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. Author manuscript; available in PMC: 2020 Aug 1.
Published in final edited form as: Cancer. 2019 Apr 12;125(15):2693–2703. doi: 10.1002/cncr.32120

AUTOPHAGY INHIBITION OVERCOMES SORAFENIB RESISTANCE IN S45F-MUTATED DESMOID TUMORS

Danielle Braggio 1,2, David Koller 1,2, Feng Jin 3, Nanda Siva 4, Abeba Zewdu 1,2, Gonzalo Lopez 1,2, Kara Batte 1,2, Lucia Casadei 1,2, Meng Welliver 3, Anne M Strohecker 2,5,6, Dina Lev 7,8, Raphael E Pollock 1,2
PMCID: PMC6625904  NIHMSID: NIHMS1018979  PMID: 30980399

Abstract

BACKGROUND:

Desmoid tumors (DTs) are rare and understudied fibroblastic lesions that are frequently recurrent and locally invasive. Desmoid tumor patients often experience chronic pain, organ dysfunction, decrease in quality of life, and even death.

METHODS:

Sorafenib has emerged as a promising therapeutic strategy, which has led to the first randomized phase III clinical trial in this disease. Concurrently, we conducted a comprehensive analysis of sorafenib efficacy in a large panel of desmoid cell strains to probe for response mechanism.

RESULTS:

We found distinctive groups of higher- and lower-responder cells. Clustering the lower-responder group, we observed CTNNB1 mutation was determinant of outcome. Our results showed that even lower dose of sorafenib was able to inhibit cell viability, migration and invasion of wild-type and T41A-mutated DTs. Apoptosis induction was observed in those cells after treatment with sorafenib. On the other hand, lower dose of sorafenib was not able to inhibit cell viability, migration, and invasion or to induce apoptosis in the S45F-mutated DTs. The investigation of autophagy showed the dependency of S45F-mutated DTs on this pathway as a part of cell survival mechanism. Significantly, when autophagy was inhibited genetically or pharmacologically in the S45F mutant cell strains, sensitivity to sorafenib was restored.

CONCLUSIONS:

Our findings suggest that the response to sorafenib differs when comparing S45F-mutated DTs and T41A-mutated or wild-type DTs. Furthermore, combination of hydroxychloroquine and sorafenib enhances the anti-proliferative and pro-apoptotic effects in S45F-mutated DT cells, suggesting that profiling β-catenin status could guide clinical management of desmoid patients considering sorafenib treatment.

Keywords: Desmoid tumors, Sorafenib, Autophagy, Hydroxychloroquine, Therapeutic combination

Precis for use in the Table of Contents:

Sorafenib treatment is more effective in wild-type and T41A-mutated DT cell strains. The resistance observed in S45F-mutated cells was driven by elevated basal autophagy and could be reversed by autophagy inhibition.

Introduction

Desmoid tumors (DTs) are rare and understudied fibroblastic monoclonal lesions that are commonly found in the extremities, abdominal wall, trunk and within the intestinal mesentery intestine 1. Although considered benign due to the lack of metastatic potential, DTs have a high risk of local recurrence and can be remarkably locally invasive, thereby causing significant morbidity and mortality 2. Most DTs are sporadic and usually associated with somatic mutations in codons 41 or 45 of exon 3 of β-catenin (CTNNB1) gene 3.

Due to the unpredictable natural history and clinical behavior of this disease, selecting a treatment strategy for DT patients can be challenging for physicians. While many therapeutic options are available, the standard treatment for desmoids remains uncertain in that due to their rarity there is a lack of randomized clinical trials to inform therapeutic comparisons. When feasible, an active surveillance has been accepted as the front-line therapy in some centers; however, the lack of a complete understanding of the natural history of desmoids makes it difficult to know which tumors will not grow and which will need a more aggressive treatment strategy 4. Other modalities for desmoid management include surgery, radiation, systemic therapies and chemotherapy 5–8. Recently, targeted therapies, such as gamma secretase inhibitor, imatinib, and sorafenib, have also been utilized 9–12; however, overall response to most treatment options remains modest, suggesting a clear ongoing need for better and more individualized approaches.

