to cooperate with other DNA-binding transcription factors to regulate gene expression [13,14]. In addition to canonical SMAD signaling, non-SMAD signaling can also be initiated downstream of TGF-β receptors [17].
TGF-β/SMAD signaling pathway TGF-β/non-SMAD signaling pathway
TGFβ TGFβ
P P
PI3K
SMAD7
RHO ?
P
TRAF4/6
SHC
GRB2
SOS
SMAD4
P
SMAD2/3
Target p15↑, p21↑, Myc ↓
BIM↑, BMF↑,
Response Cell cycle arrest
Apoptosis
Tumor suppression
ROCK
LIMK
P
PAR6
SMURF
TAK1
MKK MKK
RAS
RAF
MEK
AKT
mTOR
P
SMAD2/3 SMAD2/3 SMAD4
P
DAPK↑
SNAIL↑, SLUG↑, IL11↑, PTHrP↑, CTGF↑
EMT Osteolytic Angiogenesis
Tumor promoting
cofilin Actin
cytoskeletal
RHO Tight-
junction
P38 JNK
Transcriptional regulation
ERK
S6K Translational
control
changes resolution
Figure 1. TGF-β/SMAD and Non-SMAD Signaling Pathways. The TGF-β signaling pathway is initiated by binding of TGF-β to TβRII, which recruits and phosphorylates TβRI. Subsequently, either SMAD or non-SMAD signaling pathways can be activated. While two receptor complexes for each pathway are shown, both pathways can be activated from the same complex. In the canonical SMAD pathway TβRI phosphorylates SMAD2 and SMAD3, which subsequently assemble into heterodimeric and trimeric complexes with SMAD4. These complexes translocate to the nucleus where they regulate the expression of TGF-β signaling pathway target genes. Various non-SMAD signaling pathways can also be activated downstream of TGF-β receptors. Some of these pathways, for example RHO, p38, JNK, and ERK MAPK, are illustrated here. Abbreviations: BIM, BCL-2 interacting mediator of cell death; BMF, Bcl-2-modifying factor; CTGF, connective tissue growth factor; DAPK, death-associated protein kinase; EMT, epithelial–mesenchymal transition; ERK, extracellular signal-regulated kinase; GRB2, growth factor receptor-bound protein 2; IL-11, interleukin 11; JNK, c-Jun N-terminal kinase; LIMK, LIM kinase; MEK/MKK, mitogen-activated protein kinase kinase; mTOR, mechanistic target of rapamycin; P, phosphorylation; PAR-6, partitioning-defective protein 6; PI3K, phosphatidylinositol-4,5-bisphosphate 3-kinase; PTHrP, parathyroid hormone-related peptide; TβR, transforming growth factor (TGF)-β receptor; TRAF, TNF receptor-associated factor; ROCK, RHO-associated coiled-coil containing protein kinase; SHC, SRC homology 2 domain-containing transforming protein; SMAD, SMA- and MAD-related protein; SMURF, SMAD-specifi c E3 ubiquitin protein ligase; SOS, son of sevenless; TAK-1, TGF-β-activated kinase-1.
As illustrated in Figure 1, non-SMAD signaling can lead to the activation of various pathways such as PI3K as well as JNK, P38, and ERK MAP kinases. For example, activated TβRI can directly phosphorylate SRC homology domain 2-containing protein (SHC) on serine and tyrosine residues, which in turn then recruits the proteins GRB2 and SOS, and subsequently activates ERK MAPK signaling [18].
TGF-β is part of a larger family of structurally and functionally related proteins that include activins and bone morphogenetic proteins (BMPs). All TGF-β family members are multifunctional and their effects are highly dependent on the cellular context [19,20]. In part, this complexity is mediated by crosstalk between TGF-β family/SMAD proteins and other signaling pathways. For example, there is a large repertoire of SMAD-interacting proteins that include nuclear effectors of Wnt, Hedgehog, Hippo, cytokine/JAK, and growth factor/receptor tyrosine pathways [21]. In this review we focus on TGF-β; in studies where specific TGF-β isoforms were used and are important, this is indicated; otherwise we refer to TGF-β. The role of the three isoforms in cancer can be different. For example, in breast cancer and glioblastoma elevated expression of TGF-β1 and TGF-β2 has been linked to poor clinical outcome, whereas high TGF-β3 expression in breast cancer suggests a protective function [15,22]. For the role of other TGF-β family members in cancer see an excellent recent review [23].
