Association of activated Gαq to the tumor suppressor Fhit is enhanced by phospholipase Cβ
BMC Cancer volume 15, Article number: 775 (2015)
G proteins are known to modulate various growth signals and are implicated in the regulation of tumorigenesis. The tumor suppressor Fhit is a newly identified interaction partner of Gq proteins that typically stimulate the phospholipase C pathway. Activated Gαq subunits have been shown to interact directly with Fhit, up-regulate Fhit expression and enhance its suppressive effect on cell growth and migration. Other signaling molecules may be involved in modulating Gαq/Fhit interaction.
To test the relationship of PLCβ with the interaction between Gαq and Fhit, co-immunoprecipication assay was performed on HEK293 cells co-transfected with different combinations of Flag-Fhit, Gα16, Gα16QL, pcDNA3 vector, and PLCβ isoforms. Possible associations of Fhit with other effectors of Gαq were also demonstrated by co-immunoprecipitation. The regions of Gαq for Fhit interaction and PLCβ stimulation were further evaluated by inositol phosphates accumulation assay using a series of Gα16/z chimeras with discrete regions of Gα16 replaced by those of Gαz.
PLCβ1, 2 and 3 interacted with Fhit regardless of the expression of Gαq. Expression of PLCβ increased the affinities of Fhit for both wild-type and activated Gαq. Swapping of the Fhit-interacting α2-β4 region of Gαq with Gαi eliminated the association of Gαq with Fhit without affecting the ability of the mutant to stimulate PLCβ. Other effectors of Gαq including RGS2 and p63RhoGEF were unable to interact with Fhit.
PLCβ may participate in the regulation of Fhit by Gq in a unique way. PLCβ interacts with Fhit and increases the interaction between Gαq and Fhit. The Gαq/PLCβ/Fhit complex formation points to a novel signaling pathway that may negatively regulate tumor cell growth.
The fragile FHIT gene at the chromosomal fragile site FRA3B is often regarded as an early target of DNA damage in precancerous cells. Its gene product, the ubiquitously expressed Fhit (Fragile Histidine Triad) protein, is a member of the HIT (histidine triad) superfamily with three signature histidines in the conserved nucleoside binding motif. Fhit binds and hydrolyzes various dinucleoside polyphosphates (such as Ap3A, Ap4A, Ap3G and Cp3G) into two nucleotides where one is a nucleoside monophosphate . The preferred substrate of Fhit is Ap3A (diadenosine 5′,5‴-P1,P3-triphosphate) which is hydrolyzed to AMP and ADP. Interestingly, Fhit acts as a tumor suppressor and its down-regulation is associated with different tumors including lung cancers . Re-expression of Fhit in Fhit-deficient tumor cells can notably suppress tumor development [3–5]. Several theories of tumor suppression have been proposed for Fhit with the overarching idea of Fhit acting as a genome “caretaker” [6, 7]. Reduced Fhit expression has indeed been shown to increase DNA replication stress and genome alterations as a result of a decreased intracellular thymidine triphosphate (dTTP) level . Moreover, Fhit is apparently involved in suppressing lung tumor cell migration/invasion by down-regulating the expression of matrix metalloproteinase 2/9 . Despite considerable efforts, the precise mechanism by which Fhit exerts its tumor suppressive function remains elusive. The dinucleoside polyphosphate hydrolase activity of Fhit seemingly plays a trivial role in tumor suppression .
