Hsp90 functions to balance the phosphorylation state of Akt during C2C12 myoblast differentiation

The function of the 90-kDa heat shock protein (Hsp90) is essential for the regulation of a myriad of signal transduction cascades that control all facets of a cell's physiology. Akt (PKB) is an Hsp90-dependent serine-threonine kinase that plays critical roles in the regulation of muscle cell physiology, including roles in the regulation of muscle differentiation and anti-apoptotic responses that modulate cell survival. In this report, we have examined the role of Hsp90 in regulating the activity of Akt in differentiating C2C12 myoblasts. While long-term treatment of differentiating C2C12 cells with the Hsp90 inhibitor geldanamycin led to the depletion of cellular Akt levels, pulse-chase analysis indicated that geldanamycin primarily enhanced the turnover rate of newly synthesized Akt. Hsp90 maintained an interaction with mature Akt, while Cdc37, Hsp90's kinase-specific co-chaperone, was lost from the chaperone complex upon Akt maturation. Geldanamycin partially disrupted the interaction of Cdc37 with Akt, but had a much less significant effect on the interaction of Hsp90 with Akt. Surprisingly, short-term treatment of differentiating C2C12 with geldanamycin increased the phosphorylation of Akt on Ser473, an effect mimicked by treatment of C2C12 cells with okadaic acid or the Hsp90 inhibitor novobiocin. Furthermore, Akt was found to interact directly with catalytic subunit of protein phosphatase 2A (PP2Ac) in C2C12 cells, and this interaction was not disrupted by geldanamycin. Thus, our findings indicate that Hsp90 functions to balance the phosphorylation state of Akt by modulating the ability of Akt to be dephosphorylated by PP2Ac during C2C12 myoblast differentiation.     

Differential effects of Hsp90 inhibition on protein kinases regulating signal transduction pathways required for myoblast differentiation

As derivatives of the Hsp90-inhibitor and tumoricidal agent geldanamycin move into phase II clinical trials, its potential for triggering adverse effects in non-tumor cell populations requires closer examination. In this report, the effect of geldanamycin on the differentiation and survival of C2C12 myoblasts was investigated. Treatment of differentiating C2C12 myoblasts with geldanamycin blocked myogenin expression, inhibited myotubule formation, and led to the depletion of three Hsp90-dependent protein kinases, ErbB2, Fyn, and Akt, and induction of apoptosis. ErbB2 levels declined rapidly, while Fyn and Akt levels decreased at a slower rate. Geldanamycin blocked the interaction of Hsp90 and its "kinase-specific" co-chaperone Cdc37 with Fyn, indicating that Fyn is an Hsp90-dependent kinase. Pulse-chase experiments indicated that geldanamycin caused newly synthesized Akt and Fyn to be degraded rapidly, but geldanamycin had little effect on the turnover rate of mature Fyn and Akt. Curiously, total cellular Src (c-Src) protein levels and the turnover rate of newly synthesized c-Src were unaffected by geldanamycin. While, geldanamycin had no effect on the levels of the putative Hsp90 client protein MyoD expressed in C2C12 cells, geldanamycin disrupted the interaction of Cdc37 with MyoD. Thus, inhibition of Hsp90 caused C2C12 cells to become depleted of multiple signal transduction proteins whose functions are essential for myoblast differentiation, and muscle cell survival, suggesting that geldanamycin derivatives may have the prospective of adversely affecting the physiology of certain sensitive muscle cell populations in vivo.     

TIEG1 negatively controls the myoblast pool indispensable for fusion during myogenic differentiation of C2C12 cells

The transforming growth factor (TGF)-β inducible early gene (TIEG)-1 is implicated in the control of cell proliferation, differentiation, and apoptosis in some cell types. Since TIEG1 functioning may be associated with TGF-β, a suppressor of myogenesis, TIEG1 is also likely to be involved in myogenesis. Therefore, we investigated the function of TIEG1 during myogenic differentiation in vitro using the murine myoblasts cell line, C2C12. TIEG1 expression increased during differentiation of C2C12 cells. Constitutive expression of TIEG1 reduced survival and decreased myotube formation. Conversely, knocking down TIEG1 expression increased the number of viable cells during differentiation, and accelerated myoblast fusion into multinucleated myotubes. However, expression of the myogenic differentiation marker, myogenin, remained unaffected by TIEG1 knockdown. The mechanism underlying these events was investigated by focusing on the regulation of myoblast numbers after induction of differentiation. The knockdown of TIEG1 led to changes in cell cycle status and inhibition of apoptosis during the initial stages of differentiation. Microarray and real-time PCR analyses showed that the regulators of cell cycle progression were highly expressed in TIEG1 knockdown cells. Therefore, TIEG1 is a negative regulator of the myoblast pool that causes inhibition of myotube formation during myogenic differentiation.     