TGF-β Signaling in Cancer and Stromal Cells
In a pre-malignant stage, TGF-β secreted by tumor and/or stromal cells can induce cell-cycle arrest, promote apoptosis, and thereby play a tumor-suppressive role. Cancer cells often bypass these cytostatic/apoptotic effects by mutating key players along the TGF-β pathway. TβRII is frequently mutated in colorectal cancer (CRC) cells from a subgroup of patients with methylation or mutation of genes encoding mismatch repair proteins [microsatellite instable (MSI) patients] [24]. Interestingly, in some MSI CRCs, cells with mutated receptor remain responsive to TGF-β [25]. Inactivating mutations in the SMAD2 gene have been reported in hepatocellular carcinoma (HCC), CRC, and lung cancer [26,27]. Inactivating mutations in the SMAD4 gene are found in CRC and HCC, and SMAD4 is deleted in a high percentage of pancreatic cancers. Obviously, in cancer cells with SMAD mutations, non-SMAD signaling may still be activated upon binding of TGF-β to its receptor [17]. Recently, David et al. showed in pancreatic ductal adenocarcinoma (PDA) that loss of SMAD4 leads to the breakup of a pro- apoptotic EMT transcriptional program. In PDA cells with intact SMAD4, TGF-β-induces the expression of sex-determining region Y-box 4 (SOX4) and represses Krüppel-like factor 5 (KLF5) in a SMAD-dependent and -independent manner, respectively. KLF5 prevents SOX4-mediated apoptosis and, in the absence of SMAD4, KLF5 cooperates with SOX4 in oncogenesis [28].
Tumors that have lost core pathway components such as TGF-β receptors have a growth advantage, but may not be highly invasive [29]. Particular advanced tumors, such as melano- mas, gliomas, and breast cancer, have retained an intact TGF-β receptor/SMAD pathway but become selectively resistant to the cytostatic effects of TGF-β by acquiring oncogenic mutations in the PI3K/AKT, RAS/MAPK, or p53 pathways [1]. In these tumors the subverted TGF-β/SMAD pathway is utilized to promote EMT, invasion, and metastasis. Transcriptional repressors of E-cadherin, such as SNAIL and SLUG that mediate EMT, can be induced in these tumors by TGF-β in a SMAD-dependent manner [30]. In addition, TGF-β/non-SMAD signaling (and the activation of proto-oncogenes) can collaborate with the SMAD pathway to foster tumor progression.
TGF-β also plays important roles in regulating stromal cells in the tumor microenvironment (Figure 2). TGF-β has potent immunosuppressive effects on both innate and adaptive immune cells, including dendritic cells, macrophages, natural killer cells, and CD4+ and CD8+ T cells. Moreover, TGF-β stimulates the differentiation of immune-suppressive regulatory T (Treg) cells
Tumor suppression
Normal epithelial cells
Cell-cycle arrest
apoptosis
TGF-β
E-cadherin
ZO-1 Tumor cells
Immune cells
Immuno suppression
EMT
TGF-β
Metastatic tumor cells
Tumor growth,
metastasis, therapy resistance
Cancer-associated
fibroblasts
TGF-β TGF-β
N-cadherin
Tumor promotion
vimentin
Angiogenesis
Endothelial cells
Figure 2. Regulation of (Non-)Cancerous cells and Microenvironmental Cells by Transforming Growth Factor (TGF)-β. TGF-β is an important player in cancer progression and it is expressed by stromal cells including cancer- associated fi broblasts (CAFs) and tumor cells. TGF-β has plethora of effects, and in normal cells it induces cell-cycle arrest and/or apoptosis. However, its expression is increased in high-stage cancers and here it can stimulate the epithelial to mesenchymal transition (EMT). In this morphological process, TGF-β induces a switch from epithelial [e.g., E-cadherin and zonula occludens-1 (ZO-1)] to mesenchymal (e.g., N-cadherin and vimentin) marker expression, and promotes the formation of actin stress fi bers that replace the peripheral actin cytoskeleton. EMT is thought to foster tumor cell migration and invasion, and also plays an important role in conferring therapy resistance. Moreover, TGF-β can contribute to tumor progression by stimulating immune evasion and promoting angiogenesis.
[31]. Together with interleukin-6 (IL-6), TGF-β promotes the differentiation of IL-17-producing CD4+ T helper cells and CD8+ cytotoxic T cells [32]. Interfering with TGF-β signaling can promote antitumor immunity. In B cell acute lymphoblastic leukemia (B-ALL), tumor cells express TGF-β, which inhibits natural killer (NK) cell function, and tumor cells can thereby evade immune surveillance. Inhibition of TGF-β in B-ALL rescues NK cell function [33]. Inhibition of TGF-β in breast cancer mouse models inhibits IL-17 expression by CD8+ T cells and takes away survival signals, leading to decreased primary and metastatic tumors [34]. Combining radiation therapy with TGF-β pathway inhibitory antibodies also improves response to therapy by overcoming immunosuppression by TGF-β and by stimulating the CD8+ T cell response to tumor antigens [35]. TGF-β can also contribute to tumor suppression indirectly by affecting cytokine production by tumor cells. Inactivation of TGF-β signaling by SMAD4 deletion in T cells leads to gastroin- testinal tumors in mice [36]. SMAD4 inactivation is observed in majority of patients with juvenile polyposis syndrome [37,38]. At a young age these patients have several benign intestinal polyps, which can become malignant. Therefore, Juvenile polyposis syndrome patients have a high risk of developing CRC [38]. Deleting one allele of TβRI (Tgfbr1) in a CRC mouse models causes increased inflammation that correlates with more polyps and decreased survival [39].
