Based Apoptosis Regulator Nrh/BCL2L10

Author Manuscript Published OnlineFirst on January 12, 2018; DOI: 10.1158/0008-5472.CAN-17-0846 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Breast cancer targeting through inhibition of the endoplasmic reticulum- based apoptosis regulator Nrh/BCL2L10

Adrien Nougarede1, Nikolay Popgeorgiev1, Loay Kassem2, Soleilmane Omarjee1, Stephane Borel1, Ivan Mikaelian1, Jonathan Lopez1,3, Rudy Gadet1, Olivier Marcillat1, Isabelle Treilleux4, Bruno O.Villoutreix5, Ruth Rimokh1*, Germain Gillet1,6*

1 Univ Lyon, Université Claude Bernard Lyon 1, INSERM 1052, CNRS 5286, Centre Léon Bérard, Centre de recherche en cancérologie de Lyon, Lyon, 69008, France

2 Department of clinical oncology, Cairo University Hospitals, Al-Saray Street, Al-Maniel, 11451 Cairo, Egypt

3 Hospices civils de Lyon, Centre de Biologie Sud, Centre Hospitalier Lyon Sud, chemin du Grand Revoyet, 69495 Pierre Bénite, France

4 Biopathology department, Centre Léon Bérard, 28 rue Laennec, 69008 Lyon, France 5 Inserm-Paris 7 Unit U973, Lamarck Building, 35 Rue Hélène Brion, 75013 Paris, France

6 Hospices civils de Lyon, Laboratoire d’anatomie et cytologie pathologiques, Centre Hospitalier Lyon Sud, chemin du Grand Revoyet, 69495 Pierre Bénite, France

Running title: Nrh inhibitor for breast cancer treatment

*These authors share senior authorship

Correspondence: [email protected]; Tel.: +33469166656; Fax: +33469166660 [email protected]; Tel.: +33478782903; Fax: +33469166660

Conflict of interest

The authors declare that they have no conflict of interest.

Downloaded from cancerres.aacrjournals.org on October 6, 2021. © 2018 American Association for Cancer Research. Author Manuscript Published OnlineFirst on January 12, 2018; DOI: 10.1158/0008-5472.CAN-17-0846 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Abstract

Drug resistance and metastatic relapse remain a top challenge in breast cancer treatment. In this study, we present preclinical evidence for a strategy to eradicate advanced breast cancers by targeting the BCL2 homolog Nrh/BCL2L10, which we discovered to be overexpressed in >45% of a large cohort of breast invasive carcinomas. Nrh expression in these tumors correlated with reduced metastasis-free survival and we determined it to be an independent marker of poor prognosis. Nrh protein localized to the endoplasmic reticulum (ER). Mechanistic investigations showed that Nrh made BH4 domain-dependent interactions with the ligand-binding domain of the inositol-1,4,5-triphosphate receptor (IP3R), a type I/III Ca2+ channel, allowing Nrh to negatively regulate ER-Ca2+ release and mediate anti-apoptosis. Notably, disrupting Nrh/IP3R complexes by BH4 mimetic peptides was sufficient to inhibit the growth of breast cancer cells in vitro and in vivo. Taken together, our results highlighted Nrh as a novel prognostic marker and a candidate therapeutic target for late stage breast cancers that may be addicted to Nrh.

Keywords

Breast Cancer, Nrh, Endoplasmic Reticulum, Inositol-1,4,5-Triphosphate Receptor, Apoptosis, BH4- peptide

Main text word count: 5435 words

Introduction

Apoptosis is a protective program promoting the clearance of aberrant cells. Failure to undergo apoptosis also enables tumor cells to escape conventional chemotherapy and targeted therapies (1) (2).

Thorough understanding of the apoptotic machinery is thus critical to overcome patient relapse. Bcl-2 family proteins act as master gatekeepers of the mitochondrial pathway of apoptosis (3) by regulating outer mitochondrial membrane permeability (OMM), the increase of which is a point of non-return in apoptosis (4). Apoptosis inhibitors are known to trap the BH3 domains of apoptosis drivers, such as

Bax and Bak, which prevents their insertion into the OMM and subsequent cytochrome c release, activating caspase-dependent cell death. Regarding anticancer therapy, BH3 mimetics are currently being developed to inhibit anti-apoptotic Bcl-2 proteins, although such molecules present drawbacks and side effects limiting clinical use (5,6).

In fact several Bcl-2 homologs co-localize at the endoplasmic reticulum (ER), where they control Ca2+

2+ fluxes by interacting with Ca channels, including the inositol 1,4,5-trisphosphate receptors (IP3R)

(7,8). Indeed, Bcl-2 was found to interact with the modulatory and transducing domain (MTD) and the coupling domain (CD) of IP3R (9,10), whereas Bcl-xL preferentially interacts with IP3R CD (11,12).

The Bcl-2 homolog Nrz was reported to interact with IP3R1 ligand binding domain (LBD), and control in this way actin cytoskeletal dynamics during zebrafish development (13–15). Actually, nrz is the zebrafish ortholog of chicken nr-13, mouse diva/boo and human nrh (also referred to as bcl-2l10/bcl-

B). Although nrh is considered as a genuine anti-apoptotic bcl-2 homolog (16–20), its actual function remains enigmatic, largely owing to the fact that no phenotype could be detected in diva/boo knock- out mice (21). Furthermore, Nrh protein displays limited capacity to inhibit Bax/Bak-dependent apoptosis (22) together with restricted tissue nrh gene expression pattern (16,17).

Actually, nrh was reported to be overexpressed in breast, prostate and lung cancers, as well as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) (23,24). In such diseases, its expression was correlated with poor prognosis and chemoresistance (25). However, little is known about the involvement of Nrh protein in breast cancer. Systematic studies of the pathological parameters associated with Nrh protein expression and its role at the molecular level are also lacking.

Here, we assess the contribution of Nrh to breast tumor development and chemotherapy resistance.

Based on the analysis of a large cohort of breast cancer patients, we report that Nrh protein is present in invasive breast carcinomas and is an independent poor prognostic marker correlated with metastatic relapse. We demonstrate that Nrh protein preferentially localizes at the ER, inhibits Ca2+ release and prevents apoptosis by interacting with the LBDs of IP3R channels. Finally, we show that disruption of

Nrh/IP3Rs complexes using BH4-mimicking peptides primes Nrh-addicted cells to apoptosis and inhibits tumor growth both in vitro and in vivo. Collectively, our results reveal that Nrh is a poor prognostic marker linked with chemotherapy resistance and is a promising target for breast cancer treatment.

