Parthenolide Covalently Targets and Inhibits Focal Adhesion Kinase in Breast Cancer Cells

•Parthenolide covalently reacts with C427 of FAK1 to inhibit FAK1 activity•Parthenolide impairs FAK1 signaling in breast cancer cells•Other sesquiterpene lactone natural products also target FAK1 Parthenolide, a natural product from the feverfew plant and member of the large family of sesquiterpene lactones, exerts multiple biological and therapeutic activities including anti-inflammatory and anti-cancer effects. Here, we further study the parthenolide mechanism of action using activity-based protein profiling-based chemoproteomic platforms to map additional covalent targets engaged by parthenolide in human breast cancer cells. We find that parthenolide, as well as other related exocyclic methylene lactone-containing sesquiterpenes, covalently modify cysteine 427 of focal adhesion kinase 1 (FAK1), leading to impairment of FAK1-dependent signaling pathways and breast cancer cell proliferation, survival, and motility. These studies reveal a functional target exploited by members of a large family of anti-cancer natural products. Parthenolide, a natural product from the feverfew plant and member of the large family of sesquiterpene lactones, exerts multiple biological and therapeutic activities including anti-inflammatory and anti-cancer effects. Here, we further study the parthenolide mechanism of action using activity-based protein profiling-based chemoproteomic platforms to map additional covalent targets engaged by parthenolide in human breast cancer cells. We find that parthenolide, as well as other related exocyclic methylene lactone-containing sesquiterpenes, covalently modify cysteine 427 of focal adhesion kinase 1 (FAK1), leading to impairment of FAK1-dependent signaling pathways and breast cancer cell proliferation, survival, and motility. These studies reveal a functional target exploited by members of a large family of anti-cancer natural products. Parthenolide, a natural product found in the feverfew plant (Tanacetum parthenium), possesses myriad therapeutic activities, including anti-inflammatory and anti-cancer effects. Through covalent bond formation between its reactive α-methylene-γ-butyrolactone moiety and various protein targets, multiple cellular signaling pathways are affected (Ghantous et al., 2013Ghantous A. Sinjab A. Herceg Z. Darwiche N. Parthenolide: from plant shoots to cancer roots.Drug Discov. Today. 2013; 18: 894-905Crossref PubMed Scopus (220) Google Scholar, Kwok et al., 2001Kwok B.H. Koh B. Ndubuisi M.I. Elofsson M. Crews C.M. The anti-inflammatory natural product parthenolide from the medicinal herb Feverfew directly binds to and inhibits IkappaB kinase.Chem. Biol. 2001; 8: 759-766Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, Liu et al., 2018aLiu M. Xiao C. Sun M. Tan M. Hu L. Yu Q. 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Phase I dose escalation trial of feverfew with standardized doses of parthenolide in patients with cancer.Invest. New Drugs. 2004; 22: 299-305Crossref PubMed Scopus (104) Google Scholar). Using a biotinylated parthenolide analog, previous studies by the lab of Crews established that one of the primary targets that drives the anti-inflammatory and anti-cancer activity of parthenolide is IκB kinase β (IKK-β) wherein cysteine 179 (C179) is modified, thus impairing IKK-β and nuclear factor κB (NF-κB) signaling (Kwok et al., 2001Kwok B.H. Koh B. Ndubuisi M.I. Elofsson M. Crews C.M. The anti-inflammatory natural product parthenolide from the medicinal herb Feverfew directly binds to and inhibits IkappaB kinase.Chem. Biol. 2001; 8: 759-766Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). Additional studies have revealed other direct targets of parthenolide that may help to explain the therapeutic properties of this natural product, including targeting of specific cysteines within heat-shock protein Hsp72 and STAT3 downstream signaling targets such as Janus kinases JAK2 (Liu et al., 2018aLiu M. Xiao C. Sun M. Tan M. Hu L. Yu Q. Parthenolide inhibits STAT3 signaling by covalently targeting Janus kinases.Molecules (Basel). 