Mitochondrial ATP fuels ABC transporter-mediated drug efflux in cancer chemoresistance.

Chemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ATP binding cassette (ABC) transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of methylation-controlled J protein (MCJ) (also named DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an alternative strategy for treatment of multiple cancers.

DNAJA4 deficiency enhances NF-kappa B-related growth arrest induced by hyperthermia in human keratinocytes.

BACKGROUND: Hyperthermia is an effective treatment against cancer and human papillomavirus (HPV) infection. Previous studies have shown that heat shock proteins are crucial to the action of hyperthermia. OBJECTIVES: To examine the effects of hyperthermia in combination with DNAJA4-deficiency on human keratinocytes and Condyloma acumunatum (CA) tissues. METHODS: HaCaT cells were subjected to 44°C (compared to 37°C) waterbath for 30min for stimulation. Foreskin or CA tissues obtained from patients undergoing circumcision or pathological examination were bisected and subjected to similar treatments. DNAJA4-knockout (KO) HaCaT cells were generated with CRISPR/Cas9 technology. mRNA and protein expressions were determined using rt-qPCR and western-blotting. Cell cycle distribution, apoptosis and senescence were analyzed by flow cytometry. RESULTS: DNAJA4 was induced in HaCaT cells, foreskin and CA tissues subjected to hyperthermia at both transcriptional and translational levels. NF-kB,3 was activated by hyperthermia in HaCaT cells, and further enhanced by DNAJA4-deficiency. Transcription of TNF-α4; IL-1B,5 TNFAIP36 and IL-87 were induced in HaCaT cells subjected to hyperthermia. DNAJA4-knockout promoted transcriptions of TNF-α and IL-1B, whereas decreased that of TNFAIP3 and IL-8. Reduced cell survival, proliferation and viability were demonstrated using flow cytometry and MTS assays. Furthermore, NF-kB inhibitors reversed most of the phenotypes observed. CONCLUSIONS: Hyperthermia reduced HaCaT cell proliferation and promoted cytokine expressions responsible for anti-viral activity, mainly through a NF-kB dependent pathway. DNAJA4-deficiency enhanced the activation of NF-kB by hyperthermia in HaCaT cells, indicating that DNAJA4 may be a promising therapeutic target for use in the treatment of cutaneous HPV infections.

Neuronal ceroid lipofuscinosis with DNAJC5/CSPα mutation has PPT1 pathology and exhibit aberrant protein palmitoylation.

Neuronal ceroid lipofuscinoses (NCL) are a group of inherited neurodegenerative disorders with lysosomal pathology (CLN1-14). Recently, mutations in the DNAJC5/CLN4 gene, which encodes the presynaptic co-chaperone CSPα were shown to cause autosomal-dominant NCL. Although 14 NCL genes have been identified, it is unknown if they act in common disease pathways. Here we show that two disease-associated proteins, CSPα and the depalmitoylating enzyme palmitoyl-protein thioesterase 1 (PPT1/CLN1) are biochemically linked. We find that in DNAJC5/CLN4 patient brains, PPT1 is massively increased and mis-localized. Surprisingly, the specific enzymatic activity of PPT1 is dramatically reduced. Notably, we demonstrate that CSPα is depalmitoylated by PPT1 and hence its substrate. To determine the consequences of PPT1 accumulation, we compared the palmitomes from control and DNAJC5/CLN4 patient brains by quantitative proteomics. We discovered global changes in protein palmitoylation, mainly involving lysosomal and synaptic proteins. Our findings establish a functional link between two forms of NCL and serve as a springboard for investigations of NCL disease pathways.

Proteasome inhibition alleviates SNARE-dependent neurodegeneration.

Activation of the proteasomal degradation of misfolded proteins has been proposed as a therapeutic strategy for treating neurodegenerative diseases, but it is unclear whether proteasome dysfunction contributes to neurodegeneration. We tested the role of proteasome activity in neurodegeneration developed by mice lacking cysteine string protein-α (CSPα). Unexpectedly, we found that proteasome inhibitors alleviated neurodegeneration in CSPα-deficient mice, reversing impairment of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-complex assembly and extending life span. We tested whether dysfunctional SNARE-complex assembly could contribute to neurodegeneration in Alzheimer's and Parkinson's disease by analyzing postmortem brain tissue from these patients; we found reduced SNARE-complex assembly in the brain tissue samples. Our results suggest that proteasomal activation may not always be beneficial for alleviating neurodegeneration and that blocking the proteasome may represent a potential therapeutic avenue for treating some forms of neurodegenerative disease.

Cysteine string protein-alpha prevents activity-dependent degeneration in GABAergic synapses.

The continuous release of neurotransmitter could be seen to place a persistent burden on presynaptic proteins, one that could compromise nerve terminal function. This supposition and the molecular mechanisms that might protect highly active synapses merit investigation. In hippocampal cultures from knock-out mice lacking the presynaptic cochaperone cysteine string protein-alpha (CSP-alpha), we observe progressive degeneration of highly active synaptotagmin 2 (Syt2)-expressing GABAergic synapses, but surprisingly not of glutamatergic terminals. In CSP-alpha knock-out mice, synaptic degeneration of basket cell terminals occurs in vivo in the presence of normal glutamatergic synapses onto dentate gyrus granule cells. Consistent with this, in hippocampal cultures from these mice, the frequency of miniature IPSCs, caused by spontaneous GABA release, progressively declines, whereas the frequency of miniature excitatory AMPA receptor-mediated currents (mEPSCs), caused by spontaneous release of glutamate, is normal. However, the mEPSC amplitude progressively decreases. Remarkably, long-term block of glutamatergic transmission in cultures lacking CSP-alpha substantially rescues Syt2-expressing GABAergic synapses from neurodegeneration. These findings demonstrate that elevated neural activity increases synapse vulnerability and that CSP-alpha is essential to maintain presynaptic function under a physiologically high-activity regimen.