Materials and Methods

Patient study

A total of 393 consecutive patients with operable breast cancer who were given adjuvant/neoadjuvant treatment at the Centre Léon Bérard (CLB) between 2001 and 2003 were tested for Nrh protein expression. Axillary lymph node (LN) invasion was assessed by sentinel node and/or level I and II axillary dissection. Tumor size was defined as the maximum tumor diameter measured on the tumor specimens at the time of surgery. Tumors were considered ERα- or PR- positive if they displayed a nuclear staining in 10% or more of tumor cells, as detected by immunohistochemistry. Tumors were considered positive for HER2 expression if they had 3+ staining by immunohistochemistry or 2+ staining by HER2 amplification detected using FISH. The data exported from the patients' files for analysis are consigned in the Supplementary Table 1.

Study approval

All studies including mice and deidentified human samples were conducted according to the CIOMS ethical guidelines and approved by the ethic review board of the Centre Léon Bérard Hospital. Written informed consent was obtained from each patient and the study protocol was approved by the institutional ethics committee. This study is reported according to the REMARK criteria (26).

Immunohistochemical analysis

To analyze Nrh protein expression, a human normal tissue array (TMA-1205, Protein Biotechnologies,

Clinisciences) and paraffin-embedded breast tumor tissues containing invasive carcinomas (n = 393) were used. After deparaffinization and rehydration, endogenous peroxidases were blocked by incubating the slides in 5% hydrogen peroxide in sterile water. For heat-induced antigen retrieval, tissue sections were boiled in 10 mM citrate buffer pH 8.0 (Dako, Trappes, France). The slides were then incubated at room temperature for 60 minutes with an antibody against Nrh (1/200, Anti-Nrh rabbit HPA042222, Sigma). After washing with PBS, the bound antibody was incubated with

OmniMap anti-rabbit HRP kit (Roche), then with chromoMap DAB kit (Roche). Sections were

counterstained with hematoxylin.

Statistics

Statistical significance between two groups was analyzed using the 1-tailed Student t-test. The correlation between Nrh protein expression and the clinicopathological factors was calculated using

Pearson's Chi square test. Distant metastasis-free survival (DMFS) was defined as the time from histological diagnosis of breast cancer to the date of distant relapse or death. Survival curves were derived from Kaplan-Meier estimates and were compared using the log-rank test. Hazard ratios and

95% CIs were calculated using Cox regression model for univariate and multivariate analyses. All statistical tests regarding clinical data analysis were two-sided. A P-value equal to or under 0.05 was considered statistically significant.

Cell culture

MDA-MB-231, CAL51, MCF10A and HeLa cells were obtained from the ATCC and grown in

DMEM, supplemented with 10% FBS, 1% Penicillin/Streptomycin or for MCF10A only in

DMEM/F12 supplemented with 5% horse serum, 20 ng/mL EGF, 0.5 µg/mL Hydrocortisone, 100 ng/mL Cholera toxin, 10 µg/mL Insulin and 1% Penicillin/Streptomycin. Cell lines were routinely tested for mycoplasma contamination using MycoalertTM kit (Lonza) and authenticated by single nucleotide polymorphism profiling (Multiplexion GmbH). Experiments were carried out within 6 weeks from cell thawing. Viral production for generating CRISPR/Cas9 modified cell lines were carried out as previously described (27).

Whenever stated, MDA-MB231 cells treated with FITC TAT-Nrh 1-23 or FITC TAT-Nrh 1-23 Y16F were incubated, 24 hours after plating on NuncTM Labtek® chambers, with the corresponding peptide diluted to 10 µM in regular medium for 2 hours. Each chamber was then irradiated, or

“photoactivated” whenever stated with a fluorescent bulb (λ488nm) for 2 min to trigger peptide endosomal leakage into the cytosol. Cells were grown for another 16 hours, before being used for proximity ligation assay or apoptosis assay experiments.

For treatment with TMR (tetra-methyl-rhodamine) dfTAT-Nrh 1-23 or TMR dfTAT-Nrh 1-23 Y16F, cells were seeded, onto regular 6-well, 24-well or 96-well plates (Corning) and grown for 24 hours.

Corresponding peptides were diluted to 10 µM in Ca2+-free balanced salt solution (BSS), from a 100

µM peptide stock solution in water prepared 24 hours prior to the experiment. Cells were incubated for

1 hour in the 10 µM peptide/BSS solution, then washed and placed in regular medium before subsequent experiments.

Proliferation assay

For the MDA-MB-231 cell line, 4.5x103 cells were seeded onto a 96-well plate for 24 hours before incubation with dfTAT peptides. MDA-MB-231 cells were treated with 4 µM Etoposide or 0.04 µM

Doxorubicin. For CAL51 cell line, 8x103 cells were seeded in a 96-well plate 24 hours before incubation with dfTAT peptides for. CAL51 cells were treated with 25 µM Azacytidine or 0.1 µM

Doxorubicin. Images were acquired using an IncuCyte ZOOM TM over a 96 hours timeframe, and cell proliferation was measured as the percentage of cell density observed over this period.

Apoptosis assays

Cells were treated either with Staurosporine (1 µM for 8 hours), Thapsigargin (ranging from 0.1 to 10

µM, for 24 to 36 hours), Etoposide (50 µM for 36 hours), or Doxorubicin (5 µM for 24 hours).

For kinetic experiments, 1x104 cells were seeded onto a 96-well plate 24 hours before transfection.

Cells were transfected with 50 nM of the different siRNAs using Lipofectamine ® RNAiMAX reagent

(Invitrogen), then incubated for another 48 hours. Apoptosis was assessed by following the caspase activity using the CellPlayerTM Kinetic Caspase-3/7 Apoptosis reagent (Essen Bioscience) diluted

1:2000. Images were acquired using an IncuCyte ZOOM TM. Caspase activity was corrected to cell density to obtain % of apoptotic cells.

For endpoint experiments, 1x105 cells were seeded in a BD Falcon culture chamber 24 hours before transfection. Cells were transfected with 0.5 µg of pCS2+-EGFP vector mixed with 1 µg of the indicated vectors using XtremeGene HP transfection reagent (Roche), then grown for another 24 hours. Apoptosis was assessed at the end of the corresponding drug treatment using the SR-FLICA®

Poly Caspase Assay staining (ImmunoChemistry Technologies). Images were acquired using a Nikon

NiE microscope and green transfected cells positive for FLICA staining were counted with a custom- made macro on the ImageJ software to calculate % green cells displaying caspase activity.