Sorafenib is a multikinase inhibitor that impacts on both cell surface tyrosine kinase receptors and intracellular serine/threonine kinases 13. The most common tyrosine kinase receptors inhibited by sorafenib are the vascular‐endothelial growth factor receptor (VEGFR‐2 and VEGFR‐3) and platelet‐derived growth factor receptor (PDGFR‐β and c‐Kit) families 14. This targeted therapy is an FDA approved drug for the treatment of hepatocellular carcinoma, renal cell carcinoma, and differentiated thyroid carcinoma15–17. The antitumor activity of sorafenib has been clinically demonstrated in DT patients, and the efficacy of this drug was recently further evaluated in a phase III clinical trial 12,18. Gounder et al. showed that the progression-free survival at 2 years were 81% in the sorafenib group and 36% in the placebo group. The overall rate of objective response was 33% in the sorafenib group and 20% in the placebo group. However, a decrease in sorafenib dosage, or even treatment termination, was required in some DT patients due to drug toxicity 18. The optimal use of sorafenib in desmoid tumors remains unclear, suggesting the need for additional study. The sorafenib mechanism of action has not been elucidated, which makes it more difficult to identify the patients who may derive benefit from this drug; additional studies to identify biomarkers predictive of sorafenib response in desmoid tumors are needed. Towards that end, we seek to further elucidate molecular mechanisms of sorafenib action utilizing DT cell strain models.

Materials and Methods

Cell strains and reagents

All DT cell strains were isolated in our laboratory. This study was conducted with approval from the Ohio State University institutional review board (IRB) with written informed consent of patients. To confirm that our cell strains were desmoid tumor cells, CTNNB1 genotyping of both cell strains and corresponding tumors was performed as previously reported 19 (Table S1).

Sorafenib tosylate was purchased from Selleckchem. Bafilomycin A1 was purchased from InvivoGen and hydroxychloroquine sulfate was purchase from Sigma.

Protein analyses

For western blotting analysis, protein (8–30μg) was separated using a 4–20% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-rad) and transferred to PVDF membranes using the Trans-Blot® Turbo™ Midi PVDF Transfer Packs (Bio-rad). Membranes were incubated overnight at 4°C with the indicated antibodies: phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204), p44/42 MAPK (Erk1/2), phospho-MEK1/2 (Ser217/221), MEK1/2, phospho-AKT (Ser473), AKT, phospho-p70S6K (Thr389), p70S6K, ATG5, cleaved-PARP (Cell Signaling), phospho-PDGFR-β (Tyr 857), PDGFR-β, Flk-1 (VEGFR2), GAPDH (Santa Cruz), phospho-VEGFR2 (Tyr1054/Tyr1059) (Thermo Fisher Scientific) and LC3B/MAP1LC3B (Novus Biologicals). For Odyssey CLx imaging, blots were incubated with secondary donkey anti-rabbit or donkey anti-mouse (IRDye 800CW) and donkey anti-goat, donkey anti-mouse, or donkey anti-rabbit (IRDye 680RD) (Li-Cor). For the protein array, the cell lysates were analyzed using the PathScan RTK Signaling Antibody Array Kit (Cell Signaling Technology), according to the manufacturer’s instructions.

Cell viability and proliferation assays

Cell viability assays were done using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) after the indicated days of exposure to the specific drug or combination of drugs. The EC50 values were determined using GraphPad Prism Version 6.05 software. The capacity to form colonies was evaluated by soft agar experiments, as previously reported 20.

Flow cytometry and apoptosis analysis

Cell cycle progression was measured as previously described 21. Apoptosis was measured using Annexin V-PI staining (BD Biosciences) as previously described 21. Caspase 3/7 apoptosis activity was measured using Incucyte software (Essen Biosciences) over 15 days in culture. Apoptotic index was measured by dividing the fluorescence of caspase 3/7 substrate by total number of cells measured using Vybrant® DyeCycle™ Green stain (Life Technologies). Data were analyzed using Incucyte software (Essen Biosciences).

