Targeting bone morphogenetic protein receptor 2 sensitizes lung cancer cells to TRAIL by increasing cytosolic Smac/DIABLO and the downregulation of X-linked inhibitor of apoptosis protein
Bone morphogenetic protein
Bone morphogenetic protein receptor
- C. elegans
inhibitor of apoptosis proteins
DNA double stranded breaks
Non-small cell lung carcinomas
Reactive oxygen species
Transforming growth factor beta (TGFβ) activated kinase 1
Including tumour-necrosis factor (TNF)-related apoptosis-inducing lingand
X-linked inhibitor of apoptosis protein
Lung cancer is the leading cause of cancer death in the United States. Despite advances in cancer treatments, 85% of patients diagnosed with lung cancer will succumb to the disease. Bone morphogenetic proteins 2 and 4 (BMP2/4) are highly conserved embryonic proteins required for normal development and regulate the survival, migration, and cell fate decisions of stem cells [1, 2]. BMP signaling is not active in adult lung tissue but is reactivated in lung carcinoma and lung inflammation [2, 3]. The majority of non-small cell lung carcinomas (NSCLC) highly overexpress the BMP2 ligand . BMP signaling in lung cancer regulates cell survival, migration, proliferation, stemness, angiogenesis, and ligand overexpression and is correlated with a worse prognosis [3, 5, 6, 7, 8]. BMP signaling stimulates tumorigenesis in many carcinomas including prostate , breast [10, 11], pancreatic , melanoma, and sarcoma . The BMP receptors are expressed in all NSCLC and inactivating mutations are infrequent .
There are over 20 BMP ligands that signal through serine/threonine kinases. The BMP ligands bind to the BMP type I receptors (ALK2, ALK3, or ALK6) , which are phosphorylated by the constitutively active BMP type 2 receptors (BMPR2, ActR-IIA, ActR-IIB) . The BMP receptor complex then phosphorylates Smad 1/5 , which then translocates to the nucleus, transcriptionally regulating downstream targets including the inhibitor of differentiation proteins (ID1, ID2, and ID3) [17, 18].
The BMP signaling cascade also regulates Smad 1/5-independent mechanisms. Smad 1/5-independent signaling occurs by the binding of proteins to the cytosolic tail of the BMP receptor. BMP regulation of cancer cell survival involves the regulation of X chromosome-linked inhibitor of apoptosis protein (XIAP) and transforming growth factor beta (TGFβ) activated kinase 1 (TAK1), an evolutionary conserved Smad 1/5-independent signaling pathway [19, 20, 21]. During embryonic development, BMPR2 regulates XIAP, which leads to the activation of TAK1 . Both XIAP and TAK1 are potent inhibitors of cell death in cancer cells. XIAP inhibits apoptosis by binding to and inactivating effector caspases 3, 7, and 9 . XIAP also functions as an E3 ligase inducing the degradation of caspases via the proteasome system . TAK1 inhibits cell death by activating nuclear factor-kappa beta (NF-κB)  and inhibits reactive oxygen species (ROS) production . XIAP is being targeted as a cancer therapeutic because its inhibition of caspases promotes resistance to cancer therapeutics that induce apoptosis including tumour-necrosis factor (TNF)-related apoptosis-inducing lingand (TRAIL) and various chemotherapeutics [23, 27, 28].
Several generations of small molecule inhibitors of BMP receptors have been derived from the same pyrazolo [1,5-a] pyrimidine core [29, 30, 31]. JL5 is an analog of DMH2, with improved pharmacokinetic properties compared to DMH2, that has been demonstrated to cause tumor regression of lung cancer xenografts [14, 20]. JL5 and DMH2 both cause a decrease in the expression of XIAP and a decrease in TAK1 activity [14, 20]. The mechanism by which the inhibition of BMP signaling decreases XIAP expression has not been fully elucidated. DMH2 and JL5 cause greater inhibition of BMP signaling, induce more cell death, and decrease expression of XIAP compared to the BMP inhibitors DMH1 and LDN [14, 20]. These BMP inhibitors all have potent inhibition of BMP type I receptors. JL5 and DMH2 demonstrate inhibition of BMPR2 while DMH1 and LDN have no activity for BMPR2. It is unknown whether the enhanced activity of JL5 is caused by its inhibition of BMPR2 smad-independent signaling.
