Background
Osteosarcoma (OSA) is the most common form of malignant bone cancer in humans and dogs [
1,
2]. Multidrug chemotherapy and aggressive surgical techniques have improved survival; however, the prognosis for human patients with metastatic disease remains extremely poor with survival rates of 10-20% [
3]. The disease in dogs occurs approximately 10 times more frequently than in people and treatment with surgery and adjuvant chemotherapy results in long-term survival rates of only 10-15% [
4]. Both clinical and molecular evidence suggest that human and canine OSA share several key features including early metastasis, chemotherapy resistance, altered expression of several proteins (e.g., ezrin, Met, PTEN), and p53 mutation, among others [4-10]. Given these similarities, canine OSA serves as a relevant model in which to evaluate the potential clinical utility of novel therapeutic targets for this disease.
The transcription factor STAT3 has been implicated as a key player in several features of malignant neoplasia including tumor cell survival, metastasis, and resistance to chemotherapy [
11‐
13]. Our data and the work of others support the notion that STAT3 may be a relevant target for therapy in both human and canine OSA. In previous work, we demonstrated that human and canine OSA cell lines and tumors from canine patients exhibited constitutive activation of STAT3 [
14]. Loss of this expression after transfection with small interfering RNA targeting STAT3 or by reducing STAT3 DNA binding using LLL3 (a small molecule inhibitor) abrogated expression of STAT3 transcriptional targets and enhanced apoptosis [
14]. Increased levels of phosphorylated STAT3 have been identified in a subset of human OSA tissue samples and cell lines supportive of the role of this transcription factor in OSA [
15]. Suppression of this activated STAT3 with a dominant negative STAT3 led to decreased growth in these cell lines [
15]. Studies by Wang
et al. showed that inhibition of STAT3 expression in OSA cells by siRNA decreased proliferation and enhanced apoptosis of these cells [
11]. Treatment of multidrug resistant OSA cell lines with a synthetic oleanane triterpenoid, C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid (CDDO-Me) downregulated STAT3 phosphorylation and nuclear translocation, subsequently inducing apoptosis [
16]. Indeed, overexpression of phosphorylated STAT3 was associated with a poor prognosis in patients with OSA [
17] and high levels of STAT3 protein were associated with metastasis [
11]. Given the apparent role of STAT3 in the biology of OSA, clinically relevant therapies aimed at downregulating its activity would likely be therapeutically useful.
Curcumin (diferuloylmethane) is a naturally occurring compound found in the plant
Curcuma longa that has numerous medicinal properties including anti-inflammatory and antitumor effects [
18‐
20]. Curcumin has been investigated extensively as a potential therapeutic agent for the treatment of many different cancers, such as colorectal carcinoma [
21,
22], head and neck squamous cell carcinoma [
23], pancreatic cancer [
24], and OSA [
25,
26]. Curcumin is known to target multiple biochemical pathways, such as those mediated by Wnt/β-catenin [
26], NF-κB [
20], growth factor receptors like EGFR and HER2 [
27], and JAK/STAT [
28] enhancing its effect on cancer cells. Indeed, studies indicated that curcumin targets cellular transformation, invasion, angiogenesis, and metastasis [
27,
29‐
32]. Recent work demonstrated that curcumin induced cell cycle arrest and apoptosis, and inhibited migration in human OSA cell lines [
19,
33]. However, curcumin is not stable under physiologic conditions and is not readily absorbed after ingestion [
34]. Multiple modifications to the structure of curcumin have been investigated in an attempt to improve potency and biochemical properties [
18,
35‐
37].