2018; 23https://doi.org/10.3390/molecules23061478Crossref Scopus (11) Google Scholar, Shin et al., 2017Shin M. McGowan A. DiNatale G.J. Chiramanewong T. Cai T. Connor R.E. Hsp72 is an intracellular target of the α,β-unsaturated sesquiterpene lactone, parthenolide.ACS Omega. 2017; 2: 7267-7274Crossref PubMed Scopus (14) Google Scholar). Moreover, this natural product has also been shown to affect additional cell signaling pathways including induction of oxidative stress and apoptosis, focal adhesion kinase 1 (FAK1) signaling, hypoxia-inducible factor 1α signaling, epithelial-to-mesenchymal transition, Wnt/β-catenin signaling, mitogen-activated protein kinase signaling, and mitochondrial function (Carlisi et al., 2011Carlisi D. D’Anneo A. Angileri L. Lauricella M. Emanuele S. Santulli A. Vento R. Tesoriere G. Parthenolide sensitizes hepatocellular carcinoma cells to TRAIL by inducing the expression of death receptors through inhibition of STAT3 activation.J. Cell. Physiol. 2011; 226: 1632-1641Crossref PubMed Scopus (76) Google Scholar, Carlisi et al., 2016Carlisi D. Buttitta G. Di Fiore R. Scerri C. Drago-Ferrante R. Vento R. Tesoriere G. Parthenolide and DMAPT exert cytotoxic effects on breast cancer stem-like cells by inducing oxidative stress, mitochondrial dysfunction and necrosis.Cell Death Dis. 2016; 7: e2194Crossref PubMed Scopus (68) Google Scholar, Jafari et al., 2018Jafari N. Nazeri S. Enferadi S.T. Parthenolide reduces metastasis by inhibition of vimentin expression and induces apoptosis by suppression elongation factor α-1 expression.Phytomedicine Int. J. Phytother. Phytopharm. 2018; 41: 67-73Crossref PubMed Scopus (23) Google Scholar, Kim et al., 2017Kim S.L. Park Y.R. Lee S.T. Kim S.-W. Parthenolide suppresses hypoxia-inducible factor-1α signaling and hypoxia induced epithelial-mesenchymal transition in colorectal cancer.Int. J. Oncol. 2017; 51: 1809-1820Crossref PubMed Scopus (27) Google Scholar, Kwok et al., 2001Kwok B.H. Koh B. Ndubuisi M.I. Elofsson M. Crews C.M. The anti-inflammatory natural product parthenolide from the medicinal herb Feverfew directly binds to and inhibits IkappaB kinase.Chem. Biol. 2001; 8: 759-766Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar, Lin et al., 2017Lin M. Bi H. Yan Y. Huang W. Zhang G. Zhang G. Tang S. Liu Y. Zhang L. Ma J. et al.Parthenolide suppresses non-small cell lung cancer GLC-82 cells growth via B-Raf/MAPK/Erk pathway.Oncotarget. 2017; 8: 23436-23447PubMed Google Scholar, Zhang et al., 2017Zhang X. Chen Q. Liu J. Fan C. Wei Q. Chen Z. Mao X. Parthenolide promotes differentiation of osteoblasts through the Wnt/β-catenin signaling pathway in inflammatory environments.J. Interferon Cytokine Res. 2017; 37: 406-414Crossref PubMed Scopus (14) Google Scholar). Based on the broader scope of influence on these biological pathways and systems, parthenolide likely still possesses additional targets that are not yet fully elucidated. In previous works investigating the direct targets of parthenolide, multiple studies have revealed unique ligandable and functional cysteines within their respective proteins that could be targeted to influence cellular signaling and pathogenicity. Recent studies have shown that activity-based protein profiling (ABPP)-based chemoproteomic platforms can be utilized to uncover unique and functional druggable hotspots and modalities, which can be accessed by covalently acting small molecules and natural products that may not be obvious using standard drug-discovery paradigms (Backus et al., 2016Backus K.M. Correia B.E. Lum K.M. Forli S. Horning B.D. González-Páez G.E. Chatterjee S. Lanning B.R. Teijaro J.R. Olson A.J. et al.Proteome-wide covalent ligand discovery in native biological systems.Nature. 