Tumor angiogenesis ensures sustainable tumor growth and better diffusion of nutrients and removal of waste products. TGF-β can also have dual inhibitory or stimulatory effects on tumor vessel growth depending on the cellular context [40]. The stimulatory effect of TGF-β is mediated
by activation of the type I receptor ALK1 and the auxiliary TGF-β receptor endoglin that are selectively expressed on endothelial cells. Antagonists of ALK1 and endoglin function, such as neutralizing antibodies and ligand traps in which the extracellular domains are fused to immu- noglobulin, have been developed and show efficacy in inhibiting tumor growth and angiogene- sis. We refer to recent reviews for a discussion of clinical studies of these targeting reagents in cancer patients [41,42].
Cancer-associated fibroblasts (CAFs) are the most abundant cell type in the microenvironment of many types of tumors. Knockout of TβRII (Tgfbr2) in CAFs leads to prostate intraepithelial neoplasia and squamous cell carcinoma in mouse models [43]. Similarly, coinjection of mam- mary cancer cells with fibroblasts lacking TβRII results in enhanced tumor growth and metas- tasis [44,45]. By contrast, lung fibroblasts respond to TGF-β3 expressed by breast cancer cells by secreting factors that stimulate a pre-metastatic niche [46]. Hence, in this model, inhibition of TGF-β3 correlates with less metastasis [46]. Vermeulen and colleagues showed that a subtype of CRC patients with poor prognosis are characterized by high TGF-β target gene expression [47] (see also discussion below on biomarkers). It was suggested that CRC patients with CAFs exhibiting high TGF-β pathway activity are prone to develop metastatic lesions [3]. IL-11 produced by CAFs in response to TGF-β provides a survival advantage to tumor cells by activating the signal transducer and activator of transcription (STAT)1 pathway [48]. Activation of TGF-β signaling can also differentiate stromal mesenchymal stem cells (MSCs) into myofibro- blasts that secrete extracellular matrix proteins and fibrogenic growth factors to support tumor growth [49–51]. For example, conditioned medium from breast cancer cells and TGF-β1 expressed by prostate cancer cells differentiate MSCs into CAFs and stimulate tumor growth [50,52]. In mouse models of gastric cancer, a proportion of CAFs also originate from bone marrow-derived MSCs in a process mediated by TGF-β [53]. In addition to MSCs, endothelial cells can change into tumor-facilitating fibroblast-like cells upon activation of TGF-β pathway, via a process termed endothelial to mesenchymal transition (EndMT) [54]. Thus, TGF-β can regulate stromal cells, making this pathway an interesting but also challenging target in cancer. Increasing evidence shows that CAFs and infl ammation can promote or inhibit tumorigenesis. For instance, deletion of IKKβ (Ikbkb) in intestinal fibroblasts increases tumor formation in CRC mouse models. This is due to impaired expression of SMAD7 and SMAD-specific E3 ubiquitin protein ligase (SMURF)1, two negative regulators of TGF-β signaling, resulting in enhanced TGF-β target gene expression [55]. However, in another report, in which a different Cre recombinase driver was used to delete IKKβ in intestinal MSCs, decreased tumor growth in colitis-associated cancer, while such tumor growth was not observed in a spontaneous CRC mouse model [adenomatous polyposis coli (APC)-deficient mice] [56].
Targeting TGF-β and Its Receptors in Cancer
The potent pro-oncogenic roles of TGF-β stimulated the development of anticancer drugs targeting TGF-β signaling. Pharmacological TGF-β targeting compounds include antisense oligonucleotides (AONs), neutralizing antibodies that inhibit ligand–receptor interactions, recep- tor domain-immunoglobulin fusions that sequester ligands and prevent binding to receptors, and receptor kinase inhibitors (Figure 3A) [11]. These agents are not selective for pro-oncogenic responses and inhibit all TGF-β-induced or selective TGF-β isoform-induced signaling effects. Considering the tumor-suppressive and pleiotropic effects of TGF-β, careful screening of patients is warranted to minimize unwanted on-target side effects. Another issue is that TGF-β targeting agents by themselves are not cytotoxic to tumor cells but are aimed at curtailing the excessive TGF-β pathway activation in cancer and stromal cells that drives tumor cell invasion and metastasis [11].