Immunoprecipitation and pull-down assays

For immunoprecipitation experiments 6x106 HeLa cells were transfected with the indicated vectors and grown for another 24 hours. Cells were lysed in TNE buffer (10 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA (pH 7.4), supplemented with 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, 0.1 mM sodium pyrophosphate, 0.2 % NP-40 and a protease inhibitor, Roche). Extracts were pre-cleared with protein G-Sepharose beads (Sigma) for 2 hours at 4°C, then incubated overnight with 5 µg IP3R1 primary antibody. Afterwards, extracts were incubated with protein G-Sepharose beads for 3 hours.

Immunoprecipitated fractions were washed three times with TNE buffer, then analyzed by immunoblotting. For HA and Flag immunoprecipitation, 1x106 HeLa cells were seeded 24 hours before transfection with the indicated vectors and grown for another 24 hours. Two micrograms of primary antibody HA rabbit or Flag rabbit were used.

For pull-down experiments, 6x106 HeLa cells were transfected with the indicated vectors and grown for another 24 hours. Lysates were pre-cleared with protein Streptavidin beads (Dynabeads MyOne

Streptavidin T1, Thermo) for 2 hours at 4°C split in two and 5 µg of purified biotin-coupled Nrh 1-23

WT or Nrh 1-23 R6A/Y16F peptides were then added when indicated.

Full listing of the antibodies used can be found in the Supplementary Materials and Methods section.

Subcellular Fractionation

All steps were carried out at 4°C if not otherwise stated. 20x106 MDA-MB-231 cells were resuspended in 1mL cold MB buffer (210 mM mannitol, 70 mM sucrose, 1 mM EDTA, 10 mM

HEPES (pH 7.5)) and disrupted using a dounce tissue grinder. Extracts were centrifuged at least five time at 1500 x g for 5 min to eliminate nuclei, and then centrifuged at 10600 x g for 10 min. The pellet containing mitochondria was washed twice in MB buffer and then resuspended in TNE buffer. The supernatant was then centrifuged at 100000 x g for 1 h. The supernatant containing the cytosolic

fraction was conserved; the pellet containing ER membranes was resuspended in TNE buffer for immunoblotting.

Intracellular Ca2+ measurements

HeLa cells cultured in NuncTM Labtek® chambers were incubated with 5 µM of FluoForteTM Ca2+

2+ probe in a Ca -free Balanced Salt Solution (BSS) (121 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 6 mM NaHCO3, 5.5 mM D-glucose, 25 mM HEPES, pH 7.3) for 45 min at 37°C. After 10 seconds of measurement, 100 µM histamine in Ca2+-free medium was added. Time-lapse fluorescence values were collected using a Zeiss LSM 780 confocal microscope.

Fixed and live cell imaging

Immunofluorescence staining, transmission electron microscopy were carried out as described previously (14,15). Subcellular localization of endogenous Nrh in MDA-MB-231 cells was performed by Nrh immunogold labeling and Transmission Electron Microscopy (TEM) imaging. Uranyle acetate staining was used to allow for subcellular structure visualization in fixed cell sections. Images were collected using a JEOL 1400JEM transmission electron microscope and Zeiss LSM780 confocal microscope. Co-localization scores were calculated using Pearson’s coefficient, corrected by subtracting the background intensity in both channels (pCS2+ empty vector). The proximity ligation assay kit was used according to the manufacturer’s protocol (Olink). Images were acquired as Z-stacks and then analyzed using the ImageJ software.

Circular Dichroism

Briefly, TAT-NDP and TAT-CTRL were diluted to a concentration of 80 µM in water with 20%

2,2,2-Trifluoroethanol. Circular dichroism spectra were recorded using a Chirascan CD Spectrometer

(Applied Photophysics).

Orthotopic xenograft model

Experiments using mouse xenograft models were carried out by Antineo, Lyon, France. 6.106 MDA-

MB-231 cells were injected into the mammary gland of SCID mice (Charles River Laboratory). Mice were randomly distributed into treatment groups. When the tumors reached a mean volume of 90 mm3, peritumoral injection of the corresponding peptides or PBS (control) was performed every 2-3 days for

4 weeks. When indicated, intraperitoneal injection of Doxorubicin (1.2 mg/kg) was performed every week. Tumor size was measured twice a week using calipers and tumor volume (TV) was calculated using the formula TV= (4/3π) x r3. All animal studies were conducted in accordance with European

Union guidelines and approved by the regional ethics committee.

Vector construction, siRNA, reagents and antibodies

Full details and listing of vector constructions, siRNA, reagents and antibodies can be found in

Supplementary Materials and Methods section. Nrh constructs targeting the mitochondria, using the

ActA domain (Nrh-ActA), the endoplasmic reticulum (ER), using the cb5 ER-anchoring domain (Nrh- cb5), or a truncated Nrh lacking the BH4 domain (Nrh-BH4) were cloned into the pCS2+ vector.

Two Nrh ER-associated mutants feature with the loss of a functional BH3 binding pocket, with either a point mutation in the Bcl-2 family conserved motif NWGR of the BH1 domain (Nrh G85A) or deletion of half of the BH1 and BH2 domains (Nrh 1-97) were cloned into the pCS2+ vector.

Results

Nrh protein expression is an independent poor prognostic marker in breast cancer

In order to evaluate the possible correlation between Nrh protein expression and clinical parameters in breast cancer, we performed a systematic analysis using immunohistochemistry staining in a large cohort of patients (n = 393) with invasive breast carcinomas (Fig. 1A, Supplementary Table 1).

Accordingly, 180 patients (45.8%) displayed Nrh-positive staining while 213 patients (54.2%) were

Nrh-negative (Fig. 1B; Supplementary Table 1). Furthermore, using a panel of thirty-three human healthy tissues, we observed that Nrh is restricted to the kidneys (proximal tubules), stomach

(glandular layer), liver and testis (Leydig cells), but is not expressed in healthy breast tissues (n = 3)

(Supplementary Fig. 1A). Our results also show that Nrh expression is enhanced in liver tumors compared to normal liver tissue (Supplementary Fig. 1B). Though the presence of Nrh was significantly correlated with the premenopausal status and with larger tumors, there was no association with lymph node (LN) invasion, SBR grade, ERα, progesterone receptor (PR), HER2 status or breast cancer subtype (Supplementary Table 2). Nrh-positivity was associated with shorter distant metastasis- free survival (DMFS), as evidenced by the higher rate of metastasis recurrence at 10 years, with a

DMFS of 69.5% versus 80.6% in the Nrh-negative subgroup (P = 0.02) (Fig. 1B). Moreover, after adjusting the significant prognostic factors in the univariate model, larger tumors, a high SBR grade and Nrh protein expression were the only predictors of shorter DMFS, as shown in the final multivariate model (Supplementary Table 3). Altogether these data support the hypothesis that Nrh is a novel independent marker of poor prognosis in breast cancer, a predictor of metastatic relapse, and might be an indicator of resistance to chemotherapy.