In this study, we show that the BMP inhibitor JL5 enhances cell death of TRAIL and the Smac mimetic AEG 40730 treated lung cancer cells. JL5 enhances apoptosis by inducing the downregulation of XIAP through its inhibition of BMPR2 receptor function. Knockdown of BMPR2, but not BMP type I receptors, increase cytosolic Smac/DIABLO, which is a known inhibitor of XIAP. These studies show that BMPR2 regulates cell survival signaling pathways not mediated by type I receptors and that targeted inhibition of BMPR2 may enhance apoptotic cell death of cancer therapeutics.
Cell culture and reagents
The H1299 and A549 lung cancer cell lines (ATCC) were maintained in Dulbecco’s Modified Eagle’s medium (DMEM, Sigma Aldrich, St Louis, MO, USA) supplemented with 5% fetal bovine serum . JL5 and DMH2 were synthesized by the David Augeri laboratory, Rutgers School of Pharmacy. TRAIL was purchased from Thermo Fisher Scientific, Trolox from Cayman Chemical, Z-VAD-FMK from Selleckchem, and AEG 40730 from Tocris. DMH1 was purchased from Selleckchem (Houston, TX). Constitutively active ALK3 and ALK6 constructs were a gift from Joan Massague (Memorial Sloan Kettering Cancer Center, New York, New York) and the pcDNA3-XIAP-Myc K322/D28 was purchased from Addgene (Watertown, Massachusetts).
Western blot analysis was performed as previously reported . In brief, total cellular protein was generated using RIPA buffer and concentration was measured using the BCA assay. Protein was separated by SDS-PAGE and transferred to nitrocellulose. The blots were incubated overnight at 4 °C with the primary antibody. The primary antibodies that were used were rabbit monoclonal anti-Smac/DIABLO, rabbit monoclonal anti-cytochrome c, rabbit monoclonal anti-cIAP1, rabbit monoclonal anti-pTAK1, rabbit monoclonal XIAP, rabbit monoclonal anti-activated caspase-3, rabbit monoclonal anti-activated caspase-8, rabbit monoclonal anti-PARP (Cell signaling Technology, Danvers MA), rabbit monoclonal anti-ID1(Calbioreagents, San Mateo, CA), rabbit anti-actin, an affinity isolated antigen specific antibody (Sigma, Saint Louis, MO), and rabbit polyclonal anti-GAPDH (Sigma, St. Louis, MO).
Lung cancer cells were plated into 6-well plates and treated the next day for the designated period of time. Cells were trypsinized and the number of live and dead cells were determined using the Vi-CELL cell analyzer (Beckman Coulter), which analyzed 500 cells per sample and utilized trypan blue dye exclusion to determine dead cells.
Transient knockdown and transfection
Validated select siRNA was used to knockdown the expression of XIAP, BMPR2, ALK3, and ALK6 (Life Technologies). The ID numbers for the siRNA are: XIAP (S1456), ALK3 (s281), ALK6 (s2042), and BMPR2 (s2044 and s2045). Silencer Select negative control siRNA (4390843) was used to evaluate selectivity. Silencer Select negative controls do not target any gene product and have no effect on cell proliferation or viability. Transfections of the siRNA were performed using Lipofectamine® RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s protocol. Briefly, H1299 and A549 lung cancer cell lines were seeded for 24 h (h) up to 70–80% confluence at the time of transfection. 150 μl of Opti-MEM® Medium was used to dilute 9 μl of Lipofectamine® RNAiMAX Reagent, control siRNA and target siRNA. Diluted Lipofectamine® RNAiMAX Reagent was mixed with diluted siRNA in 1:1 ratio and incubated for 5 min (min) at room temperature to obtain the RNA-lipid complex. The cells were incubated in siRNA-lipid complexes for 24 h at 37 °C. After 24 h, the media was changed to fresh media and the transfected cells were used for further experiments. Cells were transfected with 30 nM ALK3, 20 nM ALK6, 30 nM XIAP, and 6 nM BMPR2.