DNA plasmids, virus production, and transduction

Non-targeting control vector plasmid and shRNA targeting endogenous human ATG5 transcript were obtained from Addgene and Sigma. The plasmids were packaged in Lenti-X™ 293T cell line (Clontech) by transfection with Lenti-X™ Packaging Single Shots (VSV-G) (Clontech). D168 and D23 cell strains were transduced with lentiviral particles with 8μg/mL of polybrene (Sigma) and selected with 1.5μg/mL puromycin (Gibco).

Explant tissue slice culture

Tissue cores were generated with 4-mm disposable biopsy punches (Integra Miltex) from fresh patient tissue. Tissue cores were embedded in 6% low-melting-point agarose gel (Lonza) and cut into slices (400μm) with the Vibratome Series 1000 (Technical Products International, Inc.). The tissue slices were placed in 96-well plates with 200μL of complete media. The plates were seated on a platform rocker at 37°C with 5% CO2. Tissue slices were treated with varying doses sorafenib, either alone or in combination with other therapeutic compounds. Tissue slices were analyzed for cell survival using AlamarBlue® Cell Viability Reagent (Thermo Fisher Scientific). Protein was isolated using Precellys® Evolution homogenizer, Precellys® Lysing Kit (Bertin Instruments).

Statistical analysis

Statistical significance between experimental groups was determined by unpaired two-tailed Student t test analysis. P < 0.05 was considered statistically significant.

Results

Sorafenib efficacy in desmoid tumor cell strains

We exposed a panel of DT cells to increasing concentrations of sorafenib in vitro and evaluated cell growth. Because of DT cell slow proliferation rates, growth inhibition effects were only first observable 15 days after sorafenib treatment. DT cell treatment with increasing sorafenib doses (1–10μM) induced significant growth inhibition (Fig. 1A); however, sorafenib did not induce significant growth inhibition in normal cells controls (NDF-α). Interestingly, although the EC50 values were similar in all cell strains analyzed, at the lower concentration of 1uM, desmoids harboring the CTNNB1 S45F mutation appeared to be more tolerant of sorafenib as compared to DTs harboring T41A or wild-type CTNNB1. To confirm these results, we expanded the panel of desmoid cell strains treated with sorafenib. Our results show that S45F-mutated desmoids are more tolerant to sorafenib at the lower concentration of 1μM as compared to DTs harboring T41A or wild-type CTNNB1. (P<0.001; Fig 1B). Although some researchers have shown that the therapy efficacy depends more strongly on the tumor location and patients’s age, our results showed that neither the tumor anatomical location nor the patient’s age at the time of surgery correlated with sorafenib efficacy at the lower concentration of 1μM (Fig. S1). The sorafenib half-maximal effective concentrations and the response of desmoid cell strains to 1μM of sorafenib are shown in table S2. It should be noted that the desmoid cells were analyzed by Sanger sequencing so some of these wild-type CTNNB1 DTs may indeed have a mutated CTNNB1 or a mutated gene comprising the Wnt/β-catenin pathway that could only be observed by high-throughput sequencing. We also examined the potential effect of sorafenib on DT cell migration and invasion. Treatment with 10μM sorafenib for 24 hours resulted in decreased cell migration and invasion in all cell strains analyzed (Fig. 2). CTNNB1 S45F mutated desmoid cell strains showed less decrease in migration and invasion compared to other genotypes in desmoid (P<0.001), suggesting that S45F-mutated DTs are more tolerant to lower doses of sorafenib than other DTs. To investigate if these differences were due to a differential expression of the common sorafenib targets, we evaluated basal activation of VEGFR-2 and PDGFR-β. Surprisingly, our results showed no activation of basal PDGFR-β (p-Tyr857) (Fig. S2A), nor expression of basal VEGFR-2 (Fig. S2B). To expand the panel of receptor tyrosine kinases (RTK) and their downstream pathways investigated, we analyzed the expression of 28 RTKs and 11 downstream effectors using phospho-specific antibody arrays. Our results showed no expression of any tyrosine kinase receptors; however, phospho-p70S6K (Thr421/Ser424), phospho-AKT (Ser473) and phospho-ERK1/2 (Thr202/Tyr204) were all highly expressed (Fig. S2C). To investigate if the differences in response were due to differential expression of downstream effectors, we analyzed basal activation of ERK1/2 and AKT. We did not observe a differential expression of these proteins that could be associated with β-catenin mutational status (Fig. S3A). Moreover, our results showed that inhibition of ERK and PI3K/AKT/mTOR did not differ between S45F-mutated desmoid as compared to desmoids harboring T41A or wild-type CTNNB1 (Fig. S3B and S3C). Taken together, these results suggest that while it is likely that sorafenib targets the tumor microenvironment, here we show that sorafenib exhibits significant anti-tumor activity directly on DT cells per se and that CTNNB1 S45F mutated DTs might require higher doses of sorafenib for control of disease. Also, sorafenib resistance in S45F-mutated DT cells is not associated with differential expression of tyrosine kinase receptors or downstream effectors.