Overexpression of Nrh in breast cancer cells contributes to resistance to apoptosis and tumor growth

Having shown the association of Nrh and breast cancer, we analyzed endogenous Nrh levels in a panel of breast cancer cell lines and found that the protein was present in all cell lines tested, but remained undetectable in the non-tumorigenic epithelial MCF10A and MCF12A cell lines, although this was not correlated with mRNA levels (Fig. 1C, Supplementary Fig. 1C).

We selected the MDA-MB-231 cell line to test the effect of nrh silencing, using two specific siRNAs

(Fig. 1D), on the cell response to death-inducing compounds. In contrast to Staurosporine,

Thapsigargin showed a limited death-inducing effect in MDA-MB-231 cells over the time-course of the experiment (36 hours), since only 17.1±1.1% of the cells displayed caspase activity when treated with siSCR (control siRNA). However, in nrh-silenced cells, Thapsigargin treatment triggered a significant increase in cell death, as evidenced by 43.6±1.4% (si3-Nrh) and 44.8±1.2% (si6-Nrh) of

MDA-MB-231 cells displaying caspase activity (Fig. 1E and 1F; Supplementary Fig. 2A-D).

Based on these observations, we decided to delete the nrh gene in MDA-MB-231 cells, using the

CRISPR/Cas9 technology, to evaluate its contribution to tumor growth (Supplementary Fig. 3A and

3B). In effect, nrh-/- homozygotes could not be obtained, suggesting that MDA-MB231 cells could be addicted to nrh. However, the nrh +/- cells proved to be viable and showed a markedly reduced proliferation rate after four days of treatment with Etoposide, Doxorubicin or Azacytidine (Fig. 1G), compared to control cells. Furthermore, xenograft assays revealed that partial deletion of nrh reduced tumor growth over a long time frame and increased potency of doxorubicin, (Fig. 1H-I). Of note, reduced tumor size appeared to be correlated with increased apoptosis events as shown by TUNEL assays (Supplementary Fig. 3C-E). Thus Nrh seems to be involved in a mechanism preventing

Thapsigargin-mediated cell death, and could contribute to the tumorigenicity of cancer cells in vivo.

Nrh prevents apoptosis at the ER, independently of its BH3-binding pocket

To unravel the underlying mechanisms, we evaluated the cellular localization of endogenous Nrh using subcellular fractionation and transmission electron microscopy. Remarkably, Nrh was detected at the ER but not at the mitochondria in MDA-MB-231 cells (Fig. 2A and 2B). We confirmed these findings by confocal microscopy, using ectopic expression of Flag-tagged Nrh in HeLa cells (Fig. 2C and 2D). Taken together, these results suggested that Nrh may have a predominant ER-related function. In order to assess the importance of Nrh subcellular localization to regulate apoptosis, we generated Nrh mutants, in which the C-terminus TM domain was replaced with an ER (Nrh-cb5) or mitochondria (Nrh-ActA) targeting sequence. (Fig. 2E). As shown in Fig. 2F, expression of Nrh-cb5 in HeLa cells prevented caspase activation upon Thapsigargin treatment with the same efficacy as

native Nrh, contrary to cells transfected with the Nrh-ActA construct. In addition, the BH4 Nrh deletion mutant, lacking the N-terminal -helical domain, was unable to protect HeLa cells against

Thapsigargin-induced apoptosis, suggesting that, at the ER, Nrh prevents lethal Ca2+ insults in a BH4- dependent manner.

Bcl-2-like apoptosis inhibitors are known to trap the BH3 domains of the apoptosis activators Bax and

Bak (3). To examine whether the anti-apoptotic activity of Nrh was also linked to such BH3- dependent interactions, we analyzed the effects of two Nrh mutants unable to bind Bax or Bak. These mutants, referred to as Nrh G85A and Nrh 1-97 (Fig. 2E), respectively bear a point mutation, hindering BH3 binding (28,29), or a truncation at the C-terminus, resulting in the same loss of function (15). However, both mutants were able to prevent caspase activation in HeLa cells following

Thapsigargin treatment, similarly to native Nrh (Fig. 2G). Hence, at the level of the ER, the anti-death activity of Nrh does not appear to be dependent on binding with Bax-like proteins. To further characterize the molecular events that could be affected by Nrh, we analyzed its effect on the unfolded protein response (UPR), a major ER-dependent response to stress-inducing agents, including

Thapsigargin. Accordingly, CHOP accumulation and increased eIF2α phosphorylation, typical UPR events (30,31), were observed within 4 hours in control HeLa cells, upon exposure to Thapsigargin.

Conversely, CHOP accumulation and eIF2α phosphorylation were weaker in Nrh-stably expressing cells (Fig. 2H), indicated that Nrh might behave as an inhibitor of the PERK-mediated UPR pathway.

Furthermore, the previously observed kinetic of caspase activation (within 36 hours) following

Thapsigargin exposure of nrh silenced cells appears to substantiate the reported time course of UPR- induced cell death (Supplementary Fig. 2A-C and 2H). Of note, the overall status of two other UPR pathways, as measured by ATF6 cleavage and xbp1 splicing, was not significantly changed in Nrh- stably expressing cells (Supplementary Fig. 4A and 4B). Together these data suggest that Nrh might modulate UPR initiation and subsequent cell death via an effect on ER Ca2+ fluxes.

2+ Nrh inhibits ER Ca release by interacting with the LBD of IP3R1 and IP3R3

2+ Recently we reported that Nrz, the zebrafish ortholog of Nrh, directly interacts with the IP3R1 Ca channel and controls the release of Ca2+ from the ER into the cytosol (15). We therefore investigated the ability of Nrh to interact with IP3R1 domains using co-immunoprecipitation (co-IP) assays. This revealed that Nrh interacted with the Ligand Binding Domain (LBD), as well as the MTDII domain of

IP3R1 (Fig. 3A and 3B). GST pull-down assays identified residues 227 to 448 of the LBD of IP3R1 as critical for this interaction (Supplementary Fig. 5A). Moreover, the BH4 Nrh deletion mutant was unable to bind endogenous IP3R1 in HeLa cells, indicating that the N-terminal BH4 domain mediates

Nrh/IP3R1 complex formation (Fig. 3C). Interestingly, amongst Bcl-2 homologs, Nrh appears to exhibit the highest affinity for the IP3R1 LBD (Supplementary Fig. 5B). It should also be noted that

Nrh interacted with the LBD of IP3R3, but not IP3R2 (Fig. 3D).