Cytosolic protein extraction was performed using Mitochondria/Cytosol fractionation kit (Enzo Life Sciences, NY, USA). Briefly, 750,000 cells/well were seeded in 6-well plates for 24 h. The cells were treated with TRAIL and JL5 at designated times. After treatment, the cells were trypsinized, pelleted and washed twice with cold phosphate-buffered saline (PBS) and centrifuged at 600 x g for 5 min at 4 °C. Supernatant was removed and cell pellets were resuspended in 100 μl of ice-cold Cytosol Extraction Buffer Mix containing DL-Dithiothreitol (DTT) and Protease Inhibitors. After a 10 min incubation on ice, cells were homogenized. The homogenates were collected to a fresh 1.5 ml tube and centrifuged at 700 x g for 10 min at 4 °C. The supernatant was collected in a 1.5 ml tube and centrifuged at 10,000 x g for 30 min. at 4 °C. The supernant was collected as the cytosolic fraction and used for further experiments.
Reactive oxygen species (ROS) measurements
Intracellular reative oxygen species (ROS) development after treatment of JL5 and TRAIL alone or in combination were measured by the total ROS detection kit (Enzo Life Sciences) according to the manufacturer’s protocol. Briefly, H1299 cells were seeded in a 6-well plate at a density of 750,000 cells/well. After 24 h, cells were treated with DMSO, 2.5uM JL5 and 50 ng/ml TRAIL alone or in combination for 3 h, 24 h and 48 h. At the end of the treatment, cells were trypsinized and then stained with ROS detection solution. Stained cells were incubated in the dark at 37 °C for 30 min. The results were monitored by using a flow cytometer (BD Biosciences).
Deoxyribonucleic acid (DNA) double-strand breaks (DSB) after treatment were analyzed using FlowTACS In Situ TUNEL-based apoptosis detection kit (Trevigen) according to the manufacturer’s protocol. Briefly, 750,000 cells/well were seeded in 6-well plates for 24 h. The cells were treated with JL5, DMH2, TRAIL and JL5 and TRAIL combination at designated times. After treatment, cells were trypsinized and the cell pellet was fixed with 4% formaldehyde and permeabilized with cytonin for 30 min. After washing with labeling buffer, cells were resuspended in the labeling reaction mix and incubated for 1 h at 37 °C. Then the cells were stained with strep-fluorescein solution for 10 min at 37 °C. The samples were analyzed by using flow cytometry (LSRII, BD Biosciences).
H1299 cells at 450,000 cells/well concentration were seeded for 24 h onto microscope cover glasses in a 6-well plate. Next, cells were treated with 2.0 μM DMH1 or 2.5 μM JL5 for 24 h. After treatment, cells were fixed with 4% formaldehyde and permeabilized with 0.5% triton-X. After blocking with CAS-block for 1 h, cells were stained with anti-BMPR2 antibody (Sigma-Aldrich) for 1 h at room temperature. Cells were washed with PBS and stained with Alexa Flour 488 conjugated secondary antibody for 1 h at room temperature. After washing with PBS, the nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 10 min. Coverslips were then washed with PBS, rinsed with Mili-Q water and mounted with a mounting media. After drying, cells were observed under a fluorescence microscope (Nikon eclipse TE300).
