Benjamin J. Lang

HSP90 inhibitors enhance differentiation and MITF (microphthalmia transcription factor) activity in osteoclast progenitors.

The HSP90 (heat-shock protein 90) inhibitor 17-AAG (17-allylamino-demethoxygeldanamycin) increases osteoclast formation both in vitro and in vivo, an action that can enhance cancer invasion and growth in the bone microenvironment. The cellular mechanisms through which 17-AAG exerts this action are not understood. Thus we sought to clarify the actions of 17-AAG on osteoclasts and determine whether other HSP90 inhibitors had similar properties. We determined that 17-AAG and the structurally unrelated HSP90 inhibitors CCT018159 and NVP-AUY922 dose-dependently increased RANKL [receptor activator of NF-κB (nuclear factor κB) ligand]-stimulated osteoclastogenesis in mouse bone marrow and pre-osteoclastic RAW264.7 cell cultures. Moreover, 17-AAG also enhanced RANKL- and TNF (tumour necrosis factor)-elicited osteoclastogenesis, but did not affect RANKL-induced osteoclast survival, suggesting that only differentiation mechanisms are targeted. 17-AAG affected the later stages of progenitor maturation (after 3 days of incubation), whereas the osteoclast formation enhancer TGFβ (transforming growth factor β) acted prior to this, suggesting different mechanisms of action. In studies of RANKL-elicited intracellular signalling, 17-AAG treatment did not increase c-Fos or NFAT (nuclear factor of activated T-cells) c1 protein levels nor did 17-AAG increase activity in luciferase-based NF-κB- and NFAT-response assays. In contrast, 17-AAG treatment (and RANKL treatment) increased both MITF (microphthalmia-associated transcription factor) protein levels and MITF-dependent vATPase-d2 (V-type proton ATPase subunit d2) gene promoter activity. These results indicate that HSP90 inhibitors enhance osteoclast differentiation in an NFATc1-independent manner that involves elevated MITF levels and activity.

The Scavenger Receptor SREC-I Cooperates with Toll-Like Receptors to Trigger Inflammatory Innate Immune Responses.

Scavenger receptor expressed by endothelial cell-I (SREC-I) is a class F scavenger receptor expressed by immune cells with a significant role in CD8+- and CD4+-mediated T cell immunity. This receptor can also modulate the function of toll-like receptors (TLRs), which play essential roles in innate immunity. Earlier, it was found that human monocyte/macrophage THP1 cells and bone marrow-derived macrophages from mice exhibited increased responses to polyinosine–polycytidylic acid (poly I:C, PIC) and CpG (unmethylated) DNA and enhanced production of inflammatory cytokines with overexpressed SREC-I. Our data also showed that intracellular/endocytic TLR3 and TLR9 could directly interact with SREC-I in the presence of their respective ligands. We also observed that the internalized ligand along with TLR3/TLR9 colocalized in the endosome in macrophages and THP-1 cells overexpressing these receptors. In the absence of these ligands, there was no detectable colocalization between the SREC-I and endocytic TLRs. Earlier, it was shown that SREC-I stimulated double-stranded RNA/CpGDNA-mediated TLR3/TLR9 activation of the innate immune response by triggering signaling through the NF-κB, IRF3, and MAP kinase pathways leading to transcription of cytokine genes. We also established that SREC-I can associate with plasma membrane TLRs, such as TLR2 and TLR4. We demonstrated that SREC-I–TLR4 signals more efficiently from lipid microdomain in which lipopolysaccharide (LPS) can associate with SREC-I–TLR4 complex. We also proved that SREC-I is an alternate receptor for LPS capable of internalizing the complex and for endocytic TLR ligands as well. This binding activated endocytic TLR-mediated downstream cytokine production in THP1 cells and macrophages. Finally, SREC-I could also form complexes with TLR2 and induce the release of cytokines in the presence of bacterial, viral, and fungal ligands.

Molecular Stress-inducing Compounds Increase Osteoclast Formation in a Heat Shock Factor 1 Protein-dependent Manner

Background: HSP90 inhibitors increase osteoclast formation and bone loss. Results: Altered Hsf1 activity impacts the ability of stress-inducing compounds to modulate osteoclast formation. Conclusion: Hsf1 plays an important role in stress-associated osteoclast formation, potentially via MITF. Significance: We identified a novel pathway whereby agents inducing stress can enhance osteoclast formation. Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss.