2016; 534: 570-574Crossref PubMed Scopus (421) Google Scholar, Banerjee et al., 2013Banerjee R. Pace N.J. Brown D.R. 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Quantitative reactivity profiling predicts functional cysteines in proteomes.Nature. 2010; 468: 790-795Crossref PubMed Scopus (1079) Google Scholar). ABPP uses reactivity-based chemical probes to profile proteome-wide reactive, ligandable, and functional sites directly in complex proteomes. When used in a competitive manner, covalently acting small molecules can be competed against binding of reactivity-based probes to map the proteome-wide targets of these compounds (Backus et al., 2016Backus K.M. Correia B.E. Lum K.M. Forli S. Horning B.D. González-Páez G.E. Chatterjee S. Lanning B.R. Teijaro J.R. Olson A.J. et al.Proteome-wide covalent ligand discovery in native biological systems.Nature. 2016; 534: 570-574Crossref PubMed Scopus (421) Google Scholar, Bateman et al., 2017Bateman L.A. Nguyen T.B. Roberts A.M. Miyamoto D.K. Ku W.-M. Huffman T.R. Petri Y. Heslin M.J. Contreras C.M. Skibola C.F. et al.Chemoproteomics-enabled covalent ligand screen reveals a cysteine hotspot in reticulon 4 that impairs ER morphology and cancer pathogenicity.Chem. Commun. Camb. Engl. 2017; 53: 7234-7237Crossref PubMed Google Scholar, Grossman et al., 2017Grossman E.A. Ward C.C. Spradlin J.N. Bateman L.A. Huffman T.R. Miyamoto D.K. Kleinman J.I. Nomura D.K. Covalent ligand discovery against druggable hotspots targeted by anti-cancer natural products.Cell Chem. Biol. 2017; 24: 1368-1376.e4Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, Hacker et al., 2017Hacker S.M. Backus K.M. Lazear M.R. Forli S. Correia B.E. Cravatt B.F. Global profiling of lysine reactivity and ligandability in the human proteome.Nat. Chem. 2017; 9: 1181-1190Crossref PubMed Scopus (220) Google Scholar, Roberts et al., 2017aRoberts A.M. Ward C.C. Nomura D.K. Activity-based protein profiling for mapping and pharmacologically interrogating proteome-wide ligandable hotspots.Curr. Opin. Biotechnol. 2017; 43: 25-33Crossref PubMed Scopus (58) Google Scholar, Wang et al., 2014Wang C. Weerapana E. Blewett M.M. Cravatt B.F. A chemoproteomic platform to quantitatively map targets of lipid-derived electrophiles.Nat. Methods. 2014; 11: 79-85Crossref PubMed Scopus (196) Google Scholar). Importantly, this technology allows for the interrogation of natural products in their unmodified form. In this study, we used ABPP chemoproteomic platforms to map additional targets of parthenolide in breast cancer cells, uncovering additional druggable hotspots that may contribute to the cell signaling and anti-cancer effects of parthenolide (Figure 1A). Parthenolide impaired cell proliferation and serum-free cell survival, induced cell death, thwarted early cell motility, and significantly attenuated in vivo tumor xenograft growth in estrogen receptor, progesterone receptor, and HER2 receptor-negative breast cancer (triple-negative breast cancer [TNBC]) cells—231MFP or HCC38 cells—in a time-dependent and dose-responsive manner (Figures 1B–1F and S1). The impairment in cell viability induced by parthenolide, evidenced by propidium iodide-positive and annexin-V-positive cells, may be due to various forms of cell death, including apoptosis, necrosis, or ferroptosis. We show that parthenolide leads to the activation of caspase-3/7 