Quantification of gene expression
Quantitative polymerase chain reaction (PCR) was performed for BMPR1A (ALK3), BMPR1B (ALK6), and BMPR2 following knockdown with small interfering ribonucleic acid (siRNA) as previously reported [20, 32]. In brief, RNA was extracted using the RNeasy kit (Quagen, Valencia, CA) and then treated with DNAse. cDNA was generated using Advantage RT for PCR kit (BD Bioscience, Clontech, Palo Alto). Quantitative PCR was performed with the Stratage Mx3005p (Agilent Technologies) and validated Taq-Man primers according to the manufacturer’s specifications (Applied Biosystems, Foster City, CA). Reference numbers used were: GAPDH (Hs99999905_m1), BMPR1A (ALK3) (Hs00831730_s1), BMPR1B (ALK6) (Hs00176144_m1), BMPR2 (Hs00176148_m1). Negative control included all reagents except cDNA. Expression was normalized to GAPDH using the formula 2 ΔCT.
BMPR2 (daf-4) response to JL5 in Caenorhabditis elegans (C. elegans)
Assessing BMP activity using spp-9::GFP reporter
Animals were age synchronized and treated with drug at the L1 stage at the indicated concentrations for JL5. Animals were then grown at 20 °C until the L4 stage. Live animals at the L4 stage were mounted on 2.5% (w/v) agarose and anesthetized using 10 mM levamisole. Animals were imaged at 5x magnification on a standard epifluorescent microscope. The average total intensity was calculated. Imaging quantification was performed using the open-source Fiji Software for each individual animal using the “Segmented Line” tool. A minimum of 60 animals were quantified for each condition performed twice. A one-way analysis of variation (ANOVA) was performed to compare differences in mean intensity across conditions.
Localization experiments for daf-4::GFP
Animals were age synchronized and treated with drug at the L1 stage at the indicated concentrations of JL5. Animals were then grown at 20 °C until the L4 stage. Live animals at the L4 stage were mounted on 25 (w/v) agarose and anesthetized using 10 mM Levamisole. Animals were imaged at 63x magnification on a laser spinning disc confocal microscope (Zeiss). Either the 3rd or 4th cell (from the anterior end) of the intestine was imaged. A minimum of 60 animals were quantified for each condition performed twice. An ANOVA was performed to compare differences in mean intensity across conditions.
The mean of the control group as compared to the mean of each treated group using a paired student t-test assuming unequal variances. Differences with p values < 0.05 were considered statistically significant.
JL5 enhances cell death of TRAIL treated lung cancer cells
JL5 enhances TRAIL-induced caspase-3 activation in H1299 cells
JL5 induced decrease in XIAP expression enhances TRAIL induced caspase-3 activation
XIAP inhibits caspase-3 apoptotic activity by binding the activated 19 and 17 kDa fragments as well as inhibiting its processing to the 19 and 17 kDa forms [35, 36]. Downregulating the expression of XIAP can overcome resistance to TRAIL [35, 36]. To determine whether the decreased expression of XIAP is the mechanism by which JL5 enhances apoptosis, H1299 cells were transiently transfected with XIAP that had its ubiquitination sites removed (mXIAP) , or vector control, and treated with TRAIL for 3 h. Processing of caspase-3 to its active 17 kDa form was less efficient in cells transfected with mXIAP as compared to cells transfected with vector control (Fig. 3c). Cells transfected with mXIAP also had an increased expression of pTAK1, confirming an increased activity of XIAP (Fig. 3c). The knockdown of XIAP with siRNA enhanced TRAIL induced activation of caspase-3 after 24 h compared to controls (Fig. 3d). Forced expression of mXIAP inhibited cell death induced by JL5 alone and in combination with TRAIL (Fig. 3e). These studies demonstrate that JL5 enhances apoptotic cell death induced by TRAIL and involves a decrease in the expression of XIAP.