Figure 1. Sorafenib efficacy in desmoid cell strains.

Figure 1.

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A, Desmoid tumor cells strains and normal dermal fibroblasts (NDF-α) were treated with Sorafenib (0–10μM) as indicated. B, CellTiter-Glo® Luminescent Cell Viability Assay analysis of 1μM of Sorafenib treated for 14 days.

Figure 2. Sorafenib decreases desmoid tumor cell migration and invasion.

Figure 2.

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Desmoid tumor migration and invasion in response to sorafenib treatment assessed with Boyden chamber assays. Error bars represent SD from 3 independent experiments. (*P < 0.05, **P < 0.001, Student t test).

β-catenin mutational status is predictive of sorafenib efficacy

To examine whether anti-proliferative sorafenib effects on DT cell strains were mediated via cell cycle arrest or induction of apoptosis, we performed flow cytometric cell cycle and apoptosis analyses of sorafenib–treated cells. No significant changes in cell cycle levels were observed (data not shown). Interestingly, apoptosis analysis showed an increase in the percentage of apoptotic cells in only a subset of cell strains (Fig. 3A). To confirm our results and to investigate if the observed apoptosis was caspase-dependent, we analyzed cleavages of caspases-3 and −7 with the IncuCyte imaging system using CellEvent™ Caspase-3/7 Green Detection Reagent. Our results showed an increase of cleaved caspases-3 and −7 in the same subset of cells strains that underwent apoptosis in the previous flow analysis (Fig. 3B). Our results again showed that a subgroup of cell strains, despite a decrease in viable cells, did not have apoptosis induction after sorafenib treatment. Investigating further into these differences, we observed that all the cells that were resistant to apoptosis were S45F mutated cells, suggesting that the cell death response to sorafenib differs comparing DTs harboring the CTNNB1 S45F mutation versus T41A-mutated or wild-type DTs. Caspase 3/7 appears to have a key role in triggering sorafenib-induced apoptosis only in T41A mutated or wild-type DTs.

Figure 3. Analysis of sorafenib-induced cell death in desmoid cell strains.

Figure 3.

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A, Effects of sorafenib on cell apoptosis were measured by flow cytometry. B, Representative cleaved-caspase 3/7 fluorescent dye images of 2 desmoid cell strains. Effects of sorafenib on cell caspase-dependent apoptosis were measured using automated IncuCyte imaging.