and that this cell death is significantly attenuated by the pan-caspase inhibitor Q-VD-OPh, indicating that parthenolide impairs cell viability in a caspase-dependent manner (Figure S1) and suggesting that a portion of the cell death is apoptotic. We note that we are observing anti-tumorigenic effects at a relatively low dose of 30 mg/kg, despite observing cell-viability impairments at 50-μM concentrations. This may be because of the covalent nature of parthenolide and accumulating target engagement over time. Since parthenolide irreversibly binds to their targets, the targets will stay bound to parthenolide until the protein turns over. TNBCs show the worst prognoses due to the lack of key druggable targets, and there are few targeted therapies (Dawson et al., 2009Dawson S.J. Provenzano E. Caldas C. Triple negative breast cancers: clinical and prognostic implications.Eur. J. Cancer. 2009; 45: 27-40Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). Our data suggested that parthenolide may be effective at attenuating TNBC pathogenicity. We next used ABPP methods to identify additional targets of parthenolide in breast cancer cells. To confirm that parthenolide was not completely non-specific, we first performed a competitive gel-based ABPP experiment in which we competed parthenolide against labeling of 231MFP breast cancer cell proteomes with a rhodamine-functionalized cysteine-reactive iodoacetamide (IA-rhodamine) probe. While this method is imprecise, we observed that parthenolide did not broadly inhibit global proteome-wide cysteine reactivity (Figure S1). Using a more specific, previously reported alkyne-functionalized parthenolide probe (parthenolide-alkyne) (Shin et al., 2017Shin M. McGowan A. DiNatale G.J. Chiramanewong T. Cai T. Connor R.E. Hsp72 is an intracellular target of the α,β-unsaturated sesquiterpene lactone, parthenolide.ACS Omega. 2017; 2: 7267-7274Crossref PubMed Scopus (14) Google Scholar), we observed multiple labeled proteins in 231MFP proteomes, of which some, but not all, targets were competed by parthenolide (Figure S1). Collectively, these results indicated that parthenolide does possess multiple protein targets in 231MFP proteomes but that this natural product is not completely promiscuous in its reactivity. While the parthenolide-alkyne probe could be used to identify additional targets of this natural product, we sought to map the specific amino acids within these targets that were engaged by unfunctionalized parthenolide. Thus, we next used isotopic tandem orthogonal proteolysis-enabled ABPP (isoTOP-ABPP) to identify specific ligandable sites targeted by parthenolide in 231MFP breast cancer proteomes. We competed parthenolide binding against the broadly cysteine-reactive alkyne-functionalized iodoacetamide probe (iodoacetamide-alkyne [IA-alkyne]) directly in 231MFP TNBC proteomes using previously established methods (Figure 2A and Table S1) (Backus et al., 2016Backus K.M. Correia B.E. Lum K.M. Forli S. Horning B.D. González-Páez G.E. Chatterjee S. Lanning B.R. Teijaro J.R. Olson A.J. et al.Proteome-wide covalent ligand discovery in native biological systems.Nature. 2016; 534: 570-574Crossref PubMed Scopus (421) Google Scholar, Bateman et al., 2017Bateman L.A. Nguyen T.B. Roberts A.M. Miyamoto D.K. Ku W.-M. Huffman T.R. Petri Y. Heslin M.J. Contreras C.M. Skibola C.F. et al.Chemoproteomics-enabled covalent ligand screen reveals a cysteine hotspot in reticulon 4 that impairs ER morphology and cancer pathogenicity.Chem. Commun. Camb. Engl. 2017; 53: 7234-7237Crossref PubMed Google Scholar, Grossman et al., 2017Grossman E.A. Ward C.C. Spradlin J.N. Bateman L.A. Huffman T.R. Miyamoto D.K. Kleinman J.I. Nomura D.K. Covalent ligand discovery against druggable hotspots targeted by anti-cancer natural products.Cell Chem. Biol. 2017; 24: 1368-1376.e4Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, Roberts et al., 2017aRoberts A.M. Ward C.C. Nomura D.K. Activity-based protein profiling for mapping and pharmacologically interrogating proteome-wide ligandable hotspots.Curr. Opin. Biotechnol. 