Heat-shock factor 1 both positively and negatively affects cellular clonogenic growth depending on p53 status

HSF1 (heat-shock factor 1) is the master regulator of the heat-shock response; however, it is also activated by cancer-associated stresses and supports cellular transformation and cancer progression. We examined the role of HSF1 in relation to cancer cell clonogenicity, an important attribute of cancer cells. Ectopic expression or HSF1 knockdown demonstrated that HSF1 positively regulated cancer cell clonogenic growth. Furthermore, knockdown of mutant p53 indicated that HSF1 actions were mediated via a mutant p53-dependent mechanism. To examine this relationship more specifically, we ectopically co-expressed mutant p53(R273H) and HSF1 in the human mammary epithelial cell line MCF10A. Surprisingly, within this cellular context, HSF1 inhibited clonogenicity. However, upon specific knockdown of endogenous wild-type p53, leaving mutant p53(R273H) expression intact, HSF1 was observed to greatly enhance clonogenic growth of the cells, indicating that HSF1 suppressed clonogenicity via wild-type p53. To confirm this we ectopically expressed HSF1 in non-transformed and H-Ras(V12)-transformed MCF10A cells. As expected, HSF1 significantly reduced clonogenicity, altering wild-type p53 target gene expression levels consistent with a role of HSF1 increasing wild-type p53 activity. In support of this finding, knockdown of wild-type p53 negated the inhibitory effects of HSF1 expression. We thus show that HSF1 can affect clonogenic growth in a p53 context-dependent manner, and can act via both mutant and wild-type p53 to bring about divergent effects upon clonogenicity. These findings have important implications for our understanding of HSF1s divergent roles in cancer cell growth and survival as well as its disparate effect on mutant and wild-type p53.

Modulation of Alloimmunity by Heat Shock Proteins

The immunological mechanisms that evolved for host defense against pathogens and injury are also responsible for transplant rejection. Host rejection of foreign tissue was originally thought to be mediated mainly by T cell recognition of foreign MHC alleles. Management of solid organ transplant rejection has thus focused mainly on inhibition of T cell function and matching MHC alleles between donor and host. Recently, however, it has been demonstrated that the magnitude of the initial innate immune responses upon transplantation has a decisive impact on rejection. The exact mechanisms underlying this phenomenon have yet to be characterized. Ischemic cell death and inflammation that occur upon transplantation are synonymous with extracellular release of various heat shock proteins (Hsps), many of which have been shown to have immune-modulatory properties. Here, we review the impact of Hsps upon alloimmunity and discuss the potential use of Hsps as accessory agents to improve solid organ transplant outcomes.

Histone deacetylase activity mediates acquired resistance towards structurally diverse HSP90 inhibitors

Heat shock protein 90 (HSP90) regulates multiple signalling pathways critical for tumour growth. As such, HSP90 inhibitors have been shown to act as effective anticancer agents in preclinical studies but, for a number of reasons, the same effect has not been observed in the clinical trials to date. One potential reason for this may be the presence of de novo or acquired resistance within the tumours. To investigate mechanisms of resistance, we generated resistant cell lines through gradual dose escalation of the HSP90 inhibitor 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG). The resultant resistant cell lines maintained their respective levels of resistance (7–240×) in the absence of 17‐AAG and were also cross‐resistant with other benzoquinone ansamycin HSP90 inhibitors. Expression of members of the histone deacetylase family (HDAC 1, 5, 6) was altered in the resistant cells. To determine whether HDAC activity contributed to resistance, pan‐HDAC inhibitors (TSA and LBH589) and the class II HDAC‐specific inhibitor SNDX275 were found to resensitize resistant cells towards 17‐AAG and 17‐dimethylaminoethylamino‐17‐demethoxygeldanamycin. Most significantly, resistant cells were also identified as cross‐resistant towards structurally distinct HSP90 inhibitors such as radicicol and the second‐generation HSP90 inhibitors CCT018159, VER50589 and AUY922. HDAC inhibition also resensitized resistant cells towards these classes of HSP90 inhibitors. In conclusion, we report that prolonged 17‐AAG treatment results in acquired resistance of cancer cells towards not just 17‐AAG but also to a spectrum of structurally distinct HSP90 inhibitors. This acquired resistance can be inhibited using clinically relevant HDAC inhibitors. This work supports the potential benefit of using HSP90 and HDAC inhibitors in combination within the clinical setting.

A Workflow Guide to RNA-seq Analysis of Chaperone Function and Beyond

RNA sequencing (RNA-seq) is a powerful method of transcript analysis that allows for the sequence identification and quantification of cellular transcripts. RNA-seq has many applications including differential gene expression (DE) analysis, gene fusion detection, allele-specific expression, isoform and splice variant quantification, and identification of novel genes. These applications can be used for downstream systems biology analyses such as gene ontology analysis to provide insights into cellular processes altered between biological conditions. Given the wide range of signaling pathways subject to chaperone activity as well as numerous chaperone functions in RNA metabolism, RNA-seq may provide a valuable tool for the study of chaperone proteins in biology and disease. This chapter outlines an example RNA-seq workflow to determine differentially expressed (DE) genes between two or more sample conditions and provides some considerations for RNA-seq experimental design.