A number of studies have revealed unsuspecting binding partners of Fhit that may provide linkages to processes that contribute to tumor eradication such as cellular oxidation and apoptosis. Fhit-interacting molecules include β-catenin , ferredoxin reductase , Src tyrosine kinase  and ubiquitin conjugating enzyme 9 . More recently, we have demonstrated that Fhit can distinguish between inactive and active signal transducing Gα subunits of the Gq family . This finding is intriguing as it may link Fhit to Gαq-dependent signals that modulate a variety of cellular events. Fhit-mediated suppression of epithelial-mesenchymal transition in bronchial cells involves the epidermal growth factor receptor (EGFR), Src, and extracellular signal-regulated kinase (ERK)  that have all been shown to be activated or transactivated by Gαq [17, 18]. In human colon cancer cell lines, Fhit inhibits cell proliferation by attenuating the nuclear factor κB (NFκB) pathway  which can be stimulated by Gq-coupled receptors . It is also noteworthy that sustained activation of the Gq pathway often leads to mitogenesis in a variety of cell types  with disparate mechanisms of regulating cell cycle progression . The opposing roles of Fhit and Gαq tend to suggest that they may exert counteracting actions on each other. However, Fhit neither inhibits nor enhances Gαq-induced signals  whereas its own expression becomes translationally up-regulated by activated Gαq . Given the links between Gq signaling and mitogenesis as well as those between Fhit and tumor suppression are well-established, it seems reasonable to expect that the binding of Fhit to activated Gαq would impart functional consequences. Since the canonical signaling pathway of all Gαq subfamily members (Gαq, Gα11, Gα14 and Gα15/16) is the activation of phospholipase Cβ (PLCβ), we further explored the influence of PLCβ on the formation of Fhit/activated Gαq complexes. Here, we report that different isoforms of PLCβ can also associate with Fhit in the absence of Gαq activation.
The human cDNAs of various Gα subunits were obtained from Guthrie Research Institute (Sayre, PA). Wild-type Fhit in pCMV-SPORT6 was purchased from Invitrogen (Carlsbad, CA). Gα16/z chimeras were constructed by overlapping PCR which swapped the corresponding regions of Gα16 with Gαz as described previously . Cell culture and Lipofectamine PLUS reagents, and anti-Fhit antibody were purchased from Invitrogen (Carlsbad, CA). Anti-Gα16 was obtained from Gramsch Laboratories (Schwabhausen, Germany). Anti-Gαq/11 antibody was purchased from Calbiochem (San Diego, CA). Anti-α-tubulin, anti-HA, and anti-Flag antibodies as well as anti-HA affinity gel were from Sigma-Aldrich (St. Louis, MO). Antisera against PLCβ1/2/3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Other antibodies were purchased from Cell Signaling Technology (Danvers, MA). Protein G-agarose was from Thermo Fisher Scientific (Rockford, IL). ECL kit was from Amersham Biosciences (Piscataway, NJ).
Cell culture and Co-immunoprecipitation
HEK293 cells were obtained from the American Type Culture Collection (CRL-1573, Rockville, MD). They were maintained in Eagle’s minimum essential medium at 5 % CO2, 37 °C with 10 % fetal bovine serum, 50 units/mL penicillin and 50 μg/mL streptomycin. Transfection was performed according to the manual of Lipofectamine® transfection reagent. One day later, cells were lysed in ice-cold RIPA buffer (25 mM HEPES at pH 7.4, 0.1 % SDS, 1 % Nonidet P-40, 0.5 % sodium deoxycholate, 1 mM dithiothreitol, 200 μM Na3VO4, 4 μg/mL aprotinin, 100 μM phenylmethylsulfonyl fluoride, and 2 μg/mL leupeptin). Cell lysates were incubated with a primary antiserum with rotation at 4 °C overnight, and then incubated in 30 μL protein G-agarose (50 % slurry) at 4 °C for 4 h. Alternatively, the cell lysates were incubated in 30 μL anti-Flag affinity agarose gel (50 % slurry) at 4 °C for 4 h. Immunoprecipitates were washed with ice-cold RIPA buffer (400 μL) for four times, resuspended in 50 μl RIPA buffer and 10 μl 6× sample buffer and then boiled for 5 min. Target proteins in the immunoprecipitates were analyzed by Western blots. Signal intensities of the immunoreactive bands were quantified using Image J software, version 1.38× (National Institutes of Health, USA).
Western blotting analysis
Protein samples were resolved on 12 % SDS-polyacrylamide gels and transferred to Osmonics nitrocellulose membrane. Resolved proteins were detected by their specific primary antibodies and horseradish peroxidase-conjugated secondary antisera. The immunoblots were visualized by chemiluminescence with the ECL kit from Amersham, and the images detected in X-ray films were quantified by densitometric scanning using the Eagle Eye II still video system (Stratagene, La Jolla, CA, USA).