Possible involvement of hic-5, a focal adhesion protein, in the differentiation of C2C12 myoblasts

Hic-5, a focal adhesion protein, has been implicated in cellular senescence and differentiation. In this study, we examined its involvement in myogenic differentiation. The hic-5 expression level in growing C2C12 myoblasts increased slightly on the first day and then gradually decreased until no hic-5 was detectable after 7 days of differentiation. In vivo, its expression level declined in the thigh and the calf skeletal muscle of mouse embryos after birth. The introduction of an antisense expression vector of hic-5 into C2C12 cells decreased the number of clones expressing the myosin heavy chain (MHC) upon exposure to the differentiation medium. In the cloned cells with low levels of hic-5, the efficiency of myotube formation was significantly reduced. The expression levels of MyoD, myogenin, MHC and p21 were also reduced in these clones. The results suggested that hic-5 plays a role in the initial stage of myogenic differentiation.     

Regulation of vascular endothelial growth factor receptor 2-mediated phosphorylation of focal adhesion kinase by heat shock protein 90 and Src kinase activities

Exposure of endothelial cells to vascular endothelial growth factor (VEGF) induced tyrosine phosphorylation of focal adhesion kinase (FAK) on site Tyr(407), an effect that required the association of VEGF receptor 2 (VEGFR2) with HSP90. The association of VEGFR2 with HSP90 involved the last 130 amino acids of VEGFR2 and was blocked by geldanamycin, a specific inhibitor of HSP90. Moreover, geldanamycin inhibited the VEGF-induced activation of the small GTPase RhoA, which resulted in an inhibition of phosphorylation of FAK on site Tyr(407). In this context, the inhibition of RhoA kinase (ROCK) with Y27632 or by expression of dominant negative forms of RhoA or ROCK impaired the VEGF-induced phosphorylation of Tyr(407) within FAK. In contrast to phosphorylation of Tyr(861), the phosphorylation of site Tyr(407) was insensitive to Src kinase inhibition by 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo[3,4-d] pyrimidine (PP2). We also found that the recruitment of paxillin to FAK was inhibited by geldanamycin but not by PP2, whereas both geldanamycin and PP2 inhibited the recruitment of vinculin to FAK. In accordance, the recruitment of paxillin and vinculin to FAK was inhibited in cells that express the mutant FAK-Y407F, whereas the expression of the mutant Y861F inhibited the recruitment of paxillin but not of vinculin. Importantly, cell migration was abolished in cells in which the signal from the VEGFR2-HSP90 pathway was blocked by the expression of Delta130VEGFR2, a deletant of VEGFR2 that does not associate with HSP90. Our findings underscore for the first time the key role played by the VEGFR2-HSP90-RhoA-ROCK-FAK/Tyr(407) pathway in transducing the VEGF signal that leads to the assembly of focal adhesions and endothelial cell migration.     

敲减DTX4表达抑制C2C12细胞成肌分化

DTX4（Deltex 4 homolog）蛋白属于Deltex家族成员|Deltex家族是Notch信号通路的调节因子. 已知Notch信号通路在成肌分化中发挥重要作用. 然而,DTX4是否参与调控肌肉发育尚未有报道. 本研究探索DTX4对成肌分化的影响及作用机制. 实时定量PCR和蛋白质印迹分析揭示,伴随小鼠C2C12成肌细胞（myoblast）分化为肌管（myotube）过程,成肌分化标志蛋白肌球蛋白重链（myosin heavy-chain,MyHC）、肌细胞生成素(myogenin)表达逐渐升高,DTX4 mRNA及蛋白质表达水平也逐渐升高. 通过顺序专一的siRNA敲减DTX4表达后,C2C12成肌细胞肌管面积和肌管融合指数明显减少|MyHC、肌细胞生成素蛋白表达水平明显降低|但ERK信号通路未见明显变化.上述结果表明,敲减DTX4表达抑制C2C12细胞成肌分化.我们的结果提示,DTX4可能参与C2C12细胞成肌分化.     

