Here we reveal that sustained suppression of TGF-β signaling in vivo results in the outgrowth of carcinomas that are apparently resistant to the small molecule TβR1/TβRII inhibitor, LY2109761. We demonstrate marked differences in the outcome of LY2109761 treatment of carcinoma cells in vitro, tumor allografts in vivo and primary carcinomas in situ. Furthermore, the data illustrate that, in the same in vivo model of carcinogen-induced primary tumor outgrowth and progression, the outcome of LY2109761 treatment can be either anti- or pro-tumorigenic, dependent on the precise drug-dosing regimen.
Short Term, high dose LY2109716 treatment was unable to completely reverse TGF-β induced carcinoma EMT, in either the E4 allograft or primary chemically-induced carcinoma models. This data was in contrast with our previous finding using genetic inhibition of autocrine TGF-β signaling in E4 cells in vitro
and in vivo
). Nevertheless, Short Term LY2109716 treatment did reduce the levels of α-SMA and Shox2
in the allograft and DMBA/PMA models respectively, which suggests a trend towards a less myofibroblast phenotype in mice treated continuously with drug for 10 days. Failure to definitively replicate inhibition of EMT after blockade of TGF-β signaling inhibition (23
) could be due to additional effects of LY2109761 on the tumor stroma that potentiates tumorigenesis (34
) and/or inadequate drug delivery to the tumor parenchyma, especially in the light of vascular disruption in response to LY2109761 in the tumor allograft model.
In concordance with predictions from earlier studies using genetically-manipulated TGF-β1 or TβRII (11
), we show that long term daily LY2109761 treatment during tumor outgrowth increased papilloma number, supporting a suppressive effect of TGF-β in early tumorigenesis. Analysis of the papillomas suggests that these tumor-suppressive effects may be at least in part due to the immune suppressive action of TGF-β signaling. Inflammation has been suggested to act either protectively via
immune surveillance or in a pro-tumorigenic manner, dependent on the nature of the inflammatory cell infiltrate (37
). In the mouse skin model of chemical carcinogenesis, inflammation has clearly been shown to be pro-tumorigenic (26
). In agreement with these observations, we demonstrated that Sustained LY2109761 treatment resulted in a significant increase in CD45+ neutrophilic infiltrate within the papilloma stroma.
Importantly, we demonstrated that Sustained Treatment with LY2109761 during the tumor outgrowth phase resulted in resistance to drug induced P-Smad2 down-regulation, specifically in malignant tumor cells but not in tumor stromal fibroblasts or normal tissue. The development of acquired drug resistance to both conventional chemotherapeutics and targeted therapies is a common undesirable outcome in malignant disease. The mechanisms of acquired drug resistance are varied and, for targeted small molecule therapies, might be somewhat complex (39
). However, understanding these mechanisms of drug resistance will allow modification of therapeutic strategies that lead to more efficacious treatments. Mechanisms of acquired drug resistance include amplification of the target gene, as seen in acquired resistance to Met tyrosine kinase inhibitors (41
), as well as activation of alternative signaling pathways (39
) and perturbations in the intermolecular cross talk between interacting ligands, tyrosine kinases or their kinase-inactive partners (39
There are several possible explanations for our unexpected findings of outgrowth of tumor cells with high levels of LY2109761-resistant P-Smad2. The simplest would be inadequate drug delivery, possibly as a consequence of vascular disruption. However, this is unlikely, bearing in mind that LY2109761 can down modulate P-Smad2 in the tumor stroma, even after sustained drug treatment. Moreover, the Sustained Dosing Regimen resulted in a molecular carcinoma phenotype distinct from that of either vehicle-treated or Short Term Drug-treated mice. Mutation of the ATP/drug binding site of TβRI, Acvr1b or Acvr1c may confer drug resistance (see for example (44
)), as would mutational hyperactivation of the kinase receptor (45
). Genetic alterations that influence P-Smad2 levels downstream or parallel to TβRI (27
) may also provide mechanisms to bypass LY2107961 effects. TGF-β signaling is known to be finely regulated by both negative and positive feedback mechanisms, and in tumor cells, signaling may be regulated by trans receptor interactions that might be perturbed in the presence of LY2109761. This drug is known to inhibit type I receptors that signal for activins, GDF3, nodal and myostatin. It is therefore conceivable that perturbation of one or more of these signaling pathways within the tumor cell or tumor microenvironment might lead to expansion of a more aggressive tumor type. In the current study, it was noted that the LY2109761 target, TβRI, was up-regulated more than 1.5 fold in the Sustained LY2109761-treated carcinomas. This might be due to disruption of a negative feedback loop on TβRI levels and/or indirectly caused by expansion of a cellular compartment, characterized by high TβRI expression (46
Importantly, not only was there resistance to LY2109761-mediated attenuation of carcinoma P-Smad2 levels, but these carcinomas took on a more aggressive molecular profile, with up-regulation of markers of EMT and inflammation, illustrated by delocalization of cell surface E-cadherin and expansion of a vimentin-positive tumor stroma, as well as elevated expression of pro-inflammatory markers. In contrast, carcinomas treated on the Short Term LY2109761 Dosing regimen showed the inverse trend, with down-regulation of markers of EMT, such as Msx1
. It would appear that many of the pro-tumorigenic effects of LY2109761 are likely driven by the action of this drug on the tumor stroma, leading to immune cell infiltration, stromal cell expansion, and subsequent feedback via growth factors such as HGF, that drive the outgrowth of LY2109761-resistant carcinomas, as has been seen in genetic models of TGF-β signaling inhibition (34
). There is an increasing appreciation of the importance of the interaction between the carcinoma cell and its substratum in driving tumor progression, particularly with respect to matrix density and stiffness (48
). The expansion of the tumor stroma, the prevalence of ECM components, junctional adhesion molecules and integrins in genes up regulated after sustained exposure to LY2109761, would all support the concept of a stromal-driven enhancement in aggressive molecular phenotype.
In conclusion, the current study demonstrates that, although TGF-β inhibitors might be clinically useful for short term patient exposure (50
), long term treatment with TGF-β inhibitors should be administered with caution. Dosing regimen is clearly critical, and recommended dosage might vary considerably based on tumor type and TGF-β signaling status. Further investigation of mechanism of acquired drug resistance might provide more efficacious routes to therapy.