In many preclinical studies, TGF-β targeting agents have shown potent antitumor effects. For example, the development of lung and bone metastases from breast tumors is inhibited by
(A) (B)
TGF-β targeting agents related toxicities
Fresolimumab
Lerdelimumab Fresolimumab
Cardiac toxicity: hemorrhagic, degenerative, and inflammatory lesions in heart valves
Metelimumab LY2382770
TGF β1
Skin toxicity: eruptive keratoacanthomas and hyperkeratosis, cutaneous squamous-cell carcinomas,
TGF β2
TGF β2
and basal cell carcinoma
IMC-TR1 (C) Galunisertib/LY2157299
N
H N
2
Belagen- pemutacel-L Trabedersen
TGF-β2
GW788388
P
LY2157299 Ki26894 SD208 SM16 GW788388
O
28 days cycle
2 × 150 mg
N
N
N
28 days cycle
2 × 150 mg
mRNA
P
SMAD2/3
daily treament
daily treament
SMAD4
14 days ON
14 days
OFF
14 days ON
14 days
OFF
TGF-β2 gene
P
SMAD2/3 SMAD2/3
SMAD4
P
No cardiac toxicity Antitumor activity
Figure 3. Mode of Action of TGF-β Targeting Agents.
For a Figure360 author presentation of Figure 3, see http://dx.doi.org/10.1016/j.trecan.2016.11.008#mmc2.
(A) Various TGF-β targeting agents including antibodies and small-molecule inhibitors are depicted. (B) TGF-β targeting agents related toxicities. Fresolimumab, a pan- specific TGF-β neutralizing antibody can induce skin toxicity, such as cutaneous keratoacanthomas and squamous cell carcinomas [67,116]. The small-molecule TGF-β receptor kinase inhibitor Galunisertib/LY2157299 can lead to cardiac toxicity such as increased frequency of bleedings and infl ammatory lesions in the heart valves [68]. (C) An intermittent dosing regimen with galunisertib overcomes cardiac toxicity problems observed with TGF-β inhibitors. Cancer patients were treated for 14 days with galunisertib, followed by 14 days with no treatment. The drug is given orally twice per day, in the morning and evening, to circumvent high exposures. Patients who underwent this treatment regimen schedule with galunisertib did not show any sign of cardiac toxicity but did demonstrate antitumor activity.
LY2109761 (a chemical inhibitor of TβRI and TβRII kinases) or by 1D11 (a TGF-β neutralizing antibody) [12,57]. The TβRI kinase inhibitor SM16 synergizes with an agonistic OX40 antibody to mitigate established breast cancer tumors and metastasis [58]. Treatment of mice with LY2157299 (a clinically used TβRI kinase inhibitor; galunisertib) in patient-derived xenograft (PDX) models with various tumor types demonstrated that the antitumor effect was in part dependent on the tumor microenvironment [59]. In comparison to the reported antitumor effects by TGF-β targeting agents in xenograft models with human cancer cell lines, the majority of PDX showed no or even stimulatory tumor growth response. By contrast, in immune-competent mouse models, the effect is anti-metastatic or is directed to the primary tumor cells [12,60,61]. These contradictory observations can be explained by an impaired activation of antitumor immune response in the immunocompromised animals in response to TGF-β inhibition [62]. Furthermore, studies in immune-competent mice showed that long-term inhibition of TGF-β is without adverse effects [63]. Based on the overall highly positive data obtained in animal models, clinical trials with agents targeting TGF-β were initiated (Table 1). So far, these trials have shown both positive and negative results that are summarized below.
Trabedersen, a TGF-β2 specific phosphorothioate AON, was investigated in a dose-escalation study with patients with recurrent or refractory high-grade glioma, and no treatment-related life- threatening toxicities were reported [64]. In a Phase IIb study, 2 year survival with 10 mM
Table 1. Overview of TGF-β Targeting Agents in Clinical Trials for the Treatment of Different Types of Cancers
Drug
Targets
Cancer type
Clinical trial reference numberi
Phase
Antisense oligonucleotides
Trabedersen (AP12009) TGF-β2 Recurrent or refractory anaplastic astrocytoma, or secondary glioblastoma multiforme NCT00761280 Phase III
Trabedersen (AP12009) TGF-β2 High-grade melanoma, pancreatic cancer, and CRC NCT00844064 Phase I
