Bortezomib blocks the catabolic process of. autophagy via a cathepsin-dependent mechanism, affects endoplasmic reticulum stress, and

Autophagy ISSN: 1554-8627 (Print) 1554-8635 (Online) Journal homepage: http://www.tandfonline.com/loi/kaup20 Bortezomib blocks the catabolic process of autophagy via a cathepsin-dependent mechanism, affects endoplasmic reticulum stress, and induces caspase-dependent cell death in antiestrogen sensitive and resistant ER+ breast cancer cells Sudharsan Periyasamy-Thandavan, William H. Jackson, Julia S. Samaddar, Brian Erickson, John R. Barrett, Lauren Raney, Elangovan Gopal, Vadivel Ganapathy, William D. Hill, Kapil Bhalla & Patricia V. Schoenlein To cite this article: Sudharsan Periyasamy-Thandavan, William H. Jackson, Julia S. Samaddar, Brian Erickson, John R. Barrett, Lauren Raney, Elangovan Gopal, Vadivel Ganapathy, William D. Hill, Kapil Bhalla & Patricia V. Schoenlein (2010) Bortezomib blocks the catabolic process of autophagy via a cathepsin-dependent mechanism, affects endoplasmic reticulum stress, and induces caspase-dependent cell death in antiestrogen sensitive and resistant ER+ breast cancer cells, Autophagy, 6:1, 19-35, DOI: 10.4161/auto.6.1.10323 To link to this article: https://doi.org/10.4161/auto.6.1.10323 Published online: 01 Jan 2010. Submit your article to this journal Article views: 312 View related articles Citing articles: 27 View citing articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalinformation?journalcode=kaup20

Basic Research Paper Autophagy 6:1, 19-35; January 1, 2010; 2010 Landes Bioscience Basic Research Paper Bortezomib blocks the catabolic process of autophagy via a cathepsin-dependent mechanism, affects endoplasmic reticulum stress and induces caspase-dependent cell death in antiestrogen-sensitive and resistant ER + breast cancer cells Sudharsan Periyasamy-Thandavan, 1 William H. Jackson, 1 Julia S. Samaddar, 1 Brian Erickson, 1 John R. Barrett, 1 Lauren Raney, 1 Elangovan Gopal, 2 Vadivel Ganapathy, 2,3 William D. Hill, 1 Kapil N. Bhalla 3 and Patricia V. Schoenlein 1,3, * 1 Department of Cellular Biology and Anatomy; 2 Department of Biochemistry and Molecular Biology; 3 MCG Cancer Institute; Medical College of Georgia; Augusta, GA USA Key words: tamoxifen, bortezomib, ER stress, autophagy, protein turnover, BNIP3, cathepsins Abbreviations: 4-OHT, 4-hydroxytamoxifen; E2, estradiol; ER stress, endoplasmic reticulum stress; ERa, estrogen receptor alpha; ER +, estrogen receptor positive; SERM, selective estrogen receptor modulator; TCA, trichloroacetic acid; UPR, unfolded protein response; UPS, ubiquitin-proteasome system In recent studies, we and others showed that autophagy is critical to estrogen receptor positive (ER + ) breast cancer cell survival and the development of antiestrogen resistance. Consequently, new approaches are warranted for targeting autophagy in breast cancer cells undergoing antiestrogen therapy. Because crosstalk has been demonstrated between the autophagy- and proteasome-mediated pathways of protein degradation, this study investigated how the proteasome inhibitor bortezomib affects autophagy and cell survival in antiestrogen-treated ER + breast cancer cells. Bortezomib, at clinically achievable doses, induced a robust death response in ER +, antiestrogen-sensitive and antiestrogen-resistant breast cancer cells undergoing hormonal therapy. Cleavage of PARP and lamin A was detectable as a read-out of cell death, following bortezomib-induced mitochondrial dysfunction. Prior to induction of cell death, bortezomib-treated cells showed high levels of light chain 3 (LC3) and p62, two protein markers for autophagy. The accumulation of these proteins was due to bortezomib-mediated blockade of long-lived protein turnover during macroautophagy. This novel action of bortezomib was linked to its blockade of cathepsin-l activity, which is required for autolysosomal-mediated protein turnover in ER + breast cancer cells. Further, bortezomib-treated breast cancer cells showed induction of the unfolded protein response, with upregulation of CHOP and GRP78. Bortezomib also induced high levels of the pro-apoptotic protein BNIP3. Knockdown of CHOP and/or BNIP3 expression via RNAi targeting significantly attenuated the deathpromoting effects of bortezomib. Thus, bortezomib inhibits prosurvival autophagy, in addition to its known function in blocking the proteasome, and is cytotoxic to hormonally treated ER + breast cancer cells. These findings indicate that combining a proteasome inhibitor like bortezomib with antiestrogen therapy may have therapeutic advantage in the management of early-stage breast cancer. Introduction In the United States, approximately 40,000 women die from breast cancer annually, second only to lung cancer mortality. 1 In the western world, the chance of breast cancer development in women is approximately one out of 10 during their lifetime. 2 Of these breast cancers, approximately 75% will be categorized as estrogen receptor alpha (ERα) positive, with estradiol (E2)-bound ERα as the key determinant in promoting breast cancer growth. 1,3 E2-bound ERα acts as a transcription factor that regulates the expression of target genes that drive breast cancer cell proliferation. 4 Additionally, ERα-mediated nongenomic effects have been identified and include proteinprotein interactions that can activate ERα. Typically, these *Correspondence to: Patricia V. Schoenlein; Email: pschoenl@mail.mcg.edu Submitted: 04/24/09; Revised: 09/01/09; Accepted: 10/12/09 Previously published online: www.landesbioscience.com/journals/autophagy/article/10323 www.landesbioscience.com Autophagy 19