Sorafenib resistance in S45F-mutated desmoid cell strain is associated with activation of autophagy

Autophagy is a pro-survival mechanism in normal cells and has also been associated with response to sorafenib. Therefore, we investigated if the autophagic responsiveness to sorafenib differs in DTs with differing CTNNB1 mutational status under autophagy-enhancing conditions. Western blot of microtubule-associated protein 1 light chain 3-II (LC3-II) showed that sorafenib at a concentration of 10μM was not able to induce LC3-II conversion in a T41A-mutated desmoid cell strain (D168). However, in a S45F-mutated cell strain (D23), 10μM of sorafenib significantly induced LC3-II conversion, suggesting that this concentration of sorafenib alters autophagy in S45F-mutated DTs but not in T41A-mutated DTs (Fig. 4A). To better understand the role of autophagy in desmoid-specific contexts, we treated DT cell strains with sorafenib and/or 5nM of bafilomycin, a known inhibitor of the fusion between autophagosomes and lysosomes. Interestingly, treatment with bafilomycin alone led to a significant increase of cell death in DT cells harboring the CTNNB1 S45F mutation, whereas no effect was seen in T41A-mutated DT cells. Moreover, the combination of sorafenib with bafilomycin caused more DT cell death in S45F-mutated strains when compared to either sorafenib or bafilomycin alone (Fig 4B). Furthermore, specific inhibition of autophagy by knockdown of ATG5 (Fig. 4C), a gene that encodes an essential protein for the autophagic vesicle formation, showed induction of cell death only in the S45F mutated DT cells (Fig. 4D). This was accomplished by activation of cleaved caspase 3/7 (Fig. 4E), suggesting that S45F mutated DT cells are highly dependent on autophagy as a cell survival mechanism.

Figure 4. CTNNB1 S45F mutated cells are dependent on autophagy for survival.

Figure 4.

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A, Autophagic flux of desmoid cell strains in presence and absence of Sorafenib (3 days). B, Cell survival analysis (MTT) of T41A and S45F mutated DT cell strains treated with 10μM of sorafenib +/− bafilomycin for 4 days. C, ATG5 was stably knocked-down in desmoid cell strains. GAPDH was used as a loading control. D, silencing endogenous ATG5 inhibited cell growth only in S45F mutated DT cell strain as determined by proliferation assay using automated IncuCyte imaging. E, cleaved-caspase 3/7 fluorescent dye was measured using automated IncuCyte imaging. Error bars represent SD from 3 independent experiments. *P < 0.05.

Combination of sorafenib and hydroxychloroquine enhances the anti-proliferative and pro-apoptotic effects on S45F mutated DT cells

We explored whether hydroxychloroquine (HCQ), a medically approved inhibitor of lysosomal function, would enhance sorafenib-induced inhibitory effects on DT cell strains. Concentration-response curves of sorafenib alone were first assessed and then compared to those obtained after co-treatment with HCQ after 7 days using 1μM of sorafenib and 10μM of HCQ. HCQ alone produced decreased cell growth and induction of caspase-dependent apoptosis in all S45F mutated DT cells tested but had no effect in T41A-mutated DT cells, confirming our previous results that S45F-mutated DT cell strains are highly dependent on autophagy instead of apoptosis. Furthermore, the combination of sorafenib with chloroquine showed a profound inhibition of cell growth compared to either sorafenib or HCQ treatment, but only in S45F-mutated DT cell strains. We also observed that the induction of apoptosis was greater with the combination of sorafenib and HCQ in only the S45F-mutated DT cell strains as compared to either treatments alone (Fig. 5A), suggesting that autophagy blockade by HCQ enhances the antitumor activity of sorafenib in S45F-mutated DT cells. Moreover, an explant culture assay performed using S45F-mutated DT tissues showed that the greatest decrease in cell viability was achieved after combining sorafenib with HCQ (Fig. 5B), confirming our previous results with DT cell strains. Taken together, our results show that the combination of HCQ with sorafenib might be especially beneficial to desmoid patients harboring the CTNNB1 S45F mutation.

Figure 5. Combination of sorafenib and hydroxychloroquine enhances the anti-proliferative and pro-apoptotic effects on S45F mutated DTs in vitro and ex vivo.

Figure 5.