JL5 causes the release of Smac/DIABLO into the cytosol
Increased permeability of the mitochondrial outer membrane allows the release of the proapoptotic agents Smac/DIABLO and/or cytochrome c into the cytosol. Cytosolic Smac/DIABLO binds XIAP with high affinity inhibiting its anti-apoptotic effects on activated executionary caspases-3 and 7 leading to apoptosis . Smac/DIABLO can also induce the ubiquitination and proteasomal degradation of XIAP . Since JL5 decreases expression of XIAP and enhances apoptosis, we examined whether it increases cytosolic Smac/DIABLO and/or cytochrome c. In H1299 cells, JL5 increased cytosolic Smac/DIABLO within 3 h, which persisted for up to 24 h (Fig. 3f, h). Both JL5 and TRAIL increased cytosolic cytochrome c in H1299 cells after 24 h (Fig. 3g). TRAIL did not increase cytosolic Smac/DIABLO expression after 24 h (Fig. 3h). JL5 did not increase cytosolic Smac/DIABLO or cytochrome c induced by TRAIL (Figures G-H). In A549 cells, JL5 and TRAIL had little effect on cytosolic cytochrome c or Smac/DIABLO expression in the time points examined (Fig. 3g-h). Since JL5 increases cytosolic Smac/DIABLO expression prior to the decrease in the expression of XIAP suggests that increasing cytosolic Smac/DIABLO expression may be a mechanism by which JL5 downregulates XIAP in H1299 cells.
Knockdown of BMPR2 but not BMP type 1 receptors increases cytosolic Smac/DIABLO levels
We next examined whether JL5 regulation of XIAP and the increase in cytosolic Smac/DIABLO and/or cytochrome c was mediated by the inhibition of BMPR2. Quantitative PCR showed two different siRNA decreased BMPR2 RNA expression (Fig. 4e). The siRNA caused a 70 and 50% reduction in BMPR2 protein expression in comparison to control in H1299 and A549 cells respectively (Fig. 4 f-i). Consistent with our prior report, the knockdown of BMPR2 decreased XIAP expression in both H1299 (Fig. 4f) and A549 cells (Fig. 4h). Both BMPR2 siRNAs increased cytosolic Smac and cytochrome c in A549 cells (Fig. 4j). Knockdown of BMPR2 was associated with a decrease in Smad 1/5 activity, confirming downregulation of BMP signaling (Fig. 4k). Knockdown of BMPR2 in H1299 cells also caused an increase in the expression of Smac/DIABLO in the cytosol (Fig. 4l), however, no clear increase in cytoplasmic cytochrome c was detected at this time point (Fig. 4l). These studies suggest that JL5 mediates the downregulation of XIAP and increases cytosolic Smac/DIABLO by inhibiting BMPR2.
JL5 causes cytoplasmic trapping of BMPR2 in lung cancer cells and in C. elegans
We examined whether the effects of JL5 on BMPR2 localization are conserved in C. elegans. The intracellular trafficking and recycling of the BMPR2 has been studied in great detail in C. elegans . The nematode BMPR2, daf-4, is internalized utilizing a clathrin-independent mechanism and recycles back to the plasma membrane via the recycling endosome . We first showed that BMP signaling in the worm can be inhibited with JL5 (Fig. 5b). Microarray studies conducted in C. elegans have identified BMP regulated genes . The spp-9 gene is negatively regulated by BMP signaling; mutants in the BMP signaling pathway exhibit increased spp-9::GFP expression in live animals bearing this transgene . In order to determine whether JL5 inhibits BMP signaling in C. elegans, animals bearing a germline-integrated spp-9::GFP transgene were treated with varying concentrations of the drugs from early L1 stage and spp-9::GFP expression was assayed at L4 stage worms (~ 72 h later). Similar to what was found in lung cancer cells, JL5 decreased BMP signaling as demonstrated by an increase in spp-9::GFP activity (Fig. 5b). A higher concentration of JL5 was needed in comparison to cell cultures experiments to penetrate the C. elegans tough outer cuticle covering. Given this conservation of function, we asked whether JL5 would also lead to a trafficking defect in vivo in the whole animal. Treatment with JL5 for 72H. leads to dramatic changes in the localization and trafficking of daf-4 (BMPR2) – with the receptor being trapped intracellularly within vesicles (Fig. 5c). These studies suggest that JL5 decreases BMPR2 signaling and influences trafficking of the BMPR2 and its mechanism of action likely works through a conserved trafficking pathway.