Hsp90 and Hsp40/Erdj3 are required for the expression and anti-apoptotic function of KSHV K1.

Kaposi sarcoma-associated herpesvirus (KSHV) is a member of the gammaherpesvirus family. It is the etiological agent of three different human cancers, Kaposi sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman disease. The far left end of the KSHV genome encodes a unique transmembrane glycoprotein called K1. K1 possesses the ability to transform rodent fibroblasts and block apoptosis. K1 has also been shown to activate the PI3K/Akt/mTOR pathway in different cells. Using tandem affinity purification, we identified heat shock protein 90beta (Hsp90beta) and endoplasmic reticulum-associated Hsp40 (Erdj3/DnaJB11), as cellular binding partners of K1. Interactions of K1 with Hsp90beta and Hsp40 were confirmed by co-immunoprecipitation in both directions. Furthermore, K1 also interacted with the Hsp90alpha isoform. We report that small-interfering RNAs directed against Hsp90 and Hsp40/Erdj3, as well as pharmacological inhibitors of Hsp90, dramatically reduced K1 expression, suggesting that K1 is a client protein of these chaperones. In addition, both Hsp90 and Hsp40/Erdj3 were essential for K1's anti-apoptotic function. Finally, we report that the Hsp90 inhibitors, 17-AAG and 17-DMAG, can suppress the proliferation of KSHV-positive PEL cell lines and exhibited IC(50) values of 50 nM and below.

Methylation-controlled J protein promotes c-Jun degradation to prevent ABCB1 transporter expression.

Methylation-controlled J protein (MCJ) is a newly identified member of the DnaJ family of cochaperones. Hypermethylation-mediated transcriptional silencing of the MCJ gene has been associated with increased chemotherapeutic resistance in ovarian cancer. However, the biology and function of MCJ remain unknown. Here we show that MCJ is a type II transmembrane cochaperone localized in the Golgi network and present only in vertebrates. MCJ is expressed in drug-sensitive breast cancer cells but not in multidrug-resistant cells. The inhibition of MCJ expression increases resistance to specific drugs by inducing expression of the ABCB1 drug transporter that prevents intracellular drug accumulation. The induction of ABCB1 gene expression is mediated by increased levels of c-Jun due to an impaired degradation of this transcription factor in the absence of MCJ. Thus, MCJ is required in these cells to prevent c-Jun-mediated expression of ABCB1 and maintain drug response.

CSPalpha-deficiency causes massive and rapid photoreceptor degeneration.

Cysteine string protein (CSP) alpha is an abundant synaptic vesicle protein that contains a DNA-J domain characteristic of Hsp40-type cochaperones. Previous studies showed that deletion of CSPalpha in mice leads to massive lethal neurodegeneration but did not clarify how the neurodegeneration affects specific subpopulations of neurons. Here, we analyzed the effects of the CSPalpha deficiency on tonically active ribbon synapses of the retina and the inner ear. We show that CSPalpha-deficient photoreceptor terminals undergo dramatic and rapidly progressive neurodegeneration that starts before eye opening and initially does not affect other retinal synapses. These changes are associated with progressive blindness. In contrast, ribbon synapses of auditory hair cells did not exhibit presynaptic impairments in CSPalpha-deficient mice. Hair cells, but not photoreceptor cells or central neurons, express CSPbeta, thereby accounting for the lack of a hair-cell phenotype in CSPalpha knockout mice. Our data demonstrate that tonically active ribbon synapses in retina are particularly sensitive to the deletion of CSPalpha and that expression of at least one CSP isoform is essential to protect such tonically active synapses from neurodegeneration.

Tid1 negatively regulates the migratory potential of cancer cells by inhibiting the production of interleukin-8.

Tid1 is the human homologue of the Drosophila tumor suppressor, Tid56. Reducing the expression of Tid1 in MDA-MB231 breast cancer cells enhanced their migration without affecting their survival or growth rate. From microarray screening, we discovered that after Tid1 depletion, the mRNA level of interleukin-8 (IL-8) was significantly increased in these cancer cells, which consequently increased secretion of IL-8 protein by 3.5-fold. The enhanced migration of these Tid1-knockdown cells was blocked by reducing the IL-8 expression or by adding an IL-8 neutralizing antibody to the culture medium, suggesting that enhancement of cell motility in these Tid1-deficient cells is dependent on the de novo synthesis of IL-8. Subsequently, we found that abrogating the nuclear factor kappaB binding site in the IL-8 promoter completely blocked the Tid1 depletion-induced IL-8 expression in the breast cancer cells. As increased IL-8 levels are known to promote tumor metastasis, we tested the effect of Tid1 knockdown on tumor metastasis and found that Tid1 depletion enhanced the metastasis of breast cancer cells in animals. Together, these results indicate that Tid1 negatively regulates the motility and metastasis of breast cancer cells, most likely through attenuation of nuclear factor kappaB activity on the promoter of the IL8 gene.