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A, Cell survival analysis (CellTiter Glo) of T41A and S45F mutated DT cell strains treated with 1μM of sorafenib +/− HCQ for 4 days. Apoptosis was measured by caspase 3/7 activity using IncuCyte Zoom imaging. Error bars represent SD from 3 independent experiments. *P < 0.05, **P < 0.001 (Student t test). B, Cell survival analysis (alamar blue) of T41A and S45F mutated DT tissue treated ex vivo with 1μM of sorafenib +/− chrloquine for 11 days. Error bars represent SD from 3 replicative slices of the same tissue.

Discussion

Desmoid tumors have an unpredictable natural history and clinical behavior rendering selection of treatment strategies challenging. In a recent clinical trial using sorafenib, 6 of the 49 patients included in the sorafenib group had disease progression in contrast to 22 of 35 patients included in the placebo group. Overall, 33% of patients included in the sorafenib group showed objective response in comparison to the 20% observed in the placebo group18. Although sorafenib antitumor activity has been demonstrated in DT patients, little is known about its mechanism of action. This deficit makes it difficult to prospectively identify patients who most likely will benefit from sorafenib treatment, suggesting the need to evaluate the sorafenib mechanism of action in DTs. In keeping with previous studies, our results suggest an important DT oncogenic suppression with sorafenib treatment. While it is likely that some of the effect noted in the clinic is seemingly to the effect of sorafenib on the tumor microenvironment, our results demonstrate a direct effect on DT cells themselves. Abrogation of colony formation and reduced cell growth, migration, and invasion after sorafenib treatment was apparent in all desmoid cell strains tested, supporting sorafenib as a promising therapeutic strategy for this disease. To the best of our knowledge, this is the first report demonstrating the effect of sorafenib treatment in DT cell strains.

Autophagy is an important conserved mechanism for sustaining cellular homeostasis through degradation and recycling of protein and organelles, a process controlled by autophagy-related genes (Atg) 22. Autophagy is a widely recognized resistance mechanism in multiple tumors, including desmoid tumors. Specifically, Shimizu et al. recently reported that resistance to sorafenib observed on the hepatocellular carcinoma cell line Huh7 may be associated with activation of p-AMPK and enhanced autophagic response 23. The observation that the inhibitor of lysosome function hydroxychloroquine (HCQ) enhances the proapoptotic effects of sorafenib in S45F-mutated desmoids has potential translational relevance. HCQ is currently being evaluated in human clinical trials as a single agent or in combination therapies with encouraging results 24,25. Here we provided genetic and pharmacologic evidence that inhibition of autophagy may be a powerful strategy for S45F-mutated desmoid tumors. We showed that in desmoid tumors harboring the CTNNB1 S45F mutation, sorafenib–induced autophagy acts as a mechanism of apoptotic resistance and that combining sorafenib with autophagy inhibitors can enhance anti-proliferative effects and apoptotic cell death in vitro and ex vivo. Taken together, these findings suggest that desmoids harboring the CTNNB1 S45F mutation could use autophagy to avoid the cytotoxic effects of therapeutic agents.