Tyrosine phosphorylation of HSP90 within the P2X7 receptor complex negatively regulates P2X7 receptors

The purinergic P2X7 receptor not only gates the opening of a cationic channel, but also couples to several downstream signaling events such as rapid membrane blebbing, microvesicle shedding, and interleukin-1beta release. Protein-protein interactions are likely to be involved in most of these signaling cascades; and recently, a P2X7 receptor-protein complex comprising at least 11 distinct proteins has been identified. We have studied one of these interacting proteins, HSP90, in human embryonic kidney cells expressing either human or rat P2X7 receptors as well as in rat peritoneal macrophages using biochemical (immunoprecipitation and Western blotting) and functional (membrane blebbing and currents) assays. We found that HSP90 was tyrosine-phosphorylated in association with the P2X7 receptor complex, but not in the cytosolic compartment. The HSP90 inhibitor geldanamycin decreased tyrosine phosphorylation of HSP90 and produced a 2-fold increase in the sensitivity of P2X7 receptors to agonist. Protein expression and tyrosine phosphorylation of a mutant P2X7 receptor in which a tyrosine in the C-terminal domain was substituted with phenylalanine (Y550F) were not changed, but tyrosine phosphorylation of HSP90 associated with this mutant P2X7 receptor complex was significantly greater than that associated with the wild-type complex. P2X7-Y550F receptors showed a 15-fold lower sensitivity to agonist, which was reversed by geldanamycin. We conclude that selective tyrosine phosphorylation of P2X7 receptor-associated HSP90 may act as a negative regulator of P2X7 receptor complex formation and function.     

Transactivation of hsp70-1/2 in geldanamycin-treated human non-small cell lung cancer H460 cells: involvement of intracellular calcium and protein kinase C

Geldanamycin is an antitumor drug that binds HSP90 and induces a wide range of heat shock proteins, including HSP70s. In this study we report that the induction of HSP70s is dose-dependent in geldanamycin-treated human non-small cell lung cancer H460 cells. Analysis of the induction of HSP70s specific isoform using LC-ESI-MS/MS analysis and Northern blotting showed that HSP70-1/2 are the major inducible forms under geldanamycin treatment. Transactivation of hsp70-1/2 was determined by electrophoretic mobility-shift assay using heat shock element (HSE) as a probe. The signaling pathway mediators involved in hsp70-1/2 transactivation were screened by the kinase inhibitor scanning technique. Pretreatment with serine/threonine protein kinase inhibitors H7 or H8 blocked geldanamycin-induced HSP70-1/2, whereas protein kinase A inhibitor HA1004, protein kinase G inhibitor KT5823, and myosin light chain kinase inhibitor ML-7 had no effect. Furthermore, the protein kinase C (PKC)-specific inhibitor Ro-31-8425 and the Ca2+-dependent PKC inhibitor G?-6976 diminished geldanamycin-induced HSP70-1/2, suggesting an involvement of the PKC in the process. In addition, geldanamycin treatment causes a transient increase of intracellular Ca2+. Chelating intracellular Ca2+ with BAPTA-AM or depletion of intracellular Ca2+ store with A23187 or thapsigargin significantly decreased geldanamycin-transactivated HSP70-1/2 expression. Taken together, our results demonstrate that geldanamycin-induced specific HSP70-1/2 isoforms expression in H460 cells through signaling pathway mediated by Ca2+ and PKC.     

Heat Shock Protein 90 Inhibitors Reduce Trafficking of ATP-gated P2X1 Receptors and Human Platelet Responsiveness

We have used selective inhibitors to determine whether the molecular chaperone heat shock protein 90 (HSP90) has an effect on both recombinant and native human P2X1 receptors. P2X1 receptor currents in HEK293 cells were reduced by ∼70–85% by the selective HSP90 inhibitor geldanamycin (2 μm, 20 min). This was associated with a speeding in the time course of desensitization as well as a reduction in cell surface expression. Imaging in real time of photoactivatable GFP-tagged P2X receptors showed that they are highly mobile. Geldanamycin almost abolished this movement for P2X1 receptors but had no effect on P2X2 receptor trafficking. P2X1/2 receptor chimeras showed that the intracellular N and C termini were involved in geldanamycin sensitivity. Geldanamycin also inhibited native P2X1 receptor-mediated responses. Platelet P2X1 receptors play an important role in hemostasis, contribute to amplification of signaling to a range of stimuli including collagen, and are novel targets for antithrombotic therapies. Platelet P2X1 receptor-, but not P2Y1 receptor-, mediated increases in intracellular calcium were reduced by 40–45% following HSP90 inhibition with geldanamycin or radicicol. Collagen stimulation leads to ATP release from platelets, and calcium increases to low doses of collagen were also reduced by ∼40% by the HSP90 inhibitors consistent with an effect on P2X1 receptors. These studies suggest that HSP90 inhibitors may be as effective as selective antagonists in regulating platelet P2X1 receptors, and their potential effects on hemostasis should be considered in clinical studies.     

