Danilo Maddalo

The Molecular Chaperone GRP78/BiP in the Development of Chemoresistance: Mechanism and Possible Treatment

Treatment of several types of cancer such as lung, breast, prostate, and pancreas has shown notable progresses in the past decades. However, after an initial response, tumors eventually became resistant to chemotherapy. This phenomenon, known as chemoresistance, accounts for the death of most cancer patients. Several studies in patients refractory to therapy have revealed the upregulation of the molecular chaperone GRP78/Binding Protein, BiP (BiP) both at the RNA and protein expression level. Furthermore GRP78/BiP relocates to the cell membrane in malignant but not in benign cells. In this communication we review studies on the role and the mechanism of action of GRP78/BiP during development of chemoresistance in cancer cells. In addition we discuss the possible role of GRP78 as a biomarker and as a target in cancer therapy.

The anterior gradient 2 (AGR2) gene is overexpressed in prostate cancer and may be useful as a urine sediment marker for prostate cancer detection.

AGR2 is a member of the endoplasmatic reticulum protein disulphide isomerase gene family implicated in tumor metastasis. Its expression pattern, function, and utility as a marker remains to be further investigated.

Bcl-2 associated athanogene 5 (Bag5) is overexpressed in prostate cancer and inhibits ER-stress induced apoptosis

BackgroundThe Bag (Bcl-2 associated athanogene) family of proteins consists of 6 members sharing a common, single-copied Bag domain through which they interact with the molecular chaperone Hsp70. Bag5 represents an exception in the Bag family since it consists of 5 Bag domains covering the whole protein. Bag proteins like Bag1 and Bag3 have been implicated in tumor growth and survival but it is not known whether Bag5 also exhibits this function.MethodsBag5 mRNA and protein expression levels were investigated in prostate cancer patient samples using real-time PCR and immunoblot analyses. In addition immunohistological studies were carried out to determine the expression of Bag5 in tissue arrays. Analysis of Bag5 gene expression was carried out using one-way ANOVA and Bonferroni’s Multiple Comparison test. The mean values of the Bag5 stained cells in the tissue array was analyzed by Mann-Whitney test. Functional studies of the role of Bag5 in prostate cancer cell lines was performed using overexpression and RNA interference analyses.ResultsOur results show that Bag5 is overexpressed in malignant prostate tissue compared to benign samples. In addition we could show that Bag5 levels are increased following endoplasmic reticulum (ER)-stress induction, and Bag5 relocates from the cytoplasm to the ER during this process. We also demonstrate that Bag5 interacts with the ER-resident chaperone GRP78/BiP and enhances its ATPase activity. Bag5 overexpression in 22Rv.1 prostate cancer cells inhibited ER-stress induced apoptosis in the unfolded protein response by suppressing PERK-eIF2-ATF4 activity while enhancing the IRE1-Xbp1 axis of this pathway. Cells expressing high levels of Bag5 showed reduced sensitivity to apoptosis induced by different agents while Bag5 downregulation resulted in increased stress-induced cell death.ConclusionsWe have therefore shown that Bag5 is overexpressed in prostate cancer and plays a role in ER-stress induced apoptosis. Furthermore we have identified GRP78/BiP as a novel interaction partner of Bag5.

A peptidic unconjugated GRP78/BiP ligand modulates the unfolded protein response and induces prostate cancer cell death.

The molecular chaperone GRP78/BiP is a key regulator of protein folding in the endoplasmic reticulum, and it plays a pivotal role in cancer cell survival and chemoresistance. Inhibition of its function has therefore been an important strategy for inhibiting tumor cell growth in cancer therapy. Previous efforts to achieve this goal have used peptides that bind to GRP78/BiP conjugated to pro-drugs or cell-death-inducing sequences. Here, we describe a peptide that induces prostate tumor cell death without the need of any conjugating sequences. This peptide is a sequence derived from the cochaperone Bag-1. We have shown that this sequence interacts with and inhibits the refolding activity of GRP78/BiP. Furthermore, we have demonstrated that it modulates the unfolded protein response in ER stress resulting in PARP and caspase-4 cleavage. Prostate cancer cells stably expressing this peptide showed reduced growth and increased apoptosis in in vivo xenograft tumor models. Amino acid substitutions that destroyed binding of the Bag-1 peptide to GRP78/BiP or downregulation of the expression of GRP78 compromised the inhibitory effect of this peptide. This sequence therefore represents a candidate lead peptide for anti-tumor therapy.

PEST-domain-enriched tyrosine phosphatase and glucocorticoids as regulators of anaphylaxis in mice

To cite this article: Obiri DD, Flink N, Maier JV, Neeb A, Maddalo D, Thiele W, Menon A, Stassen M, Kulkarni RA, Garabedian MJ, Barrios AM, Cato ACB. PEST‐domain‐enriched tyrosine phosphatase and glucocorticoids as regulators of anaphylaxis in mice. Allergy 2012; 67: 175–182.