On this basis, we analyzed the effect of Nrh on ER Ca2+ release by monitoring cytosolic Ca2+ levels in

HeLa cells treated with Histamine. As shown in Fig. 3E and 3F, Nrh prevented Ca2+ release from the

ER, as evidenced by a reduction in the maximum fluorescence intensity from 4.2±0.2 A.U. to 3.1±0.2

A.U., whereas BH4 Nrh (maximum fluorescence intensity of 4.2±0.2 A.U) had similar effects as the empty control vector. Using the same experimental setup, we observed that Nrh G85A and Nrh 1-97, prevented Ca2+ release from the ER with the same efficiency as native Nrh (Fig. 3G and 3H). Together

2+ these findings confirm that the inhibition of ER Ca release by Nrh depends on IP3R1, but not on

Bax-like proteins.

A core Nrh-derived 23-amino-acid sequence is able to disrupt the IP3R/Nrh interaction

2+ Since Nrh prevents cell death by lowering IP3R1 and IP3R3 Ca permeability, we speculated that the

2+ disruption of Nrh/IP3R complexes could sensitize cells to death-inducing stimuli acting on Ca homeostasis. To test this hypothesis, we analyzed the ability of BH4-derived peptides (Fig. 4A), to prevent Nrh Co-IP with IP3R1. Indeed Co-IP assays revealed that the ectopic expression in HeLa cells of the Nrh 1-23 peptide compromised the formation of the Nrh/IP3R1 complex whereas peptides harboring R6A or Y16F point mutations were less efficient in this respect (Fig. 4B). Of note, the residual Nrh/IP3R1 interaction normalized against peptide expression levels indicated that the impact

of the loss-of-function of the Y16F mutation was greater than that of R6A (Fig. 4B). As the Tyr to Phe mutation is less likely to hinder the helical structure Nrh BH4 domain, this mutation was selected as a control in downstream experiments, even if some residual activity was observed.

With respect to cell death, the Nrh 1-23 peptide, but not the Y16F mutant, increased HeLa cells response to Thapsigargin, as measured by caspase activity (control: 31%±2.1, Nrh 1-23: 60%±1.3,

Y16F 30.8%±0.1, see Fig. 4C). Moreover, Nrh 1-23, but not the Y16 mutant, suppressed the inhibiting effect of Nrh on Histamine-triggered Ca2+ release (Fig. 4D and 4E). Interestingly, potentiation of

Thapsigargin effect by Nrh 1-23 can be reversed by the PERK inhibitor GSK2606414, suggesting that

Nrh regulates UPR-mediated cell death via the PERK pathway (Supplementary Fig. 6A).

At the molecular level, we investigated whether the Nrh 1-23 peptide could compete with Nrh for

IP3R1 binding. In contrast to full length Nrh (Fig. 4F and 4G), Nrh 1-23 interacted neither with the

IP3R1 LBD nor with Beclin-1, a known interactor of Nrh (29) used here as a control. In fact, Nrh 1-23 interacted directly with the Nrh protein, suggesting that disruption of the Nrh/IP3R1 complex might be due to the association of this peptide with Nrh but not with IP3R1 (Fig. 4F and 4G). Pull-down assays revealed that this peptide could interact with a truncated form of Nrh lacking residues 98-194, hence ruling out the requirement for the BH3 binding pocket (Supplementary Fig. 6B). In addition, Nrh 1-23 peptide failed to inhibit the interaction between the autophagy regulator Beclin-1 and Nrh

(Supplementary Fig. 6C), which is in accordance with previous findings attributing this interaction to the canonical BH3 binding pocket of Nrh (29). Of note, residue Y16 also appeared to be essential for

Nrh 1-23/Nrh interaction (Fig. 4H and 4I). Thus Nrh 1-23 does not seem to act as a decoy peptide at the Nrh binding site on IP3R1, but instead binds directly to Nrh to prevent the formation of the

Nrh/IP3R1 complex.

Disruption of the endogenous IP3Rs/Nrh interaction sensitizes cancer cells to apoptosis

Having highlighted the effect of Nrh 1-23 in transfected HeLa cells, we wondered whether a synthetic membrane-permeable peptide could have a similar effect in breast cancer cells. We used the PLA technology on MDA-MB-231 cells to directly detect endogenous Nrh/IP3R1 complexes in cellulo. For

calibration purposes naive and nrh-silenced cells were used as positive and negative controls, respectively (Supplementary Fig. 7A).

A FITC tag linked to a TAT sequence was added to the N-terminus of the 1-23 Nrh-derived peptide

(NDP), hence called TAT-NDP, the Y16F mutant being used as a control (TAT CTRL peptide) (Fig.

5A). Of note, both peptides adopted an -helical conformation in solution (Fig. 5B). To prevent the trapping of the internalized TAT-NDP peptide in the endosomes, we used a light source at 488 nm to excite the FITC fluorophore and induce endosomal leakage (32), thus increasing cytosolic availability of the peptide (Supplementary Fig. 7B). Under such conditions, Nrh/IP3R1 interactions were clearly detected in control MDA-MB-231 cells (vehicle) with 80.3±5.6 dots per cell, as well as in cells incubated with TAT-CTRL (65.2±4.4 dots per cell), whereas only a weak PLA signal (11.8±1.3 dots per cell) was detected in cells incubated with TAT-NDP (Fig. 5C and 5D). Furthermore, we showed that Thapsigargin triggered caspase activation in 62.4±10 % of the MDA-MB-231 cells incubated with

TAT-NDP, compared to only 11.5±4.4 % of the cells incubated with TAT-CTRL (Fig. 5E). Altogether these data support the hypothesis that disruption of the Nrh/IP3R1 complex by TAT-NDP may enhance the cell response to death-inducing agents, in particular agents that disturb Ca2+ homeostasis.