Mutations in CTNNB1 are found at high rates (~85%) in sporadic desmoid tumors, leading to nuclear β-catenin accumulation, which results in aberrant gene expression 19. Desmoid CTNNB1 mutational spectra consists essentially of three different point mutations in exon 3 (T41A, S45F, and S45P). The implications of these mutations are still debated, but several studies have shown that the mutation S45F correlates with a higher risk of DT recurrence 26. Analogously, we also observed a different treatment response behavior in CTNNB1 S45F-mutated DTs compared to those harboring T41A or the WT CTNNB1 gene. DT cell strains harboring the CTNNB1 S45F mutation appear to be more resistant to sorafenib at the lower concentration of 1μM as compared to DTs harboring T41A or the WT CTNNB1 gene. Moreover, caspase 3/7 seemingly has a key role in triggering sorafenib-induced apoptosis in T41A -mutated or wild-type DTs, whereas sorafenib-induced cell death in S45F-mutated DTs appears to be associated with altered autophagy signaling pathways. Furthermore, our in vitro and ex vivo results show that S45F-mutated DTs are dependent on autophagy as an anti-apoptotic mechanism, and that 1μM sorafenib and 5μM HCQ combined have enhanced anti-proliferative and pro-apoptotic effects on these tumors. To date, the mutational analysis of CTNNB1 is not routinely assessed as part of differential diagnosis for DTs. However, our results suggest that CTNNB1 mutation status may be relevant to DT treatment decisioning. Indeed, The Desmoid Tumor Working Group highly recommends the analysis of CTNNB1 mutation status to be performed with the purpose of confirming diagnosis and guiding the clinical management 4. Gounder et al. observed that adverse events led to a significantly higher rate of treatment termination in the sorafenib group when compared to the placebo group (20% vs. no patients), showing that sorafenib can be quite a toxic drug, especially in the higher doses. In fact, the authors suggest that dose flexibility may be necessary to balance toxicity and benefit18, endorsing the application of our findings. Knowing the mutational status of patients at diagnosis could enable clinicians to decide between a low dose of sorafenib alone (T41A and WT DTs) versus a low dose of sorafenib in combination with an autophagy inhibitor (S45F DTs), which could obviate some of the undesirable adverse effects observed in the higher doses of sorafenib. In contrast to our results, Kasper et al observed a positive correlation between S45F-mutated patients and imatinib progression arrest rate. However, this study had an overrepresentation of S45F mutations and a remarkably lower wild-type DTs percentages 27. Interestingly, in contrast to Kasper et al study, Heinrich et al results suggest, even though not statistically significant, a trend of a poorer response to imatinib in S45F-mutated desmoids 28, corroborating our results. Therefore, additional studies are needed to further evaluate CTNNB1 mutation analysis as a useful tool for the management of different DT therapeutic regimens.

The most common tyrosine kinase receptors inhibited by sorafenib are the vascular‐endothelial growth factor receptor (VEGFR‐2 and VEGFR‐3) and platelet‐derived growth factor receptor (PDGFR‐β and c‐Kit) families 13,14. We attempted to identify the potential molecular basis for the desmoid tumor cell strain response to sorafenib, but were unable to demonstrate constitutive activation of the most common TKRs inhibited by sorafenib. Nor could we identify inhibition of any tyrosine kinase receptor after sorafenib treatment using a phospho-specific antibody array that is able to detect activation of 28 RTKs. Similar to the study by Heinrich et al, we observed an abundant expression of PDGFR-β protein levels in all DT cell strains but no phosphorylated PDGFR-β 28. It is possible that PDGFR-β and VEGFR-2 activation in the cells comprising the desmoid tumor microenvironment, such as endothelial cells and fibroblasts, may contribute to sorafenib response. Our results showing that sorafenib efficacy is not associated with the target kinases expression are in accordance with previous studies that showed no correlation of kinase receptors/downstream effectors expression and clinical response in desmoid patients treated with imatinib 28–30. To date, there are no conclusive biomarkers of response to tyrosine kinase inhibitors (TKIs). Confirming previous results found in other types of cancer 14,31, we did observe an inhibition of the ERK and PI3K/AKT/mTOR pathway after sorafenib treatment. Therefore, presumably the inhibition of a yet unknown TKR may participate in desmoid cell strain response to sorafenib. Additional studies are needed to identify biomarkers that predict sorafenib response in desmoid tumors, and these are currently underway.

Taken together, we confirmed that sorafenib has a potent anti-desmoid tumor activity by inhibiting proliferation, colony formation, migration, and invasion. We also showed that the mechanism of cell death in response to sorafenib differs between desmoids harboring the CTNNB1 S45F mutation and the desmoid harboring T41A or the wild-type CTNNB1. Moreover, our results suggest that desmoid tumors harboring the CTNNB1 S45F mutation are highly dependent of autophagy as an anti-apoptotic mechanism and that the combination of sorafenib and HCQ enhances the anti-proliferative and pro-apoptotic effects on these tumors. Although the limitation of having only a cohort of desmoid cell strains available for testing suggests caution, these findings support further investigation of sorafenib combined with autophagy inhibitors in desmoid patients harboring the CTNNB1 S45F mutation, a novel strategy which could contribute to meaningful clinical options for those patients burdened by desmoid tumors with a genetically determined more aggressive behavior.