Trabedersen (AP12009) TGF-β2 Recurrent high-grade glioma NCT00431561 Phase II
Belagenpumatucel-L (Lucanix) TGF-β2 Non-small cell lung cancer NCT00676507 Phase III
Belagenpumatucel-L (Lucanix) TGF-β2 Non-small cell lung cancer NCT01279798 Phase III
Belagenpumatucel-L (Lucanix) TGF-β2 Non-small cell lung cancer NCT01058785 Phase II–IV
Antibodies
Fresolimumab (GC-1008) TGF-β1 + TGF-β2 Relapsed malignant glioma NCT01472731 Phase II
Fresolimumab (GC-1008); TGF-β1 + TGF-β2 Metastatic breast cancer NCT01401062 Phase II
Fresolimumab (GC-1008) TGF-β1 + TGF-β2 Early-stage non-small cell lung cancer NCT02581787 Phase I/II
Fresolimumab (GC-1008 TGF-β1 + TGF-β2 Renal cell carcinoma and malignant melanoma NCT00356460 Phase I
Fresolimumab (GC-1008) TGF-β1 + TGF-β2 Malignant melanoma NCT00923169 Phase I
Fresolimumab (GC-1008); TGF-β1 + TGF-β2 Pleural malignant mesothelioma NCT01112293 Phase II
IMC-TR1 TβRII Advanced solid tumors NCT01646203 Phase I
PF-03446962 ALK1 Refractory metastatic colorectal cancer NCT02116894 Phase I
PF-03446962 ALK1 Relapsed or refractory urothelial cancer NCT01620970 Phase II
PF-03446962 ALK1 Malignant pleural mesothelioma NCT01486368 Phase II
PF-03446962 ALK1 Advanced solid tumors NCT00557856 Phase I
TβR kinase inhibitors/small-molecule inhibitors
LY2157299 (Galunisertib) TβRI Hepatocellular carcinoma NCT02240433 Phase I
LY2157299 (Galunisertib) TβRI Pancreatic neoplasms NCT02154646 Phase I
LY2157299 (Galunisertib) TβRI Metastatic prostate Cancer NCT02452008 Phase II
LY2157299 (Galunisertib) TβRI Rectal adenocarcinoma NCT02688712 Phase II
LY2157299 (Galunisertib) TβRI Advanced solid tumors NCT01722825 Phase I
LY2157299 (Galunisertib) TβRI Advanced hepatocellular carcinoma NCT02178358 Phase II
LY2157299 (Galunisertib) TβRI Advanced hepatocellular carcinoma NCT01246986 Phase II
LY2157299 (Galunisertib) TβRI Recurrent glioblastoma NCT01582269 Phase II
LY2157299 (Galunisertib) TβRI Advanced solid tumors NCT01373164 Phase I/II
LY2157299 (Galunisertib) TβRI Metastatic breast cancer NCT02538471 Phase II
LY2157299 (Galunisertib) TβRI Recurrent malignant glioma NCT01682187 Phase I
LY2157299 (Galunisertib) TβRI Triple-negative metastatic breast cancer NCT02672475 Phase I
Table 1. (continued)
Drug Targets Cancer type Clinical trial reference numberi Phase
LY2157299 (Galunisertib) TβRI Metastatic pancreatic cancer NCT02734160 Phase I
LY2157299 (Galunisertib) TβRI Advanced solid tumors NCT02423343 Phase I/II
LY2157299 (Galunisertib) TβRI Advanced solid tumors NCT02304419 Phase I
LY2157299 (Galunisertib) TβRI Metastatic pancreatic cancer NCT02734160 Phase I
TEW-7197 TβRI Advanced solid tumors NCT02160106 Phase I
trabedersen was 39% compared to 22% with standard chemotherapy treatment [65]. AONs with improved selectivity and chemical properties are being generated for cancer and other indicationsii. The allogenic tumor cell vaccine belagenpumatucel-L is generated from four non- small-cell lung cancer (NSCLC) cell lines that were transfected with a TGF-β2 antisense vector. A Phase III study demonstrated that it was well tolerated and superior in improving the survival of NSCLC patients compared to the placebo control arm. However, this effect was limited to patients who had completed their chemotherapy within 12 weeks [66].
Fresolimumab (GC1008) is a pan-TGF-β antibody that was tested in a Phase I trial of 28 patients with malignant melanoma and one patient with renal cell carcinoma [67]. Seven patients showed partial response or stable disease, four patients had reversible cutaneous keratoacanthomas and squamous-cell carcinomas, and one patient had hyperkeratosis. LY3022859 (IMC-TR1), an antibody that targets TβRII, suppresses primary tumor and metastasis in xenograft models by antagonizing the effects of TGF-β on tumor cells as well as on surrounding stromal cells in breast, pancreatic, and colon cancer models [62]. LY3022859 is being tested in Phase I clinical trials for solid cancers, but the outcome has not yet been published.