Enhanced Inhibition of Prostate Tumor Growth by Dual Targeting the Androgen Receptor and the Regulatory Subunit Type Iα of Protein Kinase A in Vivo

Progression to castration resistance is a major problem in the treatment of advanced prostate cancer and is likely to be driven by activation of several molecular pathways, including androgen receptor (AR) and cyclic AMP-dependent protein kinase A (PKA). In this study, we examined the therapeutic efficacy of a combined inhibition of the AR and the regulatory subunit type Iα (RIα) of protein kinase A with second generation antisense oligonucleotides (ODNs) in androgen-sensitive LNCaP and castration-resistant LNCaPabl tumors in vivo. We found that targeting the AR alone inhibited LNCaP, as well as LNCaPabl tumors. Combined inhibition resulted in an improved response over single targeting and even a complete tumor remission in LNCaPabl. Western blot analysis revealed that both ODNs were effective in reducing their target proteins when administered alone or in combination. In addition, treatment with the ODNs was associated with an induction of apoptosis. Our data suggest that dual targeting of the AR and PKARIα is more effective in inhibiting LNCaP and LNCaPabl tumor growth than single treatment and may give a treatment benefit, especially in castration-resistant prostate cancers.

Intracellular refolding assay.

This protocol describes a method to measure the enzymatic activity of molecular chaperones in a cell-based system and the possible effects of compounds with inhibitory/stimulating activity. Molecular chaperones are proteins involved in regulation of protein folding and have a crucial role in promoting cell survival upon stress insults like heat shock, nutrient starvation and exposure to chemicals/poisons. For this reason chaperones are found to be involved in events like tumor development, chemioresistance of cancer cells as well as neurodegeneration. Design of small molecules able to inhibit or stimulate the activity of these enzymes is therefore one of the most studied strategies for cancer therapy and neurodegenerative disorders. The assay here described offers the possibility to measure the refolding activity of a particular molecular chaperone and to study the effect of compounds on its activity. In this method the gene of the molecular chaperone investigated is transfected together with an expression vector encoding for the firefly luciferase gene. It has been already described that denaturated firefly luciferase can be refolded by molecular chaperones. As normalizing transfection control, a vector encoding for the renilla luciferase gene is transfected. All transfections described in this protocol are performed with X-treme Gene (Roche) in HEK-293 cells. In the first step, protein synthesis is inhibited by treating the cells with cycloheximide. Thereafter protein unfolding is induced by heat shock at 45°C for 30 minutes. Upon recovery at 37°C, proteins are re-folded into their active conformation and the activity of the firefly luciferase is used as read-out: the more light will be produced, the more protein will have re-gained the original conformation. Non-heat shocked cells are set as reference (100% of refolded luciferase).

The E3 ubiquitin ligase MID1/TRIM18 promotes atypical ubiquitination of the BRCA2-associated factor 35, BRAF35

MID1/TRIM18 is a member of the TRIM family of ubiquitin E3 ligases characterized by the presence of a conserved RING-containing N-terminal tripartite motif. Mutations in the MID1 gene have been associated with the X-linked form of Opitz Syndrome, a developmental disorder characterized by midline defects and intellectual disability. The effect of MID1 E3 ligase activity within the cell and the role in the pathogenesis of the disease is still not completely unraveled. Here, we report BRAF35, a non-canonical HMG nuclear factor, as a novel MID1 substrate. MID1 is implicated in BRAF35 ubiquitination promoting atypical poly-ubiquitination via K6-, K27- and K29-linkages. We observed a partial co-localization of the two proteins within cytoplasmic bodies. We found that MID1 depletion alters BRAF35 localization in these structures and increases BRAF35 stability affecting its cytoplasmic abundance. Our data reveal a novel role for MID1 and for atypical ubiquitination in balancing BRAF35 presence, and likely its activity, within nuclear and cytoplasmic compartments.

Abstract A62: The Bcl-2 associated athanogene 5 binds the molecular chaperone GRP78/BiP and increases chemoresistance in prostate cancer cells