Recent work on improving both the target specificity and stability of curcumin by the College of Pharmacy at The Ohio State University produced the novel small molecule STAT3 inhibitor, FLLL32. As a diketone analog of curcumin, FLLL32 is more selective in its targeting than the parent compound due to the replacement of two hydrogen atoms on the central carbon of curcumin with a spiro-cyclohexyl ring [
38]. Improved interaction of FLLL32 with the Src homology-2 (SH2) domain of STAT3, a region instrumental in its dimerization and nuclear translocation, as well as greater stability, was predicted with these modifications as compared to curcumin [
38,
39]. In subsequent work, FLLL32 was shown to promote apoptosis in multiple human cancer cell lines, inducing downregulation of STAT3 phosphorylation and DNA binding [
38‐
40]. In human hepatocellular cancer cells, FLLL32 inhibited IL-6-induced STAT3 phosphorylation [
41]. FLLL32 was found to be more potent than some existing STAT3 inhibitors, including Stattic, S3I-201, and curcumin in colorectal, glioblastoma, multiple myeloma, rhabdomyosarcoma, and liver cancer cell lines [
39,
42]. Together, these data demonstrate that FLLL32 exhibits improved efficacy at abrogating STAT3 functional activity and its effects in enhancing tumor cell survival in many cancer cell lines as compared to curcumin and other STAT3 inhibitors. Therefore, the purpose of this study was to explore the biologic activity of FLLL32 against canine and human OSA cell lines
in vitro, delineate the mechanism of action of FLLL32, and compare the efficacy of FLLL32 to curcumin.
Discussion
Curcumin has a long history of use as a medicinal compound and is known to have multiple anti-inflammatory and anti-cancer properties; however, blood levels that can be achieved after oral administration are low, which limits its potential clinical value [
20,
27,
34]. Curcumin also affects a broad range of cellular targets including STAT3 [
53,
54] in addition to a host of other signaling molecules such as Wnt/β-catenin [
26], NF-κB [
20], and HER2 [
27], and the proteasome [
51]. Given the number of targets affected by curcumin and its poor bioavailability, efforts have been directed at improving its chemical properties by complexing it with lipids/phospholipids [
55,
56] and developing more specific derivatives [
35,
57‐
59]. Interestingly, many of these analogues have demonstrated greater stability and more potent activity against several tumor cell lines, including those derived from breast, prostate, pancreas, and colon cancers when compared to curcumin [
35,
57‐
59]. Curcumin has been found to be well-tolerated in healthy individuals and OSA patients [
18], most recently when given as a solid lipid particle formulation. However, peak plasma levels reached only 22.43 ng/mL (approximately 60 nM), well below concentrations known to have biologic effects against OSA cells
in vitro.
During the development of novel curcumin analogs, our collaborators determined that one of these compounds, FLLL32, was particularly effective at suppressing the growth of pancreatic and breast cancer cells [
38]. To produce FLLL32, the two hydrogen atoms on the central carbon of curcumin were replaced with a spiro-cyclohexyl ring. It was proposed that this alteration would confer greater stability and specificity for STAT3 than curcumin [
38]. Recent work with FLLL32 showed that it induced apoptosis in human melanoma, multiple myeloma, glioblastoma, pancreatic, breast, and colorectal cancer cell lines and inhibited STAT3 phosphorylation and DNA binding [
38‐
40]. The compound also exhibited higher potency at inhibiting proliferation and STAT3 DNA binding activity than curcumin and other JAK/STAT3 inhibitors in human rhabdomyosarcoma cells [
42]. Indeed, FLLL32 has been shown to be more potent than other STAT3 inhibitors in promoting growth inhibition of multiple cancer cell lines, and the drug is improved in its specificity as demonstrated by kinase profile assays that revealed almost no activity against tyrosine kinases such as Lck, Syk, Lyn, Yes, and Abl-1 [
39]. Given the superior specificity and efficacy of FLLL32 as compared to curcumin in a variety of cancer cell lines, the purpose of this study was to evaluate the biologic activity of this compound against OSA cell lines.
Previous studies have explored the activity of curcumin against OSA both
in vitro and in human clinical trials [
18,
20,
27]. OSA cell lines experienced cell cycle arrest, reduced proliferation, and underwent apoptosis following treatment with curcumin [
19,
33,
60]. Prior work in our laboratory demonstrated that STAT3 is constitutively activated in OSA cell lines and that inhibition of STAT3 through STAT3 siRNAs or the small molecule STAT3 inhibitor LLL3 resulted in loss of proliferation and apoptosis [
14]. Data presented in this study showed that FLLL32 inhibited proliferation of OSA cell lines and promoted apoptosis via caspase 3/7 activation at lower concentrations than curcumin. This is consistent with recent work demonstrating apoptosis via caspase activation in human multiple myeloma, glioblastoma, liver cancer, colorectal, and melanoma cell lines after FLLL32 exposure [
39,
40]. Cleavage of PARP, an indicator of caspase-3-mediated apoptosis, was also seen in many of these human cancer cell lines upon treatment with FLLL32 [
39]. Interestingly, loss of messenger RNA and protein expression of survivin, an inhibitor of apoptosis, as well as decreased STAT3 DNA binding activity was observed in human rhabdomyosarcoma cells treated with FLLL32 [
42]. The concurrent reduction in STAT3 transcriptional activity of targets such as survivin through decreased DNA binding and loss of STAT3 phosphorylation likely both played a role in the reduced survival of OSA tumor cells observed following exposure to FLLL32.