干扰Sema7A抑制C2C12成肌细胞的增殖和分化

为研究脑信号蛋白家族（Semaphorins）成员Sema7A对成肌细胞增殖和分化的影响,本文设计并合成了Sema7A基因的小干扰RNA（small interfering RNA,siRNA）,用此siRNA转染C2C12成肌细胞．通过Hoechst核染和流式细胞术检测细胞增殖情况,免疫荧光检测肌管的形成情况,real-time qPCR和Western印迹技术检测成肌标记基因的变化.结果显示,干扰Sema7A后,C2C12成肌细胞增殖减慢,处在G2和S期的细胞所占的比例明显下降,而G1期细胞的比例升高．免疫荧光检测结果显示,干扰Sema7A后,肌管的直径及MyHC+细胞所占比例均显著降低．Real-time qPCR和Western印迹结果也显示,肌肉分化标志基因MyoD、MyoG、MyHC的mRNA及蛋白质表达均下降．进一步检测Sema7A受体下游信号通路发现,干扰Sema7A后,其下游信号分子PI3K和AKT的磷酸化水平被下调．以上结果表明,Sema7A可以调节C2C12成肌细胞的增殖和分化,可能是通过其受体作用于PI3K/AKT信号通路实现的,这为进一步研究Sema7A在骨骼肌发育中的作用提供实验基础.     

Subnuclear localization and differentiation-dependent increased expression of DGK-zeta in C2C12 mouse myoblasts

Diacylglycerol kinases (DGKs) catalyze phosphorylation of diacylglycerol (DG) to yield phosphatidic acid (PA). Previous evidence has shown that the nucleus contains several DGK isoforms. In this study, we have analyzed the expression and subnuclear localization of DGK-zeta employing C2C12 mouse myoblasts. Immunocytochemistry coupled to confocal laser scanning microscopy showed that both endogenous and green fluorescent protein-tagged overexpressed DGK-zeta localized mostly to the nucleus. In contrast, overexpressed DGK-alpha, -beta, -delta, and -iota did not migrate to the nucleus. DGK-zeta was present in the nuclear speckle domains, as also revealed by immuno-electron microscopy analysis. Moreover, DGK-zeta co-localized and interacted with phosphoinositide-specific phospholipase Cbeta1 (PLCbeta1), that is involved in inositide-dependent signaling pathways important for the regulation of cell proliferation and differentiation. Furthermore, we report that DGK-zeta associated with nuclear matrix, the fundamental organizing principle of the nucleus where many cell functions take place, including DNA replication, gene expression, and protein phosphorylation. Nuclear DGK-zeta increased during myogenic differentiation of C2C12 cells, while DGK-zeta down-regulation by siRNA markedly impaired differentiation. Overall, our findings further support the importance of speckles and nuclear matrix in lipid-dependent signaling and suggest that nuclear DGK-zeta might play some fundamental role during myogenic differentiation of C2C12 cells.     

Tumor cell pseudopodial protrusions. Localized signaling domains coordinating cytoskeleton remodeling, cell adhesion, glycolysis, RNA translocation, and protein translation