Purpose of the study: the purpose of this study is to investigate the role of the Bcl-2 associated athanogene 5 (Bag5) protein in stress tolerance and chemoresistance development in prostate cancer cells. Introduction: the Bcl-2 associated athanogene (Bag) family of proteins consists of six members sharing a common Bag domain that mediates the interaction with the molecular chaperone Hsp70. Bag family members like Bag1 and Bag3 have been already reported to be involved in the development of several cancers like breast prostate and lung cancer. However, the role of Bag5 in cancer has not been described yet. Considering the involvement of the Bag proteins in stress tolerance, we therefore investigated the role of Bag5 upon stress induction and its ability to induce tolerance to stress-inducing chemotherapeutic agents. Experimental procedures: for gene expression analysis real-time PCR assay was used. Immunofluorescence and cell fractionation were carried to analyze subcellular protein localization. Co-immunoprecipitation and GST-pull down assays were employed for protein-protein interaction analysis. GRP78 enzymatic activity was measured by in vitro ATPase assay and by intracellular refolding experiment. Clonogenic assay was used to determine cell growth upon chemotherapeutic agents treatment. Western blot experiment and Caspase-3 cleavage measurement were used to analyze stress induced cell death and activation of the unfolded protein response. Results: In order to determine if Bag5 plays a role in stress response, Bag5 gene and protein expression levels were analyzed upon treatment with the stress inducers thapsigargin and tunicamycin in 22Rv.1 and PC3 prostate cancer cell lines. In both cases we could appreciate a significant increase of Bag5 levels indicating its stress inducibility. Intriguingly we could observe that Bag5 accumulates into the endoplasmic reticulum once stress is induced. Moreover we could determine that Bag5 binds to the ER chaperone GRP78/BiP, enhancing its enzymatic activity. Since GRP78 plays a major role in the regulation of the unfolded protein response (UPR), we wanted to determine if Bag5 levels could influence UPR activation. Stable transfection of Bag5 in 22Rv.1 cells attenuated UPR activation as well as apoptosis compared to the control upon stress induction. Consistently with this observation, when Bag5 gene levels were reduced, cells became more sensitive to stress induction. Since UPR activation and stress tolerance represent the basic mechanism for chemoresistance development, we analyzed the effect of Bag5 overexpression when cells were treated with a range of chemotherapeutic agents including fenretinide, casodex, (−)epigallocatechingalle and taxol. Colony forming assay and Caspase-3 cleavage measurement showed in all the treatments an increased survival in Bag5 overexpressing cells compared to the vector control, indicating that Bag5 indeed plays a role in prostate cancer cell survival and might be responsible for chemoresistance development. Conclusions: In this work we showed for the first time that Bag5 can interact with the molecular chaperone GRP78/BiP and influence UPR activation. In addition, we provided the very first evidence that Bag5 could play a role in development of stress tolerance in prostate cancer cells.

Abstract 5403: Pro-apoptotic properties of Bag-1 peptides in the control of prostate cancer cell growth

Purpose of the study: The purpose of the study is to identify sequence(s) in Bag-1 proteins responsible for growth inhibition and to eventually derive a short peptide for cancer therapy. Introduction: Bag-1 family of proteins consists of four members (Bag- 1L, 1M, 1 and 1S) originating from different translation-initiation sites on the same mRNA. Clinical reports have shown a dual role of the Bag-1 proteins in tumor progression and in the inhibition of tumor growth. In this work we have identified a 19 amino acid sequence in the Bag-1 proteins that reduces prostate cancer cell growth ex vivo and in vivo. Experimental procedures: Inhibition of prostate cancer cell growth was analyzed in clonogenic assay and in athymic nude mouse xenograft models. Histological analysis for apoptosis was carried out on paraffin-embedded tumor samples. Structural analyses were carried out by NMR and circular dichroism. Interaction partners of the Bag-1 peptides were analyzed by GST-pull down and by co-immunoprecipitation assays. Significance of cellular levels of glucose-regulated proteins (GRP) 75 and 78 for the growth inhibitory action of the peptide was demonstrated by RNA interference experiments. Results: Overexpression of Bag-1 proteins in 22Rv.1 prostate tumor cells reduced the growth of these cells in a colony forming assay. A study making use of several deletion mutants identified a 69 amino acid Bag-1 sequence as responsible for the growth inhibition. NMR and circular dichroism studies showed this peptide to be unstructured but it nonetheless interacted with the substrate binding domain of GRP 75 and 78. The cellular levels of the GRPs were shown by RNA interference studies to be essential for the action of the peptide. Overexpression of the peptide in the 22Rv1 cells decreased growth in proliferation assay and in xenograft tumor models. The decreased growth in vivo was demonstrated to be due to apoptosis of the Bag-1 peptide expressing cells as showed by immuohistochemical analysis. The growth inhibitory activity could be narrowed down to a 7 amino acid core within a 19 amino acid Bag-1 peptide sequence. Amino acid exchanges in the core region impaired binding of the peptide to the GRPs and inhibition of tumor cell growth. Colony formation assay using a wide panel of prostate cells showed that the 19 amino acid peptide inhibits growth of only malignant but not benign cells and this correlates with the level of expression of the two GRP target proteins in the cells. Conclusion: Together these results demonstrate that a short peptide sequence derived from Bag-1 proteins that binds GRP75/78 specifically inhibits the growth of prostate cancer cells. 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 5403. doi:10.1158/1538-7445.AM2011-5403