Multiple TβRI kinase inhibitors have been identified, of which some also inhibit TβRII kinase activity. However, few have been tested in clinical trials because they elicited on-target cardiac toxicity in animals. This observation also severely hampered the clinical use of galunisertib [68], the furthest along in the clinical development of the many TβRI kinase inhibitors with distinct chemical structures that have been described [12]. The success of moving galunisertib forward was largely based on the development of an intermittent dosing schedule by predictive pharmacology, pharmacodynamic markers, and preclinical toxicology in animals (Figure 3B) [69,70]. The optimal therapeutic efficacy/safety window was seen in patients treated with iterative cycles (one cycle being 28 days) of 2 weeks of treatment with galunisertib followed by 2 weeks with no drug. A fi rst-in-human dose (FHD) study tested galunisertib using this intermittent protocol mainly in patients with glioma, and reported no adverse cardiac events or other toxicities [70–72]. Antitumor responses were observed, with some patients demonstrating persistent tumor control as measured by radiography. Analysis of tissues of a subset of patients revealed that patients with IDH1 mutation responded to galunisertib [72]. Glioblastoma patients with IDH1 mutations have secondary or low-grade tumors [73] that are associated with a mesenchymal phenotype [74]. A Phase II study compared galunisertib as monotherapy or combined with lomustine (chemotherapy) to monotherapy with lomustine in patients with recurrent glioblastoma, and reported no difference in median survival between these three groups [75]. In part, these negative results can be explained by the lack of preselection of patients to the trial – for example, selection based on a mesenchymal phenotype. Another possibility could be related to the effect of the chemotherapy. Lomustine is as an alkylating agent
that affects the survival of immune cells such as lymphocytes. Depleting lymphocytes in combination with galunisertib may have removed an important immune response. Galunisertib has also been tested in Phase II clinical trials for the treatment of pancreatic cancer and HCC. Treatment of unresectable pancreatic cancer patients with galunisertib in combination with gemcitabine (104 patients) or gemcitabine in combination with placebo (52 patients) showed improved overall survival (10.9 vs 7.2 months) in the galunisertib/gemcitabine group compared to the gemcitabine/placebo group [76] (NCT01373164i). In another Phase II study, 40 HCC patients who had progressed or were ineligible to receive sorafinib were treated with intermittent dosing of galunisertib. The 74% patients that showed a decline of >20% of TGF-β1 biomarker levels demonstrated a median overall survival of 21.8 months vs 7.91 months for patients with a reduction in TGF-β1 levels of less than 20% [77] (NCT01246986i). TEW-7197 is another TβRI kinase inhibitor whose efficacy is currently being tested in clinical trials in various cancers, including breast cancer, melanoma, HCC, and glioblastoma (NCT02160106i).
Resistance and Combination Therapy
Reversing Therapy Resistance
Profiling of clinical samples from cancer patients indicated an association between the mesen- chymal phenotype of tumor cells with resistance to chemo- and targeted therapy in CRC [78]. Interestingly, several recent studies have implicated overactivation of TGF-β as the underlying causative mechanism. Bernards and colleagues elucidated that TβRII activation induced by mediator complex subunit (MED)12 knockdown can cause resistance to receptor tyrosine kinase (RTK) inhibitors and chemotherapy in multiple cancer cells. MED12 sequesters TβRII into the cytoplasm such that this receptor cannot engage in signaling at the plasma membrane [78,79]. Treatment with LY2157299 sensitized MED12 knockdown cells to RTK inhibitors [79]. A shRNA screen identified the SOX10 gene as the cause of vemurafenib (mutant B-RAF kinase inhibitor) resistance in melanomas. Similarly to MED12, SOX10 knockdown led to enhanced TGF-β pathway activation and resistance to vemurafenib [80] (Figure 4A).
Combinatorial Synergy
Arteaga and coworkers analyzed pre- and post-chemotherapy RNA expression in primary breast cancer, revealing increased TGF-β signaling in the post-chemotherapy samples. Treat- ment of triple-negative breast cancer xenografts with LY2157299 increased the efficiency of paclitaxel by mitigating cancer stem cell expansion and release of IL-8 (Figure 4B) [81]. Furthermore, Serova et al. treated tumor slices from HCC with sorafenib (tyrosine kinase inhibitor) or with sorafenib in combination with galunisertib, and found that combining the two drugs increased apoptosis and decreased proliferation [82]. In another study, Erlotinib (EGF receptor kinase inhibitor)-resistant NSCLC cells displayed increased expression of TGF- β2. Dual treatment with the TβRI inhibitor LY364947 and erlotinib suppressed the motility of these NSLCL cells [83].