Supplementary Material

Supp TableS1-2
Supp figS1-3

Supplemental Figure 1. Sorafenib efficacy in desmoid cell strains in relation to anatomical location and age. CellTiter-Glo® Luminescent Cell Viability Assay analysis of 1μM of Sorafenib treated for 14 days in relation to A, anatomical location and B, age. *EA stands for extra-abdominal; IA stands for intra-abdominal; A stands for abdominal wall; CW stands for chest wall.

Supplemental Figure 2. Canonical Sorafenib targets. Western blot analysis of phosphorylated A, PDGFR-β and total PDGFR-β; B, phosphorylated VEGFR2 and total VEGFR-2 protein levels in desmoid cells. The cells were serum-starved overnight prior to sorafenib treatment. C, Representative fluorescence image of tyrosine kinase array in two desmoid tumor cell strains.

Supplemental Figure 3. Sorafenib inhibits ERK and AKT phosphorylation in desmoid tumor cell strains. A, Western blot analysis of phosphorylated ERK (p-ERK), total ERK; B, phosphorylated AKT and total AKT protein levels in desmoid cell strains treated with sorafenib at the indicated concentrations for 96h. The cells were serum-starved overnight prior to sorafenib treatment. C, Western blot analysis of phosphorylated ERK and AKT and total ERK and AKT protein levels in desmoid cells. The cells were serum-starved overnight prior to sorafenib treatment.

Acknowledgments:

We thank the Sarcoma Research Lab for helpful discussion and for critical reading of the manuscript.

Funding Support: This manuscript was supported in part by a Desmoid Tumor Research Foundation Seed Grant, and the following grants from the National Cancer Institute of the National Institutes of Health: U54CA168512 (REP), 1K22CA187931 (AMS) and a grant from the National Institutes of Health to The OSU Comprehensive Cancer Center (P30 CA016058).

Footnotes

Conflict of Interest Disclosures: None

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Associated Data

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Supplementary Materials

Supp TableS1-2
NIHMS1018979-supplement-Supp_TableS1-2.docx (19.8KB, docx)
Supp figS1-3

Supplemental Figure 1. Sorafenib efficacy in desmoid cell strains in relation to anatomical location and age. CellTiter-Glo® Luminescent Cell Viability Assay analysis of 1μM of Sorafenib treated for 14 days in relation to A, anatomical location and B, age. *EA stands for extra-abdominal; IA stands for intra-abdominal; A stands for abdominal wall; CW stands for chest wall.

Supplemental Figure 2. Canonical Sorafenib targets. Western blot analysis of phosphorylated A, PDGFR-β and total PDGFR-β; B, phosphorylated VEGFR2 and total VEGFR-2 protein levels in desmoid cells. The cells were serum-starved overnight prior to sorafenib treatment. C, Representative fluorescence image of tyrosine kinase array in two desmoid tumor cell strains.

Supplemental Figure 3. Sorafenib inhibits ERK and AKT phosphorylation in desmoid tumor cell strains. A, Western blot analysis of phosphorylated ERK (p-ERK), total ERK; B, phosphorylated AKT and total AKT protein levels in desmoid cell strains treated with sorafenib at the indicated concentrations for 96h. The cells were serum-starved overnight prior to sorafenib treatment. C, Western blot analysis of phosphorylated ERK and AKT and total ERK and AKT protein levels in desmoid cells. The cells were serum-starved overnight prior to sorafenib treatment.

NIHMS1018979-supplement-Supp_figS1-3.pdf (1MB, pdf)

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