The pseudopodial protrusions of Moloney sarcoma virus (MSV)-Madin-Darby canine kidney (MDCK)-invasive (INV) variant cells were purified on 1-microm pore polycarbonate filters that selectively allow passage of the pseudopodial domains but not the cell body. The purified pseudopodial fraction contains phosphotyrosinated proteins, including Met and FAK, and various signaling proteins, including Raf1, MEK1, ERK2, PKBalpha (Akt1), GSK3alpha, GSK3beta, Rb, and Stat3. Pseudopodial proteins identified by liquid chromatography tandem mass spectrometry included actin and actin-regulatory proteins (ERM, calpain, filamin, myosin, Sra-1, and IQGAP1), tubulin, vimentin, adhesion proteins (vinculin, talin, and beta1 integrin), glycolytic enzymes, proteins associated with protein translation, RNA translocation, and ubiquitin-mediated protein degradation, as well as protein chaperones (HSP90 and HSC70) and signaling proteins (RhoGDI and ROCK). Inhibitors of MEK1 (U0126) and HSP90 (geldanamycin) significantly reduced MSV-MDCK-INV cell motility and pseudopod expression, and geldanamycin treatment inhibited Met phosphorylation and induced the expression of actin stress fibers. ROCK inhibition did not inhibit cell motility but transformed the pseudopodial protrusions of MSV-MDCK-INV cells into extended lamellipodia. Dominant negative Rho disrupted pseudopod expression and, in serum-starved cells, L-alpha-lysophosphatidic acid (oleoyl) activation of Rho induced pseudopodial protrusions or, in the presence of the ROCK inhibitor, extended lamellipodia. RNA was localized to the actin-rich pseudopodial domains of MSV-MDCK-INV cells, but the extent of colocalization with dense actin ruffles was reduced in the extended lamellipodia formed upon ROCK inhibition. Rho/ROCK activation in epithelial tumor cells therefore regulates RNA translocation to a pseudopodial domain that contains proteins involved in signaling, cytoskeleton remodeling, cell adhesion, glycolysis, and protein translation and degradation.     

Cyclic mechanical stretch stimulates the proliferation of C2C12 myoblasts and inhibits their differentiation via prolonged activation of p38 MAPK

Mitogen-activated protein kinases (MAPKs) play an indispensable role in activation of the myogenic program, which is responsive to mechanical stimulation. Although there is accumulating evidence of mechanical force-mediated cellular responses, the role of MAPK in regulating the myogenic process in myoblasts exposed to cyclic stretch is unclear. Cyclic stretch induced the proliferation of C2C12 myoblasts and inhibited their differentiation into myotubes. In particular, it induced persistent phosphorylation of p38 kinase, and decreased the level of phosphorylation of extracellular-signal regulated kinase (ERK). Partial inhibition of p38 phosphorylation increased cellular levels of MyoD and p-ERK in stretched C2C12 cells, along with increased myotube formation. Treatment with 10 microM PD98059 prevented myogenin expression in response to a low dose of SB203580 (3 microM) in the stretched cells, suggesting that adequate ERK activation is also needed to allow the cells to differentiate into myotubes. These results suggest that cyclic stretch inhibits the myogenic differentiation of C2C12 cells by activating p38-mediated signaling and inhibiting ERK phosphorylation. We conclude that p38 kinase, not ERK, is the upstream signal transducer regulating cellular responses to mechanical stretch in skeletal muscle cells.     

Differentiation of C2C12 myoblasts is critically regulated by FAK signaling

This study examined whether focal adhesion kinase (FAK) plays a role in the differentiation of C(2)C(12) myoblasts into myotubes. Differentiation of C(2)C(12) myoblasts induced by switch to differentiation culture medium was accompanied by a transient reduction of FAK phosphorylation at Tyr-397 (to approximately 50%, at 1 and 2 h), followed by an increase thereafter (to 240% up to 5 days), although FAK protein expression remained unchanged. FAK and phosphorylated FAK were found at the edge of lamellipodia in proliferating cells, whereas the later increase in FAK phosphorylation in differentiating cells was accompanied by its preferential location at the tip of well-organized actin stress fibers. Hyperexpression of FAK autophosphorylation site (Tyr-397) mutant (MT-FAK) reduced FAK phosphorylation at Tyr-397 in proliferating cells and was accompanied by reduction of cyclin D1 and increase of myogenin expression. These cells failed to progress to myotubes in differentiation medium. In contrast, hyperexpression of a wild-type FAK construction (WT-FAK) increased baseline and abolished the transient reduction of FAK phosphorylation at Tyr-397 in serum-starved C(2)C(12) cells. Cells transfected with WT-FAK failed to reduce cyclin D1 and to increase myogenin expression, as well as to progress to terminal differentiation in differentiation medium. These data indicate that FAK signaling plays a critical role in the control of cell cycle as well as in the progression of C(2)C(12) cells to terminal differentiation. Transient inhibition of FAK phosphorylation at Tyr-397 contributes to trigger the myogenic genetic program, but its later activation is also central to terminal differentiation into myotubes.     