Sensitizing Radiotherapy
Tumor-initiating cells (TICs) drive tumor progression in many solid tumors. Upon injection, these cells form tumors in xenografts that recapitulate human malignancy. Compared to their differ- entiated progeny cells, TICs are highly resistant to chemotherapy [84,85]. It is crucial to target these highly tumorigenic and therapy-resistant cells to achieve a sustained therapy response. TGF-β-induced EMT can generate cancer cells with the properties of TICs [86]. Galunisertib can target CD44high/ID1high glioma-initiating cells in glioblastoma [87]. Hardee et al. [88] showed that TβRI inhibition led to increased radiosensitivity in glioma cell lines (Figure 4C). Interestingly, GBM- derived TICs that were pretreated with LY364947 before radiation showed decreased formation of primary and secondary neurospheres, suggesting that this combination treatment targets TICs in glioblastoma. Glioblastoma TICs express higher TGF-β levels compared to cells that are not grown under TIC culture conditions, and TGF-β secretion promotes stemness and
(A)Reversing therapy resistance
WT MED12 WT SOX10
MED12 or SOX10 mutations or loss
MED12 or SOX10 mutations or loss
TGF-β
TGF-β pathway normal
Cell death
TGF-β pathway activation
Cell survival
inhibitor
TGF-β pathway inhibited
Cell death
(B)Combinatorial synergy
TGF-β
Tumor cell
pathway activation
Tumor cell
Pacli
taxel
(triple-negative breast cancer)
Pacli
taxel
IL-8
(triple-negative breast cancer)
TGF-β
Cell inhibitor
survival Cell death
(C)Sensitizing radiotherapy
Decreased
Tumor cell (GBM)
TGF-β inhibitor
DNA damage response
Radio-
+
therapy Decreased
self-renewal
GBM TIC Cell death
Figure 4. Transforming Growth Factor (TGF)-β Targeting Agents Increase the Efficacy of Cancer Therapy. (A) Genetic alterations leading to inactivation of the mediator complex subunit 12 (MED12) or SRY-Box 10 (SOX10) genes enhance TGF-β signaling. As a result, the cells become resistant to therapy. Inhibition of TGF-β resensitizes the cells to therapy. (B) Upon treatment of breast cancer cells with paclitaxel, cells overactivated the TGF-β signaling pathway, leading
(Figure legend continued on the bottom of the next page.)
radioresistance in glioblastoma TICs. Overall, these results provide a strong rationale for combinatorial targeting of chemo-/radiotherapy with TGF-β inhibitors. More (pre)clinical studies are needed, for example using biomarkers and toxicity assays, to define whether to administer the drugs simultaneously or sequentially.
Personalized Medicine and Biomarkers
It is of crucial importance to fi nd ways to select patients who will benefi t from treatment with TGF-β inhibitors and to predict therapy responses. Several reports indicate that plasma levels of TGF-β ligands correlate with disease progression. High plasma TGF-β1 correlates with reduced overall survival in CRC and breast cancer patients [48,89], and are associated with disease progression in patients with glioblastoma and HCC [90,91]. Gene expression profi ling of tumor biopsies showed that signatures indicative of high TGF-β signaling are of prognostic value for breast cancer patients [92,93]. In addition, the expression levels of a variety of TGF-β target genes can predict outcome in HCC patients [94]. Stratifi cation studies of clinical samples demonstrated that, among CRC patients, tumor relapse mainly occurs in the mesenchymal subgroup of patients characterized by high TGF-β target gene expression (Figure 5) [47,95]. In an experimental organoid model system for sessile serrated adenomas (SSAs), TGF-β was shown to play a key role in directing SSAs to a poor-prognosis mesenchymal phenotype [96,97]. Together, these fi ndings suggest a worse prognosis among patients with high expression of TGF-β target genes. Interestingly, in addition to CRC, mesenchymal subtypes have been identifi ed in many other cancers including glioblastoma [98], pancreatic [99], breast [100], ovarian [101], and NSCLC [102]. These patients may particularly benefi t from treatment with drugs targeting TGF-β.
It is also possible that specific regulators of the TGF-β pathway are misexpressed and confer high TGF-β signaling activity in tumor cells. The deubiquitinating enzyme ubiquitin-specific peptidase 15 (USP15) is amplifi ed at the gene level in various cancers, including glioblastoma, breast, and ovarian cancer. USP15 deubiquitinates and thereby stabilizes TβRI, leading to enhanced TGF-β signaling. In xenograft studies, Eichhorn et al. [103] reported that USP15 depletion in glioblastoma TICs resulted in decreased TGF-β signaling and tumor-forming capacity. With USP15 amplification endowing poor prognosis to glioblastoma patients, the mesenchymal-subtype patients with elevated USP15 expression may in particular benefit from TGF-β targeted agents.
Immunohistochemical staining for phospho-SMAD2 (pSMAD2), which is indicative of TGF-β activity, can also provide insight into TGF-β signaling levels in tumors. Staining of glioma tumor biopsies demonstrated that pSMAD2 levels correlated with the grade of disease, and patients with high pSMAD2 levels showed significantly lower overall survival [104]. pSMAD2 intensity has a similar prognostic value in other cancers, including breast cancer [105]. Notably, pSMAD2 levels are decreased in isolated peripheral blood mononuclear cells (PBMCs) and tumor cells upon treatment with the TGF-β inhibitor galunisertib, demonstrating an on-target effect of this drug [71]. However, pSMAD2 is not an ideal biomarker because it has a small dynamic range. Determining TGF-β signatures or TGF-β target gene expression appears to be a more reliable biomarker. However, it is not always possible to obtain (repeated) biopsies for determining the TGF-β target gene expression or TGF-β signatures. For these patients liquid biopsies can be of a great value. Liquid biopsies have received significant attention over the past decade, and this
to increased expression of interleukin (IL)-8. This cytokine increases the tumor-initiating cell (TIC) population. TGF-β targeting agents block paclitaxel-induced activation of TGF-β signaling and IL-8 expression, thereby blocking TIC expan- sion. (C) Exposure of glioblastoma cells to agents targeting TGF-β before radiation therapy inhibits DNA damage response in these cells and leads to a decreased self-renewal capacity of glioblastoma TICs. Consequently, cells become more sensitive to radiotherapy. Abbreviations: GBM, glioblastoma multiforme; WT, wild type.