IP3Rs/Nrh complex destabilization enhances response to chemotherapeutic agents

Next, we assessed whether disrupting Nrh/IP3R complexes could enhance the potency of apoptosis inducers used in the clinic, since these drugs are also known to alter intracellular Ca2+ trafficking

(33,34). To this end, we used a dimeric TAT peptide (Fig. 6A and Supplementary Fig. 8), since such peptides, here referred to as dTAT-NDP, display enhanced endosomal escape capabilities (35). First, using Co-IPs in HeLa cells, we validated the activity of the newly formulated dTAT-NDP on the

Nrh/IP3R1 BD interaction (Fig. 6B). The peptides were then used in cell lines having a high level of nrh expression (MDA-MB-231 and CAL-51) or under-expressing nrh (MCF10A and MDA-MB-231

Nrh +/-) (Fig. 1C and 1G). We found that dTAT-NDP was able to circumvent Thapsigargin resistance in MDA-MB-231 cells, but not in MCF10A or MDA-MB-231 Nrh +/- cells (Fig. 6C), indicating that the observed sensitization was not an off-target effect of the peptide. Furthermore, the dTAT-NDP, but not the dTAT-CTRL, challenged MDA-MB-231 cells upon exposure to high doses of Etoposide (50

µM) or Doxorubicin (5 µM) (Fig. 6D and 6E). We confirmed these results using a proliferation assay in CAL-51 cells, and observed that in these cells, dTAT-NDP was a strong enhancer of Azacytidine

(25 µM) and Doxorubicin (0.1 µM) activity (Fig. 6F and 6G). Furthermore, a limited effect of dTAT-

NDP alone was observed in CAL-51 cells, although the combination with either Azacytidine or

Doxorubicin led to higher death-inducing effects (Fig. 6F and 6G; Supplementary Movie 1). Thus, these results suggest that dTAT-NDP might be effective to inhibit cancer cell growth even when used as a single agent therapy. In addition, dTAT-NDP but not dTAT-CTRL was shown to greatly enhance

Etoposide activity and prevent colony growth of MDA-MB-231 cells in soft agar (Fig. 7A and 7B).

Finally, having demonstrated the potency of dTAT-NDP-TMR in vitro, we conducted xenograft experiments to assess its anti-tumor effects in vivo. We injected MDA-MB-231 cells into the mammary gland of female SCID mice, and showed that continuous peritumoral injection of dTAT-

NDP at 2.5 or 10 mg/kg, as soon as the tumors reached a volume of 90 mm3, strongly decreased tumor growth and final tumor weight (Fig. 7C and 7D). In contrast, an exponential increase in the tumor mass was observed in the control group treated with vehicle alone or dTAT-CTRL. It should be noted that in this series of experiments, the dTAT-CTRL peptide appeared to partially affect tumor growth.

This is presumably due to residual activity as revealed by Co-IPs (see above Fig. 4B). TUNEL assays showed that dTAT-NDP increased apoptosis events in tumors, but not dTAT-CTRL. However, no significant effect could be detected with specific markers for proliferation (KI67), UPR (CHOP) or vascularization (CD31) (Supplementary Fig. 9A and 9B). Together these results indicate that the dTAT-NDP has a potent anti-tumor effect, and that the 2.5 mg/kg dosage is sufficient to reach the desired effect on tumor growth. Presumably, lower doses of peptides might be used to find the minimal effective dose, although no significant difference in body weight could be observed between the different groups, suggesting that the peptide has no apparent toxic side effects, and is well tolerated at the concentration used (Supplementary Fig. 9C)

Discussion

Although Nrh protein expression was correlated to chemoresistance and poor patient prognosis in hematopoietic malignancies (24), the present work provides the first systematic study with a large cohort of patients regarding tumorigenesis and clinical parameters in breast carcinomas. Here, we provide evidence that Nrh is an independent marker of poor prognosis associated with shorter DMFS, hence highlighting the link between Nrh expression and therapy resistance. These observations are in line with the fact that Nrh is more abundant in invasive breast carcinoma than in in situ carcinoma

(23). Together, our results highlight the clinical value of Nrh as a prognostic marker in breast cancer that could prove to be a worthy target for next generation therapies. Despite being a genuine Bcl-2-like protein, Nrh actual roles remained poorly characterized. Here, we show that Nrh localizes mainly at

2+ the ER in breast cancer cells. We also report that Nrh interacts with the IP3R1 Ca channel via its BH4 domain and decreases ER Ca2+ release, following Histamine treatment. Of note, Nrh activity at the ER is Bax-independent, in accordance with low affinity for Bax (18). Furthermore, we observed that Nrh jeopardizes UPR initiation, and subsequent slowly arising cell death. In fact, this study provides the first evidence that a Bcl-2 homolog can control the UPR pathway upstream of the mitochondria, by preventing the release of Ca2+ from the ER lumen via IP3Rs (Fig. 7E).

Based both on i) the restricted localization of Nrh at the ER, where it specifically interacts with

IP3R1&3 and ii) the fact that nrh silencing, or Nrh protein inhibition, resulted in the sensitization of these cells to Thapsigargin and death-inducing agents used in the clinic, we speculated that disrupting

Nrh/IP3Rs complexes could challenge Nrh-expressing tumor cells. Indeed, disruption of the Nrh/IP3R1 complex in vitro, using the Nrh BH4-derived peptide, appears to sensitize in vitro cultured cells to conventional chemotherapeutic agents. Moreover, using an in vivo SCID mouse xenograft tumor model, we report that the Nrh-BH4 mimicking peptide alone compromises tumor growth, without apparent side effects. Together our data support that Nrh/IP3Rs complexes represent a novel target for breast cancer treatment. As a matter of fact, Nrh-BH4 might have similar effects on other types of cancers overexpressing Nrh (23–25). Surprisingly, the Nrh-BH4 peptide appears to disrupt the

Nrh/IP3R1 complex by binding to Nrh instead of the IP3R1 BD. Additional structural analyses must be conducted to identify the Nrh domains involved in this interaction.

Interestingly, the Bcl-2/IP3R2 complex was also reported to be a potential target in the context of B- cell cancers. Therefore, as proposed by Bultynck and colleagues (36), the systematic evaluation of

IP3Rs expression levels in tumor cells -referred to as “IP3R profiling”, as opposed to “BH3 profiling”

(37)- may help identify the most appropriate BH4 domain to target for patient therapy. Indeed, high

IP3R2 levels would suggest focusing on Bcl-2, whereas high IP3R1 or IP3R3 levels may point towards

Nrh. In addition, in the present study, Nrh protein could be detected in only four out of thirty-three tested healthy tissues, suggesting that pharmacological inhibition of Nrh may have few off-target effects, contrary to the inhibition of ubiquitously expressed proteins such as Bcl-2 or Bcl-xL.