A role of Rab7 in stabilizing EGFR-Her2 and in sustaining Akt survival signal

Rab7 plays an important role in regulating endocytic traffic. In view of an emerging role of membrane traffic in signaling and diseases, we have examined the possible role of Rab7 in oncogenesis. The role of Rab7 was investigated using shRNA‐mediated knockdown in A431 and MCF7 cancer cells. To our surprise, Rab7 knockdown effectively suppressed anchorage‐independent growth of cancer cells in soft agar. Anoikis (matrix‐detachment triggered apoptosis) was enhanced, while the level of phosphorylated (active) Akt (which is a key survival factor) was significantly reduced. Also intriguing was the observation that EGFR and Her2 levels were significantly reduced when Rab7 was knocked‐down. More robust reduction of EGFR and Her2 levels was observed when knocked‐down cells were treated with HSP90 inhibitor geldanamycin (GA). Low concentration of GA (50–100 nm)‐induced apoptosis of the Rab7 knocked‐down cells but not control cells, suggesting that Rab7 and HSP90 together contribute to the optimal stability of EGFR and Her2 as well as to protect cancer cells from apoptosis. Rab7 seems to protect EGFR and Her2 from proteosome‐mediated degradation. These results suggest that Rab7 is likely involved in protecting EGFR and Her2 from being degraded by the proteosome and in maintaining optimal Akt survival signal (especially during cell detachment or when HSP90 is inhibited). Rab7 is potentially a novel target for combinatory therapy with Hsp90 inhibitors. J. Cell. Physiol. 227: 2788–2797, 2012. © 2011 Wiley Periodicals, Inc.     

Effects of dimethyl sulphoxide and dexamethasone on mRNA expression of myogenesis- and muscle proteolytic system-related genes in mouse myoblastic C2C12 cells

We examined the time course of mRNA expression of myogenic cell differentiation- and muscle proteolytic system-related genes in cultures of C2C12 cells during differentiation from myoblasts to myotubes. Furthermore, we treated C2C12 myotubes with dimethyl sulphoxide (DMSO) and dexamethasone (Dex), and examined changes in these mRNA levels. Myogenin (Myog), Atrogin1, forkhead box O1 (Foxo1) and Capn1 mRNA levels increased in C2C12 cells differentiating from myoblasts to myotubes, whereas Myf5 mRNA levels decreased. Although genes such as MRF4, Foxo3a, UbB, Capn1 and MuRF1 mRNAs in the myotubes were affected by DMSO exposure, mRNA levels of other genes were not markedly affected by exposure to 0.02% or 0.5% DMSO. Myf5, MRF4, Atrogin1, Foxo3 and MuRF1 mRNA levels were elevated by Dex at all time points, Cbl and Capn1 mRNA levels were significantly elevated by Dex at 8 h, and Myog mRNA levels were significantly elevated by Dex at 24 h. However, CtsH mRNA levels decreased significantly with Dex at 24 h. This study provides a useful database of gene profiles that are differentially expressed throughout myogenic cell differentiation and the muscle proteolytic system.     

Pharmacological inhibition of HSP90 and ras activity as a new strategy in the treatment of HNSCC

Advanced head and neck squamous cell cancer (HNSCC) is currently treated with taxane‐based chemotherapy. We have previously shown that docetaxel (DTX) induces a ras‐dependent survival signal that can be antagonized by farnesyl transferase inhibitors (FTI) such as tipifarnib (TIP). Here we show that the synergistic TIP/DTX combination determines synergistic apoptotic conditions but, at the same time, it modulates the expression of the components of the multichaperone complex that is, in turn, involved in the regulation of the stability of members of the ras‐mediated pathway. Therefore, we have stably transfected HNSCC KB and Hep‐2 cells with a plasmid encoding for HSP90. The expression of the protein was increased in both transfected cell lines but its activation status was increased in Hep‐2 clones and decreased in KB clones. On the basis of these results, we have treated both parental and HSP90‐transfected cells with a HSP90 inhibitor geldanamycin (GA). We have found that the antiproliferative activity of GA is dependent upon the activation status of HSP90 and that it is strongly synergistic when added in combination with TIP but not with DTX in cells overexpressing HSP90 and even more in cells with increased HSP90 activity. These data were paralleled by the decreased expression and activity of the components belonging to the ras→mediated signal transduction pathway. The present results suggest that multichaperone complex activation could be a resistance mechanism to the anti‐proliferative and apoptotic effects induced by TIP and that the combination of FTIs such as TIP with GA could be a suitable therapeutic strategy in the treatment of HSP90‐overexpressing HNSCC. J. Cell. Physiol. 228: 130–141, 2013. © 2012 Wiley Periodicals, Inc.