Recent work has shown that expression of high levels of STAT3 in human OSA tumor samples correlated to poor differentiation, metastasis, and lower rates of overall and relapse-free survival [
11]. Overexpression of phosphorylated STAT3 in OSA has also been linked to poor prognosis [
17]. STAT3 is known to enhance tumor cell invasion, metastasis, and angiogenesis through enhanced expression of VEGF and MMP2 [
61]. Human patients with OSA whose tumors had higher VEGF expression as shown by immunohistochemistry had a significantly worse prognosis and had lung metastasis [
62,
63]. Previous work revealed that treatment of OSA cell lines with curcumin inhibited their migration [
26]. Mouse xenograft models of pancreatic and colorectal cancer treated with curcumin exhibited suppression of tumor angiogenesis and tumor growth inhibition [
20]. In more recent studies, FLLL32 inhibited vascularity and tumor growth in chicken embryo xenografts and reduced tumor volume in mouse xenografts of breast cancer [
38]. Our data demonstrate that in the OSA cell lines we tested, VEGF mRNA and protein and MMP2 mRNA were expressed and treatment with 10 μM FLLL32 downregulated the expression of these STAT3 transcriptional targets following 24 hours of drug exposure. Interestingly, VEGF mRNA expression appeared to increase over baseline in both the OSA8 and SJSA lines after curcumin exposure, although this did not correlate with the findings obtained by Western blotting in which VEGF protein was absent in OSA8 cells and unchanged in SJSA cells. The mechanism for this observed discrepancy is not clear, although there are several possible explanations. Curcumin may somehow interfere with translation of VEGF mRNA, directly enhance degradation of VEGF protein, or alternatively, given its diversity of cellular targets, affect proteins other than STAT3 that in turn alters VEGF expression. Further investigation of these potential mechanisms is needed. Given the putative role of both VEGF and MMP2 in the process of tumor growth and metastasis and recent data demonstrating the ability of FLLL32 to abrogate breast cancer xenograft growth in mice, future work assessing the effects of FLLL32 in mouse models of OSA is warranted.
Treatment of OSA cell lines with FLLL32 promoted loss of both pSTAT3 and total STAT3. This loss of STAT3 correlated with the presence of mono- and polyubiquitinylated STAT3, indicating that proteasome-mediated degradation was likely responsible for the observed decrease in protein. Interestingly, curcumin has been shown to inhibit activities of the proteasome in certain cancer cells [
51]; however we detected no evidence for this activity after treating the OSA cell lines with curcumin or FLLL32 at the doses and time points examined. Although modulation of STAT3 protein levels is known to occur in part through caspase cleavage [
52] a pan-caspase inhibitor did not affect the observed loss of STAT3 after FLLL32 treatment. Additionally, we did not see a significant decrease in STAT3 mRNA 24 hours after FLLL32 treatment, indicating that loss of STAT3 mRNA could not be primarily responsible for the protein downregulation that occurs after FLLL32 exposure. These data support the assertion that in addition to blocking STAT3 function, FLLL32 acts to promote downregulation of STAT3 protein, thereby enhancing the functional consequences of this small molecule inhibitor.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
SF designed and carried out molecular experiments on OSA cell lines and drafted the manuscript. MB participated in RT-PCR design and performance. JL, CL, ES, PL, and JRF designed and produced the small molecule STAT3 inhibitor FLLL32. JF designed and carried out the quantitative real time PCR experiments. WK assisted in experimental design. CL conceived of the study, assisted in experimental design, and helped draft the manuscript. All authors read and approved the final manuscript.