Finally, most strategies targeting pro-survival Bcl-2 proteins have focused so far on BH3 domains. In this respect, the Bcl-2 inhibitor ABT-199 (Venetoclax) was recently approved by the FDA for CLL

(38). However, the high functional redundancy between Bcl-2 anti-apoptotic proteins may represent the limit of current therapeutic strategies using BH3 mimetics, as tumor cells may adapt and resist to a single specific inhibitor and as the use of non-selective molecules proved to be impractical in trials (5).

In fact, amid the Bcl-2 family, the high level of diversity observed among BH4 domains is an opportunity to identify specific inhibitors targeting non-redundant activities amongst Bcl-2 homologs.

In this regard, the uncovered cytoprotective function of Bcl-2-BH4 was recently exploited to successfully develop new drug candidates (39,40). Interestingly, Bcl-2-BH4 appears to inhibit apoptosis by preventing Bax activation (41), whereas we show here that Nrh-BH4 accelerates the cell death by disrupting Nrh/IP3R1 complexes. Actually, BH4-derived molecules, may offer better specificity, and presumably lower residual toxicity, providing a unique opportunity to anticipate BH3 mimetic shortcomings (6). Overall, this study provides a first comprehensive model for Nrh mechanism of action in breast cancer, together with a rationale for developing Nrh inhibitors for clinical applications.

Acknowledgments

We thank Elisabeth Errazuriz-Cerda and Denis Resnikoff (CIQLE-SFR Lyon-Est), Amélie Colombe and Laetitia Odeyer (CLB) for technical assistance and Brigitte Manship (CRCL) for manuscript editing. This work is supported by AFM telethon, Ligue contre le cancer, Fondation ARC (to G.

Gillet) and Cancéropole Auvergne Rhônes-Alpes (to R. Rimokh).

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Figure Legends

Figure 1 – Nrh expression is correlated with decreased metastasis-free survival and contributes to tumorigenesis

(A) Nrh detection, immunohistochemistry. Two representative tumors are displayed among n = 393 samples. Scale bar: 50 µm (B) Cox regression plot from Nrh Negative (n = 213) and Nrh Positive (n =

180) groups of diagnosed breast cancers, distant metastasis-free survival (14-year survey). (C)

Western blotting. Nrh levels in breast cancer cells lines (MM standing for MDA-MB), MCF10A and

HeLa cells. (D) Nrh endogenous levels in MDA-MB-231 cells transfected with indicated siRNAs for

48 hours (E-F). Apoptosis quantification (caspase 3/7) in MDA-MB-231 transfected with indicated siRNAs and treated with either (E) Staurosporine (mean ± SD; n = 3; n.s. P > 0.1) or (F) Thapsigargin

(mean ± SD; n = 3; ***, P < 0.001). (G) Proliferation (cell density at 96h) of 231 empty and 231 Nrh

+/- cells treated with Etoposide (Eto), Doxorubicin (Doxo) or Azacytidine (Aza) (mean ± SD; n = 3;

***, P < 0.001; **, P < 0.01; *, P < 0.05). (H) Tumorigenic capacity of 231 empty and 231 Nrh +/- cells in SCID mice (mean ± SEM; n = 8 mice per condition). (I) Tumor weight at day 35 of experiment shown in (H) (mean ± SEM; n = 8 mice per condition; **, P < 0.01; *, P < 0.05).

Figure 2 - ER-associated Nrh prevents apoptosis independently of BH3-binding pocket

Subcellular localization of Nrh. (A) Western blotting detection of endogenous Nrh in MDA-MB-231 fractions. Whole-cell lysates (Total), mitochondria (Mito), endoplasmic reticulum (ER), cytosol

(S100). (B) Detection of endogenous Nrh in MDA-MB-231 cells. Nrh immunogold labeling. Asterisk:

ER. Arrows: gold coated beads. Scale bar: 100 nm. (C) Nrh detection in HeLa cells transfected with pCS2+Flag-Nrh (immunofluorescence). Scale bar: 20 µm. Co-localization was assessed using pCS2+GFP-cb5 (ER-GFP) or TOM20 immunostaining for ER and mitochondrial localization, respectively. (D) Quantification of ER vs mitochondria Flag-Nrh co-localization in HeLa (Pearson coefficient) (mean ± SD; n = 6; ***, P < 0.001). (E) Structure of the Nrh constructs showing BH and membrane anchoring domains. (F-G) Quantification of apoptosis in HeLa (caspase activity, FLICA)

transfected with above constructs and treated with10 µM Thapsigargin (mean ± SD; n = 3; ***, P <

0.001; **, P < 0.01). (H) Kinetics of UPR markers in presence of 1 µM Thapsigargin in HeLa stably- transfected by pLCPxFlag-Nrh or empty vector (pLCPx). Vinculin and Flag were used as loading controls.

2+ Figure 3 - Nrh interacts with the IP3-binding domain of IP3R1 and IP3R3 to inhibit Ca release

(A) Structure of Inositol 1,4,5-triphosphate receptor type 1 (hIP3R1). SD (Suppressor Domain), IP3BD

(IP3 ligand Binding Domain), MTD (Modulatory and Transducing Domain), CFD (Channel Forming

Domain), CD (Coupling Domain). (B-C) Interaction between Nrh and IP3R1 domains as well as (D)

IP3R1-3 ligand binding domains (BD) in HeLa. Antibodies for Co-IPs and western blotting are shown on the left. Transfected constructs are indicated. (E) Ca2+ release following 100 µM Histamine stimulation in HeLa transfected either with the empty vector (pCS2+), WT Nrh or Nrh BH4 expressing vectors. FluoForte cytosolic Ca2+ dye. (F) Quantification of the maximum peaks recorded in (E) (mean ± SEM; n = 9; ***, P < 0.001; n.s. P > 0.1). (G) Ca2+ release following 100 µM

Histamine stimulation in HeLa cells transfected either with empty vector (pCS2+), or vectors encoding

WT Nrh, Nrh G85A or Nrh 1-97. FluoForte cytosolic Ca2+ dye. (H) Quantification of the maximum peaks recorded in (G) (mean ± SEM; n = 9; ***, P < 0.001; **, P < 0.01).