March1-dependent modulation of donor MHC II on CD103 + dendritic cells mitigates alloimmunity

In transplantation, donor dendritic cells (do-DCs) initiate the alloimmune response either by direct interaction with host T cells or by transferring intact donor MHC to host DCs. However, how do-DCs can be targeted for improving allograft survival is still unclear. Here we show CD103+ DCs are the major do-DC subset involved in the acute rejection of murine skin transplants. In the absence of CD103+ do-DCs, less donor MHC-II is carried to host lymph nodes, fewer allogenic T cells are primed and allograft survival is prolonged. Incubation of skin grafts with the anti-inflammatory mycobacterial protein DnaK reduces donor MHC-II on CD103+DCs and prolongs graft survival. This effect is mediated through IL-10-induced March1, which ubiquitinates and decreases MHC-II levels. Importantly, in vitro pre-treatment of human DCs with DnaK reduces their ability to prime alloreactive T cells. Our findings demonstrate a novel therapeutic approach to dampen alloimmunity by targeting donor MHC-II on CD103+DCs.Donor-derived dendritic cells (do-DC) in the graft can contribute to the induction of alloimmunity and tissue rejection, but how do-DC can be targeted for improving graft survival is unclear. Here the authors show that reducing MHC-II expression on do-DCs by DnaK pre-treatment can decrease the priming of alloimmunity and prolong graft survival in mouse models.

Genotoxic stress induces Sca-1 expressing metastatic mammary cancer cells.

We describe a cell damage‐induced phenotype in mammary carcinoma cells involving acquisition of enhanced migratory and metastatic properties. Induction of this state by radiation required increased activity of the Ptgs2 gene product cyclooxygenase 2 (Cox2), secretion of its bioactive lipid product prostaglandin E2 (PGE2), and the activity of the PGE2 receptor EP4. Although largely transient, decaying to low levels in a few days to a week, this phenotype was cumulative with damage and levels of cell markers Sca‐1 and ALDH1 increased with treatment dose. The Sca‐1+, metastatic phenotype was inhibited by both Cox2 inhibitors and PGE2 receptor antagonists, suggesting novel approaches to radiosensitization.

Abstract 729: Acquired resistance to Hsp90-inhibitors and cancer progression

Heat shock protein 90 (HSP90) is a molecular chaperone required for the stability and function of many proteins. The chaperoning of mutated and over-expressed oncoproteins by HSP90 enhances survival, growth and invasive potential of cancer cells. Many HSP90 inhibitors, including the benzoquinone ansamycin 17-allylamino-17-demethoxygeldanamycin (17-AAG), are currently in clinical evaluation. However the mechanisms and implications of acquired resistance to this class of drug remain largely unexplored. We have generated isogenic human breast cancer cell lines that are resistant to 17-AAG by continued culturing in the compound. High level of resistance was maintained in the 17-AAG resistant cells after cessation of treatment. Cross resistance to other benzoquinone ansamycins such as geldanamycin and 17-DMAG was observed, as well as to compounds structurally unrelated to the benzoquinones such as radicicol, VER50589 and CCT018159. Gene expression profiling and western blot analyses revealed that bone marrow stromal cell antigen 2 (BST2), previously linked to increased bone metastasis, is elevated significantly in the resistant cells. An inverse correlation between the expression of the enzyme NAD(P)H/quinone oxidoreductase 1 (NQO1) and resistance to 17-AAG was also observed. The resistant cells demonstrated significant increase in chemotactic migration and accelerated wound closure. This was coupled by a decrease in growth both in anchorage-dependent and -independent conditions. In vivo study using xenograft mouse model showed decreased mammary tumour formation by the resistant cells. Decreased metastasis of the resistant cells to the lungs was observed following intracardiac inoculation. However, x-ray analysis revealed that nude mice inoculated with resistant cells had enhanced hindlimb bone lesions compared to the parental group. In addition, 17-AAG was also shown to increase the formation of bone resorbing osteoclasts in vitro. Pharmacological inhibition of the transcription factor HSF1 using quercetin and KNK437 suppressed 17-AAG-induced stress response and 17-AAG-enhanced osteoclast formation. These results indicate that resistance to Hsp90 inhibition is accompanied by changes in cancer cell biology that leads to decreased primary tumour formation and lower metastatic tumour burden in vivo. However, severity of bone lesion generated by the resistant cells is greater. 17-AAG also enhances osteoclast formation through a mechanism dependent on HSF1-mediated stress response. Findings from this study furthers our understanding of Hsp90 inhibition on cancer progression and increases our understanding of the potential or lack of, clinical efficacy of Hsp90-directed therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 729. doi:10.1158/1538-7445.AM2011-729