Inositol phosphates accumulation assay
HEK293 cells were seeded at a density of 2×105 cells/well into 12-well plates. Various cDNAs at a concentration of 0.5 μg/well were transiently transfected into the cells using Lipofectamine® transfection reagents. One day after transfection, cells were labeled with inositol-free Dubecco’s modified Eagle’s medium (DMEM; 750 μL) containing 5 % FBS and 2.5 μCi/mL myo-[3H]inositol overnight. The labeled cells were then washed once with IP3 assay medium (20 mM HEPES, 5 mM LiCl, serum-free DMEM) and then incubated with 500 μl IP3 assay medium at 37 °C for 1 h. Reactions were stopped by replacing the assay medium with 750 μL ice-cold 20 mM formic acid and the lysates were kept in 4 °C for 30 min before the separation of [3H]inositol phosphates from other labeled species by sequential ion-exchange chromatography as described previously .
Data were expressed as the mean ± S.E. of at least three independent sets of experiments. The probability of an observed difference being a coincidence was evaluated by Dunnett t test. Differences at values of P < 0.05 were considered significant (* P < 0.05).
We have previously shown that Fhit directly interacts with activated members of the Gαq family (Gαq, Gα14, and Gα16) via their α2-β4 region without affecting Gαq-induced PLCβ activation . As PLCβ also interacts with the α2 region of the activated Gαq , we asked whether PLCβ can compete with Fhit for the activated Gαq in co-immunoprecipitation assays. HEK293 cells were co-transfected with Flag-tagged Fhit and wild-type or activated Gα16 (Gα16QL) with or without PLCβ1, PLCβ2 or PLCβ3. Because activated Gαq signaling always increase the expression levels of Fhit , we adjusted the Fhit cDNA amount for transfection to obtain similar Fhit expression levels. In order to facilitate the assessment of expression and to minimize interference by endogenous Gαq subunits, we have opted for using Gα16 as a representative Gαq member. In vector transfected cells, Fhit pulled down detectably more Gα16QL than wild-type Gα16 (Fig. 1), as reported previously . Overexpression of PLCβ1, PLCβ2 or PLCβ3 increased the affinities of both wild-type and activated Gα16 for Fhit (cf lanes 1, 2 and lanes 4, 5 in the three panels of the second row of Fig. 1). Thus PLCβs did not appear to compete with Fhit for activated Gα16. Instead, the presence of PLCβs apparently enhanced or stabilized the association of Fhit and Gα16. More interestingly, all three isoforms of PLCβ were also detected in the Fhit-immunoprecipitates (Fig. 1). To test if Fhit can form complexes with PLCβs, HEK293 cells were transfected with vector or PLCβ1-3 in combination with Flag-Fhit or Flag vector. All three isoforms of PLCβ were able to co-immunoprecipitate with Fhit (Fig. 2a). Since HEK293 cells endogenously express PLCβ1 (Fig. 2a, upper left panel), Flag-Fhit might be able to pull down endogenous PLCβ1. Longer exposure of the anti-PLCβ1 blot indeed revealed the presence of endogenous PLCβ1 in the immunoprecipitates of Fhit (Fig. 2b). Moreover, as compared to the Flag vector control, Fhit could pull down endogenous wild-type Gαq when one of the PLCβ isoforms was overexpressed (cf lane 1 and lanes 3, 5, and 7 of the fourth row on the right in Fig. 2a). These findings indicate that PLCβs may interact with Fhit and increase the association between Fhit and Gαq. We have previously reported that increased cell proliferation by Gαq activation is suppressed in the presence of Fhit . Not surprisingly, we also found that PLCβ3 overexpression alone was sufficient to trigger a higher cell growth rate and Fhit co-expression significantly decreased PLCβ3-induced cell proliferation (data not shown). Therefore, Fhit appears to be capable of suppressing Gq-PLCβ mediated cell proliferation.
If PLCβ binds to Fhit via activated Gαq, other proteins known to associate with Gαq may also co-immunoprecipitate with Fhit. Besides PLCβs, activated Gαq also interacts with other proteins such as RGS2  and p63RhoGEF . Unlike PLCβs, Fhit did not interact with HA-tagged RGS2 in the co-immunoprecipitation assay using anti-Flag or anti-HA affinity gel (Fig. 3a). Similarly, in transfected HEK293 cells expressing various combinations of Flag-Fhit, myc-tagged p63RhoGEF, Gαq and constitutively active GαqRC, myc-p63RhoGEF pulled down GαqRC but not Fhit (Fig. 3b). Moreover, the expression of Fhit did not affect the interaction between GαqRC and p63RhoGEF (Fig. 3b). These results suggest that the ability of PLCβs to associate with Fhit and increase the interaction between Fhit and Gαq may be specific.