BMP inhibitors increase the production of ROS and DNA-DSB)
JL5 enhances cell death by the Smac/DIABLO mimetic, AEG40730
The BMP signaling cascade regulates several pro-survival signaling pathways in cancer cells including XIAP, TAK1, and the inhibitor of differentiation proteins (ID1-ID3) [6, 14, 20, 32, 44]. XIAP inhibits caspases by binding activated fragments, inhibiting the processing of executioner caspases into their active forms, and triggering caspase degradation through the ubiquitin proteasome pathway [23, 45]. XIAP inhibition of caspases promotes resistance to many cancer therapeutics including radiation, chemotherapeutics, and TRAIL [28, 35] The inhibition of BMPR2 decreases the expression of XIAP and inhibits the activity of TAK1 [20, 22, 46]. TAK1 is a BMP regulated protein that is a very potent inhibitor of cell death [25, 47, 48]. BMP signaling regulates TAK1 activity, at least, in part by its regulation of XIAP . TAK1 has also been shown to generate resistance to cancer therapeutics . BMP-Smad-1/5 signaling is one of the most potent transcriptional activators of ID1, ID2, and ID3 [17, 18]. ID1-ID3 are also tumorigenic as they stimulate cell survival, proliferation, migration/invasion of cancer cells, inhibition of senescence, and promotion of immortalization of normal cells. Our studies suggest the potential use of BMP small molecule inhibitors to augment cell death of cancer therapeutics that induce cell death by apoptosis.
This is the first report demonstrating that inhibition of a BMP receptor increases cytosolic cytochrome c and Smac/DIABLO. The increased release of cytochrome c and Smac/DIABLO is specific for BMPR2 and not the type 1 BMP receptors. This has significant implications regarding targeting BMP receptors as potential cancer therapeutics. Thus far, all the BMP receptor inhibitors developed target predominantly the type 1 BMP receptors. The BMP receptor inhibitors JL5 and DMH2 have been shown to cause a greater decrease in the expression of the BMP signaling proteins ID1, XIAP, and TAK1, and induce more cell death of lung cancer cell lines than the BMP inhibitors DMH1 and LDN . All these inhibitors have potent inhibition of BMP type 1 receptors but only JL5 and DMH2 inhibit BMPR2 . We hypothesized that the enhanced potency of JL5 in comparison to DMH1 was due to its inhibition of BMPR2. This hypothesis was supported by our studies showing that the knockdown of BMPR2 but not the BMP type 1 receptors decreased XIAP expression and increased cytosolic Smac/DIABLO and cytochrome c. Although JL5 only has an IC50 of 8 μM  for BMPR2, it was able to induce the internalization and trapping of BMPR2 in cytoplasmic vesicles, which did not occur with DMH1. BMPR2 must traffick back to the plasma membrane to remain active and trapping in cytoplasmic vesicles leads to inhibtion [39, 40, 41]. These studies demonstrate the importance of specifically targeting BMPR2 as a strategy to induce cell death in lung cancer and support further development of more potent BMPR2 inhibitors. Future studies will be needed in animal tumor models to validate this hypothesis.
BMP receptors mediate Smad-dependent and Smad-independent signaling. The cytoplasmic tail of BMPR2 is longer than that of the type 1 receptors, which mediate Smad-independent signaling. XIAP binds the cytosolic tail of BMPR2 and this binding is thought to stabilize XIAP, leading to increased expression . Our studies suggest that BMPR2 regulation of XIAP involves the release of Smac/DIABLO into the cytosol, presumably from the mitochondria where Smac/DIABLO is localized normally. Cytosolic Smac/DIABLO binds and inactivates inhibitor of apoptosis proteins . Cytosolic Smac/DIABLO or Smac3 is also reported to induce the degradation of XIAP and other inhibitor of apoptosis proteins through the ubiquitin proteasome pathway [23, 38]. Prior studies only showed that XIAP was bound to BMPR2 and that the knockdown of BMPR2 resulted in a decreased expression of XIAP. Prior reports did not reveal the mechanisms by which BMPR2 led to the “stabilization” of XIAP. The knockdown of BMPR2 causes an increase in cytosolic Smac/DIABLO, a well-known inhibitor of XIAP. This suggests that BMPR2 regulation of XIAP involves more than just promoting its stabilization. Our studies do not rule out that the downregulation of BMPR2 may also regulate the expression of XIAP through other pathways. The activation of proteolytic cell death pathways cathepsins and/or caspases can also induce the degradation of XIAP .