2017; 43: 25-33Crossref PubMed Scopus (58) Google Scholar, Weerapana et al., 2010Weerapana E. Wang C. Simon G.M. Richter F. Khare S. Dillon M.B.D. Bachovchin D.A. Mowen K. Baker D. Cravatt B.F. Quantitative reactivity profiling predicts functional cysteines in proteomes.Nature. 2010; 468: 790-795Crossref PubMed Scopus (1079) Google Scholar). This analysis revealed three highly engaged targets of parthenolide that showed isotopically light vehicle-treated to heavy parthenolide-treated probe-modified peptide ratios of greater than 10, indicating >90% engagement of these sites—focal adhesion kinase 1 (FAK1) C427, paraoxonase 3 (PON3) C240, and DNA-protein kinase (DNA-PK or PRKDC) C729. FAK1 C427 was the top target showing the highest ratio, and thus we placed subsequent focus on investigating the role of FAK1-dependent effects of parthenolide in breast cancer cells (Figure 2A and Table S1). While the role of PON3 in cancer cells is unclear, FAK1 and PRKDC are known to be important drivers of cancer cell signaling and DNA repair, respectively. Notably, FAK1 and DNA-PK inhibitors have been shown to impair both cell survival and cell proliferation in cancer cells and are being pursued in the clinic (Helleday et al., 2008Helleday T. Petermann E. Lundin C. Hodgson B. Sharma R.A. DNA repair pathways as targets for cancer therapy.Nat. Rev. Cancer. 2008; 8: 193-204Crossref PubMed Scopus (1237) Google Scholar, Sulzmaier et al., 2014Sulzmaier F.J. Jean C. Schlaepfer D.D. FAK in cancer: mechanistic findings and clinical applications.Nat. Rev. Cancer. 2014; 14: 598-610Crossref PubMed Scopus (852) Google Scholar). Since C427 of FAK1 was the most highly engaged target in this study, we focused our attention on investigating the FAK1-dependent effects of parthenolide in TNBC cells. We validated the interaction of parthenolide with C427 of FAK1 using several complementary approaches. We first validated the interaction of parthenolide with FAK1, whereby we showed parthenolide prevention of pure human FAK1 kinase domain cysteine reactivity with a rhodamine-functionalized iodoacetamide probe (IA-rhodamine) by gel-based ABPP (Figure 2A). Based on previous studies, we conjectured that parthenolide reacted covalently with C427 of FAK1 through a homo-Michael addition involving the α/β unsaturated lactone (Figure 2B) (Kwok et al., 2001Kwok B.H. Koh B. Ndubuisi M.I. Elofsson M. Crews C.M. The anti-inflammatory natural product parthenolide from the medicinal herb Feverfew directly binds to and inhibits IkappaB kinase.Chem. Biol. 2001; 8: 759-766Abstract Full Text Full Text PDF PubMed Scopus (436) Google Scholar). Second, we demonstrated that parthenolide covalently reacts with C427 of FAK1 by identifying this parthenolide adduct on human FAK1 kinase domain by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Figure 2C). We also demonstrated that IA-rhodamine labeling of pure human FAK1 was abrogated in the C427A mutant and that no additional inhibition of remaining IA-rhodamine labeling of FAK1 was observed with parthenolide treatment (Figure 2D). Using a parthenolide-alkyne probe, we further showed that this probe labeled wild-type FAK1 protein, and that this labeling was prevented by parthenolide or in the C427A mutant FAK1 protein (Figure 2E). Previous studies have shown that FAK1 is amplified or overexpressed across a large fraction of breast tumors wherein FAK1 activity and expression is correlated with poor prognosis. FAK1 has been shown to be important in breast cancer cell survival, proliferation, and migration (Luo and Guan, 2010Luo M. Guan J.