Since overexpression of PLCβs appeared to enhance the interaction between Fhit and Gα16 (Fig. 1), we assessed whether Fhit can reciprocally enhance the interaction of PLCβ with Gαq members. We performed co-immunoprecipitation assay with anti-PLCβ3 antiserum because it has the best specificity among the different anti-PLCβ antisera. Gα16QL as well as Fhit was co-immunoprecipitated with PLCβ3 (Fig. 4). Co-expression of Fhit appeared to weaken the interaction between Gα16QL and PLCβ3 (Fig. 4 cf lanes 2 and 4 of row two). The reduction of Gα16QL in the PLCβ3-immunoprecipitates was not due to variations in the expression levels or pull down efficiency, as these parameters were essentially similar in the different samples (Fig. 4 rows one and five).
By using a series of Gα16/z chimeras with discrete regions of Gα16 replaced by those of Gαz (a Gαi subfamily member which does not interact with Fhit), we have previously identified the α2-β4 region of Gα16 as critical for Fhit interaction . Interestingly, the α2 region of Gαq is seemingly involved in binding to PLCβ . To further investigate the associations among Gαq, PLCβ and Fhit, we constructed two new chimeras named zα2β4 (Gα16 backbone with α2β4 region from Gαz) and 16α2β4 (Gαz backbone with α2β4 region from Gα16), wherein the α2-β4 region of Gα16 or Gαz was swapped with each other (Fig. 5a). As shown in Fig. 5b, both wild-type and constitutively active mutants of the zα2β4 and 16α2β4 chimeras can be expressed in HEK293 cells to levels that were comparable to those of Gα16, Gαz, or C128 (a previously characterized chimera with the C-terminal 128 residues of Gα16 swapped with the corresponding sequences of Gαz). Because the Gα-specific antibodies are N-terminal targeting, the zα2β4 and 16α2β4 chimeras were recognized by anti-Gα16 and anti-Gαz antisera, respectively. When examined for their ability to stimulate PLCβ, activated zα2β4QL efficiently stimulated the formation of inositol phosphates to an extent similar to that of Gα16QL (Fig. 5b). The constitutively active GαzQL did not activate PLCβ because Gαz belongs to the Gαi subfamily (Fig. 5b). Since neither 16α2β4QL nor C128QL was able to stimulate PLCβ (Fig. 5b), it indicated that the α2-β4 region of Gα16 alone was not sufficient to stimulate PLCβ.
A hallmark of Fhit/Gαq interaction is the enhanced association with the activated Gαq subunits over their wild-type counterparts . Activation state-dependent interaction with Fhit was reproducibly observed in the present study (Fig. 1 and Additional file 1: Figure S1) and this feature was therefore used as an indicator of Fhit association with the chimeras. In co-immunoprecipitation assays, both the wild-type and constitutively active mutant of zα2β4 were pulled down by Flag-Fhit to similar extents (Fig. 5c), despite the fact that zα2β4QL was fully capable of stimulating PLCβ (Fig. 5b). Thus the α2β4 region of activated Gα16 is required by Fhit association but not by PLCβ activation. In the control groups, more Gα16QL was detected in the Fhit-immunoprecipitates than wild-type Gα16 whereas both Gαz and GαzQL were hardly detected (Fig. 5c). These control groups produced the same results as in our previous report . The presence of 16α2β4 or 16α2β4QL in the Fhit-immunoprecipitates was even weaker than those Gαz (Fig. 5c), suggesting that this chimera could not be recognized by Fhit. It should be noted that activation state-dependent association with Fhit has previously been demonstrated for the C128 chimera , while C128QL was not able to stimulate PLCβ (Fig. 5b). According to the above results of zα2β4, 16α2β4 and C128, the capability of activated Gα16QL to associate with Fhit did not affect its ability to stimulate PLCβ and it may explain why PLCβ enhanced the association of Fhit and Gαq instead of competing with Fhit for Gαq,
The diversity of pathways downstream of Gq has endowed mammalian cells with a complex signaling network for the delicate regulation of a multitude of biological effects, with some responses being cell type-specific. One example is the fact that activation of Gq leads to proliferation in some cells while it induces apoptosis in other cell types . The tumor suppressor Fhit taps into the Gq signaling network through its ability to associate with activated Gαq [15, 23]. Interestingly, Fhit suppresses Gq-mediated cell growth in H1299 lung cancer cells via an unknown mechanism [15, 23]. Here, we showed that the canonical Gαq effector PLCβ can form a complex with activated Gαq and Fhit, and can increase the overall association of the latter two proteins. Many tripartite or even higher order complexes involving Gαq subunits are known to exist . For instance, Gαq can simultaneously bind to p63RhoGEF and RhoA , to G protein-coupled receptor kinase 2 (GRK2) and Gβγ , as well as to ADP-ribosylation factor 6 (ARF6) and ARNO (a GEF for ARF6) . Hence, the existence of a Gαq/PLCβ/Fhit complex seems plausible.