CMS1 CMS2 CMS3 CMS4
3
0
–3
Key:
TGF-β signature positive correlation TGF-β signature negative correlation
Figure 5. A Subset of Colorectal Cancer (CRC) Patients Display High Correlation with Transforming Growth Factor (TGF)-β Signature. Recently, four consensus molecular subtypes (CMS1–4) of CRC were identifi ed [2]. A heatmap shows the expression of TGF-β target genes [92] in the various CMS categories. CMS1 consists of predominantly microsatellite instable (MSI) CRC. These tumors are demonstrated to have high immune infi ltration and activation. In contrast to CMS1, CMS2 refl ects chromosome instable (CIN) tumors and these tumors have high Wnt target gene expression. A high percentage of RAS mutations, often combined with PI3KCA mutations, are observed in CMS3 tumors. These tumors do not show high Wnt target gene expression but have a metabolic activation profi le. CMS4 tumors are suggested to have a mesenchymal phenotype, express epithelial–mesenchymal transition (EMT) genes, and have an overactive TGF-β signaling pathway [2]. In line with this, we show here that the CMS4 subtype correlates highly with elevated TGF-β signaling activity using a previously published TGF-β signature [92]. Importantly, CRC patients classifi ed as CMS4 have poor prognosis and show a relatively limited response to EGFR targeting therapy [2]. Figure courtesy of Dr L. Vermeulen (AMC, Amsterdam, The Netherlands).
topic has been recently reviewed [106–108]. Isolation of tumor cells or free tumor DNA from blood provides insight into minimal residual disease and tumor biology, including tumor-specifi c alterations. Circulating tumor cells (CTCs) are released from primary tumors and/or metastases [106]. CTC counts in blood are correlated with metastatic relapse and are a prognostic marker in various cancers. In particular, patients with high CTC counts after chemotherapy have a worse prognosis [109–111]. Mutational profiles of CTCs reveal that mutations detected in primary tumors and metastases are also found in CTCs [112], and this could be useful for therapeutic decisions. Repeated analysis of CTCs in breast cancer patients suggested that a high level of mesenchymal CTCs, with a gene expression profile indicative of high TGF-β pathway activation, correlated with disease progression. Moreover, the long-term monitoring of epithelial/mesen- chymal characteristics of CTCs in one patient demonstrated that therapy response and ensuing
tumor recurrence correlated with decreased or increased levels of mesenchymal CTCs, respec- tively [113]. Circulating tumor DNA can also be useful biomarker. Blood and other fluids can contain short DNA fragments called cell-free DNA (cfDNA). When cfDNA is derived from apoptotic or necrotic tumor cells, it is called circulating-tumor DNA (ctDNA) [114] and can be used to test for mutations in the tumor. This can be highly useful, especially in cases where tumor tissue is not available [115].
Concluding Remarks
While TGF-β targeting agents, such as galunisertib, have shown dramatic therapeutic effects in animal cancer models and in some cancer patients, it is still not clear how the therapeutic effect in cancer patients is achieved (see Outstanding Questions). Unraveling these mechanisms will allow more-effective therapies. While in the past many studies have focused on the pro- tumorigenic of TGF-β on tumor cells, the role of TGF-β to promote immune evasion has been somewhat overlooked. Because several immune checkpoint inhibitors are showing promising results in clinical trials and have been approved by the FDA, the role of TGF-β as an immune regulator is becoming more actively investigated. In breast cancer and in melanoma mouse models the effect of immune checkpoint inhibitors, such as antibody against cytotoxic T lymphocyte-associated protein 4 (CTLA4), can be enhanced by combining it with TGF-β targeting agents such as galunisertib [61]. Clinical trials of combinatorial targeting immune checkpoints and the TGF-β pathway have been initiated and the results are eagerly awaited (NCT02423343 and NCT02734160i).
Acknowledgments
We are grateful to Michael M. Lahn (Incyte) for critical reading of our review and Louis Vermeulen, Amsterdam Medical Center (AMC), The Netherlands, for providing Figure 5. Research on the targeting of TGF-β in cancer is supported in our laboratory by the Cancer Genomics Centre Netherlands and the Swedisch Cancerfonden.
Resources
ihttp://clinicaltrials.gov
iiwww.isarna-therapeutics.com
Supplemental Information
Supplemental information associated with this article can be found online at doi:10.1016/j.trecan.2016.11.008.
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