A549 cells have an activating K-ras mutation and are resistant to TRAIL and AEG. They are less responsive to JL5 compared to H1299 cells. Alterations or the inhibition of the TRAIL receptors can prevent the activation of caspase-8 and promote resistance . TRAIL requires the activation of caspase-3 to induce cell death. TRAIL activation of caspase-3 in some cells requires an amplification step at the mitochondria with the release of cytochrome c . TRAIL can also activate caspase-3 in some cells without mitochondrial amplification . XIAP promotes resistance to TRAIL through its inactivation of caspase-3 . In A549 cells, TRAIL did not activate caspase-8 which may be the reason why we did not find enhanced cell death with the combination of TRAIL and JL5. Our data suggests that to enhance TRAIL induced cell death with JL5, there needs to be activation of caspase-8 by TRAIL and a downregulation of XIAP by JL5. In H1299 cells, TRAIL activated caspase-8 and induced increased cytosolic cytochrome c but did not induce cell death. TRAIL did not fully activate caspase-3 as demonstrated by the lack of processing to the 17 kDa fragment. Our studies show that JL5 minimized the resistance to TRAIL by decreasing the expression of XIAP. In A549 cells, JL5 did not decrease XIAP expression, increase cytosolic Smac/DIABLO, or increase cell death. Interestingly, the knockdown of BMPR2 in A549 cells did decrease XIAP expression and increase cytosolic Smac/DIABLO and cytochrome c. It is possible that a more potent small molecule inhibitor of BMPR2 is needed to increase mitochondrial permeability in A549 cells. Genetic alterations of BMPR2 are infrequent in lung adenocarcinomas. Review of the Cancer Genome Atlas of 135 primary lung adenocarcinomas revealed only 3 missense mutations and 1 truncating mutation of BMPR2 , suggesting BMPR2 could be targeted with small molecules.
These studies provide further evidence that the BMP signaling cascade in cancer cells promotes survival. Our studies support that BMP survival mechanisms in cancer cells are mediated predominantly by BMPR2. These data support the development of a more potent and specific BMPR2 inhibitor for potential use to enhance the effects of cancer therapeutics.
YP wants to acknowledge the Rutgers Cancer Institute of New Jersey Biomedical Informatics Shared Resource with funding from NCI-CCSG P30CA072720, and the computational resources made possible through the access to the Perceval Linux cluster operated by OARC under NIH 1S10OD012346-01A1.
RN did cell proliferation studies, siRNA knockdown, Western blots, and editing of manuscript; AM did immunofluoresent imaging studies and participating in writing the manuscript; MV did the C elegans studies, DG performed Western blots, LL discussed and interpreted data; MS did language editing of the manuscript; SJ did language editing of the manuscript; YP did language editing of the manuscript and interpreted data, CR assisted with the design and interpretation of the C. elegans studies, DA synthesized JL5 used for these studies, JL designed experiments, interpreted data, and wrote the body of the manuscript. All authors read and approved the final manuscript.
This work was supported by grants National Institute of Health R01 CA225830-01A1 and Radiation Oncology Pilot Award (Rutgers Cancer Institute of New Jersey).
Ethics approval and consent to participate
Consent for publication
All authors have agreed to publish this manuscript.
A patent application has been issued for DMH2. A provisionary patent is pending for JL5. There are no active or pending financial agreements regarding these patent applications nor has any money been received or is pending.
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