Given the prior demonstration that direct binding of Fhit to activated Gαq does not affect PLCβ activity , the ability of PLCβ to form a complex with Fhit/Gαq is rather surprising. All three PLCβ isoforms (PLCβ1, 2 and 3) tested as well as the endogenously expressed PLCβ1 could be detected in the immunoprecipitates of Fhit in the absence of Gαq overexpression (Fig. 2). Therefore, it is possible that PLCβ can directly interact with Fhit. Although the PLCβ association with Fhit may also occur via binding to endogenous Gαq subunits, the interaction between PLCβ and Fhit appears to be specific because other Gαq effectors such as RGS2 and p63RhoGEF did not interact with Fhit even in the presence of activated Gαq (Fig. 3). PLCβs have weak affinities for inactive Gαq subunits (EC50 at ~10 μM range)  but this basal interaction between PLCβ3 and inactive Gαq could be detected (Fig. 4). The increased affinity of Fhit with inactive Gαq upon overexpression of PLCβs (Figs. 1 and 2b) may result from the basal interactions between PLCβs and inactive Gαq and the interaction between PLCβ and Fhit. It remains to be demonstrated if PLCβ can directly interact with Fhit.
The α2-β4 region of Gαq is essential for the binding of Fhit  and other regions may also be required (Fig. 5b). According to the structure of Gαq-PLCβ3 complex, PLCβ3 interacts with the α2 and α3 region of Gαq by a helix-turn-helix domain  and a similar interaction domain is also present in the Gαq/p63RhoGEF complex . Different from PLCβ, p63RhoGEF could not interact with Fhit irrespective of whether activated Gαq was present (Fig. 3b). This tends to suggest that when activated Gαq is bound to an effector other than PLCβ, Fhit is precluded from interacting with Gαq. The complete lack of evidence on Fhit and PLCβ competing for activated Gαq further supports the existence of a tripartite complex of Fhit/Gαq/PLCβ. A prerequisite for the simultaneous binding of Fhit and PLCβ to activated Gαq is that the two molecules should not use identical regions on Gαq for interaction. This notion is indirectly supported by the results pertaining to the zα2β4QL chimera, which stimulated PLCβ activity as robustly as Gα16QL (Fig. 5b) but failed to interact with Fhit beyond that of wild-type zα2β4 (Fig. 5c). It would appear that the conformation of activated zα2β4QL can be recognized by PLCβ but not by Fhit. Although PLCβ has additional contact points (e.g., the α3-β5 region) on Gαq , the lack of detrimental effect upon the replacement of the α2-β4 region in the zα2β4QL chimera is rather intriguing and warrant some discussion. In the α2-β4 region of Gαq, nine of the ten residues (Q209-K215, H218, C219 and E221) for PLCβ3 interaction are the same with members of Gαi subfamily while the exceptional residue on Gαi subunits corresponding to R214 of Gαq is identical to that of Gα16 . Hence, it is possible that substitution of α2-β4 region of Gα16 (a Gαq member) with the corresponding region of Gαz (a Gαi subfamily member) would still allow the zα2β4QL chimera to interact productively with PLCβ (Fig. 5b). In contrast, the mere presence of the α2-β4 region of Gα16 in the 16α2β4QL chimera was insufficient to support efficient interaction with PLCβ or Fhit. Collectively, these results indicate that the substitution-induced conformational changes on the binding interface of Gαq are tolerated by PLCβ but not by Fhit.