-L. Focal adhesion kinase: a prominent determinant in breast cancer initiation, progression and metastasis.Cancer Lett. 2010; 289: 127-139Crossref PubMed Scopus (214) Google Scholar, Sulzmaier et al., 2014Sulzmaier F.J. Jean C. Schlaepfer D.D. FAK in cancer: mechanistic findings and clinical applications.Nat. Rev. Cancer. 2014; 14: 598-610Crossref PubMed Scopus (852) Google Scholar). To determine whether any of the observed parthenolide-mediated proliferative, survival, or migration impairments were dependent on FAK1, we assessed parthenolide effects on these phenotypes under FAK1 knockdown in 231MFP breast cancer cells (Figures 2F and 2G). FAK1 knockdown confers significant resistance to parthenolide-mediated impairments in cell proliferation, serum-free cell survival, and cell migration, particularly at early time points, compared with control cells (Figures 2F and 2G), demonstrating that FAK1 contributes to the anti-cancer effects of parthenolide. Because parthenolide rapidly impairs cell proliferation and survival, we note that the migration phenotypes shown here are likely confounded by reduced cell viability from parthenolide treatment. Interestingly, FAK1 knockdown by small interfering RNA (siRNA) did not impair basal cell proliferation, survival, or migration. We postulate that this lack of effect may be due to either the multi-target polypharmacological nature of parthenolide, or potential adaptation to FAK1 knockdown during the inherently slower process of siRNA-mediated knockdown compared with acute inhibition of FAK1. We later show evidence for the latter hypothesis. We next sought to determine whether parthenolide functionally inhibits FAK1 activity and signaling. On the basis of previously reported crystal structures of FAK1, C427 resided in a loop region proximal to the ATP site, indicating that covalent modification of this site may be inhibitory (Iwatani et al., 2013Iwatani M. Iwata H. Okabe A. Skene R.J. Tomita N. Hayashi Y. Aramaki Y. Hosfield D.J. Hori A. Baba A. et al.Discovery and characterization of novel allosteric FAK inhibitors.Eur. J. Med. Chem. 2013; 61: 49-60Crossref PubMed Scopus (42) Google Scholar). Consistent with this premise, we showed that FAK1 activity was inhibited by parthenolide in vitro with pure human FAK1 kinase domain in a substrate activity assay (Figure 2H). While this paper was under revision, an elegant study describing the first structure-guided design, synthesis, and characterization of a FAK1 inhibitor that also covalently targeted C427 of FAK1 and inhibited its function was reported (Yen-Pon et al., 2018Yen-Pon E. Li B. Acebrón-Garcia-de-Eulate M. Tomkiewicz-Raulet C. Dawson J. Lietha D. Frame M.C. Coumoul X. Garbay C. Etheve-Quelquejeu M. et al.Structure-based design, synthesis, and characterization of the first irreversible inhibitor of focal adhesion kinase.ACS Chem. Biol. 2018; 13: 2067-2073Crossref PubMed Scopus (19) Google Scholar). Importantly, this report gives further credence to our hypothesis of the functional relevance of this cysteine and its effects on cancer cell proliferation. FAK1 is activated through membrane recruitment by growth factors, extracellular matrix, and integrin signaling followed by subsequent autophosphorylation at Y397. This produces an SH2-binding domain, which in turn recruits Src and promotes semi-autophosphorylation of Y576/577 of FAK1. The fully active FAK1/Src complex can now recruit, phosphorylate, and activate numerous targets including p130Cas/Bcar1 and paxillin (PXN) to drive cell motility and cytoskeletal modifications (Frame et al., 201