Figure 4 – A Nrh-derived peptide disrupts IP3Rs/NRH interaction

(A) Diagram of Nrh BH4 sequence. Targeted residues for mutagenesis are in red. (B) Interaction between the BD of IP3R1 and Nrh mutants. Co-IPs in HeLa cells. Antibodies used for Co-IPs and western blotting are shown on the left, transfected constructs are indicated on top. Peptide efficiency scores were normalized and expressed as a product of the respective peptide expression levels, whenever possible “Peptide effiency (%)”. (C) Apoptosis quantification (% caspase positive cells,

FLICA) in HeLa transfected with empty vector or plasmids encoding Nrh 1-23, Nrh 1-23 Y16F or Nrh

1-23 C20A. Cells were treated with Thapsigargin for 24 hours (mean ± SD; n = 3; ***, P < 0.001). (D)

Detection of Ca2+ release (FluoForte) following 100 µM Histamine stimulation in HeLa cells

transfected either with the empty vector (pCS2+) or plasmids encoding Nrh 1-23 WT, Nrh 1-23 Y16F or Nrh 1-23 C20A. (E) Quantification of the maximum peaks recorded in (D) (mean ± SEM; n = 9; *,

P < 0.05; n.s. P > 0.1). (F) Streptavidin pull-down detection of interactions between Nrh 1-23 and potential partners. Biotin-coupled Nrh 1-23 was used on lysates from HeLa cells transfected with a plasmid encoding Flag-Nrh, HA-tagged hBD-IP3R1 or HA-tagged Beclin-1 (HA-BECN1). (G)

Control of expression levels of Flag-Nrh, HA-tagged hBD-IP3R1 and HA-tagged BECN1 constructions (whole cell lysates) used for pull-down experiments described in (F). (H) Streptavidin pull-down detection of interactions between Nrh and Nrh 1-23. Biotin-coupled Nrh 1-23 or Nrh 1-23

Y16F peptides were used on lysates from HeLa cells transfected with plasmids encoding Flag-Nrh or

Flag-Nrz. (I) Control of Flag-Nrh and Flag-Nrz expression levels from pull-down experiments in (H).

Figure 5 - Disruption of endogenous IP3Rs/NRH interaction sensitizes cells to apoptosis

(A) Representation of the FITC-labelled TAT-Nrh 1-23 synthetic peptide sequence (TAT-NDP) including TAT cell-penetrating domain, linker, Nrh 1-23 sequence without methionine. TAT-CTRL control was designed using the same sequence as TAT-NDP but with the Y16F mutation. (B) Circular dichroism spectra of TAT-NDP and TAT-CTRL showing typical alpha-helix signature. (C)

Endogenous interactions between IP3R1 and Nrh in MDA-MB-231. Proximity Ligation Assay (PLA) using TAT-NDP or TAT-CTRL peptides either photoactivated (λ488nm [+]) or not (λ488nm [-]). (D)

Quantification of PLA experiments (mean ± SD; n = 3; ***, P < 0.001; n.s. P > 0.1). Scale bar: 20 µm.

(E) Apoptosis in MDA-MB-231 cells (% caspase positive cell, FLICA) treated with TAT-NDP or

TAT-CTRL either photoactivated (λ488nm [+]) or not (λ488nm [-]). Cells were treated with 10 µM

Thapsigargin (mean ± SD; n = 3; ***, P < 0.001; n.s. P > 0.1).

Figure 6 - Disruption of the IP3Rs/NRH interaction enhances response to chemotherapeutic agents

(A) Sequences of dTAT-1-23 (referred to as NDP) and control dTAT-CTRL both encompassing the

N-terminal dimerization sequence (CK) for endosome escape capability. (B) Co-IP detection of the

Flag-Nrh/HA-hBD IP3R1 complex. Antibodies used for IP and detection are indicted on the left.

Transfected plasmids are shown on top. Cells were pre-incubated or not with vehicle or dTAT peptides as indicated. (C) Apoptosis quantification (% caspase 3/7 positive cells) in MCF10A, MDA-

MB-231 or MDA-MB231 Nrh +/- cells incubated with 10 µM dTAT-NDP or sterile water (vehicle) in

Ca2+-free BSS and treated with 1 µM Thapsigargin (mean ± SD; n = 3; ***, P < 0.001; *, P < 0.05).

(D) Apoptosis quantification (% caspase 3/7 positive cells at 36h) in MDA-MB-231 cells incubated for 1 hour with the indicated dTAT peptides in Ca2+-free BSS and treated with Etoposide (mean ± SD; n = 3; ***, P < 0.001; n.s. P > 0.1). (E) Apoptosis quantification (% caspase positive cells) in MDA-

MB-231 cells incubated for 1 hour at 37°C with 10 µM of the indicated dTAT peptides in Ca2+-free

BSS and treated with Doxorubicin (mean ± SD; n = 3; **, P < 0.01). (F) Proliferation (% cell density) of CAL-51 cells incubated for 1 hour with 10 µM of the indicated dTAT peptides in Ca2+-free BSS and treated with Azacytidine (mean ± SD; n = 3; **, P < 0.01; ***, P < 0.001). (G) Proliferation (% cell density at 96h) of CAL-51 cells incubated for 1 hour with 10 µM of the indicated dTAT peptides in Ca2+-free BSS and treated with Doxorubicin (mean ± SD; n = 3; *, P < 0.05; **, P < 0.01). See also

Movie S1.

Figure 7 - Disruption of IP3Rs/Nrh interactions compromises tumor growth

(A) Colony formation assay. MDA-MB-231 cells were pre-incubated with the indicated dTAT peptides in Ca2+-free BSS, treated with 1 µM Etoposide for 24 hours, and then cultivated in soft agar for another 14 days (magnification x100). (B) Colony number at 14 days (mean ± SD; n = 3; **, P <

0.01; n.s. P > 0.1). (C) Evaluation of the tumorigenic capacity of MDA-MB-231 cells in SCID mice.

Mice were treated with either dTAT-CTRL or dTAT-NDP (peritumoral injection), or vehicle alone

(PBS). Tumor volume was measured twice a week (mean ± SEM; n = 5 mice per condition). (D)

Tumors weight at day 38 of the experiment shown in (C) (mean ± SEM; n = 5 mice per condition; **,

2+ P < 0.01; *, P < 0.05). (E) Model for Nrh action. Nrh interacts with IP3R1 and dampens ER Ca

2+ release. Upon Nrh downregulation or Nrh/IP3R1 complex disruption, Ca homeostasis is compromised, leading to excessive ER Ca2+-release into the cytosol. Cytosolic Ca2+ increase might lead to Ca2+ import to the mitochondria, triggering MOMP and apoptosis, whereas ER Ca2+ depletion might activate ER-stress sensors, including PERK, triggering apoptosis as well.

Breast cancer targeting through inhibition of the endoplasmic reticulum-based apoptosis regulator Nrh/BCL2L10

Adrien Nougarede, Nikolay Popgeorgiev, Loay Kassem, et al.

Cancer Res Published OnlineFirst January 12, 2018.

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