PLCβ is a key molecule in transducing activated Gq protein signal to its downstream signal pathways, and similar to Gq protein, it also plays complicated roles in regulating cell growth. PLCβ2 expression level is positively correlated with breast cancer  and it promotes mitosis and migration of breast tumor cells . On the other hand, in human erythroleukemia cells, PLCβ1 suppresses proliferation probably through regulating cyclin D3 . PLCβ3-deficiency in mice leads to lymphoma and other tumors . The interaction between PLCβs and Fhit as well as the complex formation among Fhit, Gαq and PLCβ reveals new pathway(s) of cell growth inhibition by Gαq and PLCβ.
Activated Gαq binds to Fhit through the α2-β4 region of Gαq (Fig. 6a and ), and it binds to PLCβ through multiple regions of Gαq including the α2 region (Fig. 6b and ). The overlapping PLCβ and Fhit binding domain on the activated Gαq is the α2 region (Fig. 6c). As substitution of α2-β4 region of Gα16 with the corresponding region of Gαz did not affect the activated Gα16-induced PLCβ activation (Fig. 5b), Fhit may bind to this overlapped interacting region on Gαq and change the binding interface between Gαq and PLCβ without affecting the PLCβ activity. The altered binding interfaces of activated Gαq and PLCβ may trigger Gαq and PLCβ to form a ‘clamp’ around Fhit. Beside protein interactions that were found between two protein pairs among Gαq, PLCβ and Fhit, PLCβs increased the interaction between Gαq and Fhit. But Fhit or Gαq did not enhance the interaction between PLCβ and Gαq or Fhit, respectively (Fig. 6d). One possibility for increased affinities of Fhit to Gαq by PLCβ is that PLCβ may interact with and stabilize the complex of Fhit and Gαq. Another possibility is that when binding to Gαq, PLCβ may provide direct binding sites on itself for Fhit which also leads to the formation of a heterotrimeric protein complex. In both possibilities, the activated Gαq recruits PLCβ which acts as a positive regulator for the association of Fhit with activated Gαq. In the future, the involvement of PLCβ in the regulation of Fhit by Gαq and their possible roles on cancer therapy should be demonstrated.
We showed that PLCβ could interact with Fhit, and the expression of PLCβ increased the interaction between Gαq and Fhit. This regulatory effect appears to be unique to PLCβ because other Gαq effectors such as RGS2 and p63RhoGEF could not interact with Fhit. Substitution of the α2-β4 region of Gαq with Gαi did not affect Gαq-induced PLCβ activation but eliminated the interaction between Gαq with Fhit. This new Gαq/PLCβ/Fhit signaling complex represents a novel pathway of Gαq regulation on tumor suppression.
- IP3 :
Regulator of G protein signaling
Total cell lysate
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This work was supported in part by grants from the National Key Basic Research Program of China (2013CB530900), the Research Grants Council of Hong Kong (HKUST 661808), and the Hong Kong Jockey Club.
The authors declare that they have no competing interests.
HZ performed the experiments, participated in the design of the study and wrote the manuscript. YHW participated in the design of the study and the writing of the manuscript. All authors read and approved the final manuscript.
Fhit preferentially associates with activated Gα16 in HEK293 cells co-expressing PLCβ. HEK293 cells were co-transfected with different combinations of pFlag-CMV2 (V), Flag-Fhit (F), Gα16, Gα16QL, PLCβ1, PLCβ2 or PLCβ3. After co-immunoprecipitation assay with anti-Flag affinity gel, PLCβ1, 2, 3, Gα16, Fhit and α-tubulin were determined by Western blotting. Flag-Fhit pulled down detectably more Gα16QL than Gα16 (cf lanes 1 and 3 of row two), but wild-type Gα16 became able to interact with Fhit upon overexpression of PLCβs (cf lanes 1 and 2 of row two). All three forms of PLCβ were co-immunoprecipitated with Flag-Fhit but the co-expression of Gα16QL did not enhance the interaction between PLCβs and Fhit, rather such interactions were attenuated (Gα16 versus Gα16QL lanes). (PDF 185 kb)
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Zuo, H., Wong, Y.H. Association of activated Gαq to the tumor suppressor Fhit is enhanced by phospholipase Cβ. BMC Cancer 15, 775 (2015). https://doi.org/10.1186/s12885-015-1802-z