interactions involve growth factor receptors and/or signal transduction pathways. 5 Blocking ERα action by selective estrogen receptor modulators (SERMs) has been the most common treatment strategy for breast cancer patients. 6 Tamoxifen, the most commonly used SERM, exerts its effect by binding the estrogen receptor complex and altering its conformation, affecting both its transcriptional activity and ability to undergo protein-protein interactions that are required for the nongenomic ERα effects. Despite the significant benefits from tamoxifen treatment, almost all patients with metastatic disease and as many as 40% of the patients receiving adjuvant tamoxifen therapy do not respond, or acquire resistance during treatment. 6 In postmenopausal women, treatment options for ER + breast cancer now include the use of aromatase inhibitors which block the synthesis of estrogen from steroid precursors produced by the adrenal gland. 7,8 Aromatase inhibitors are superior to ERα blockade by SERMs. 9-12 However, resistance to aromatase inhibitors can occur and is predicted to be an impediment to successful breast cancer therapy. 13 Based on the common emergence of breast cancer resistance to SERMs and aromatase inhibitors, novel strategies are needed to improve therapeutic efficacy. To this end, studies for the past several decades have been focused on understanding the biological mechanisms underlying intrinsic and acquired SERM resistance in breast cancer. Resistance mechanisms to tamoxifen have been the best described and include multiple pathways, including crosstalk between ERα and the EGFR/HER2 and insulin-like growth factor receptor (IGFR) pathways, 14-17 mutations in ERα, 15 and alterations in the ERα complex such that co-activator and/or co-repressor recruitment or function is altered. 17 In recent studies, including studies conducted in our laboratory, autophagy (also referred to as macroautophagy) has been identified as a survival mechanism for breast cancer cells challenged with chemotherapy 18 or 4-hydroxytamoxifen (4-OHT), the active metabolite of tamoxifen. 19-21 Our recent work also links autophagy to the development of antiestrogen resistance. 20,22 Importantly, blockade of autophagy promoted apoptosis in hormonally-treated, antiestrogen-resistant and -sensitive breast cancer cells. 19,20 These studies support the current view of autophagy as an important prosurvival mechanism of cancer cells and a potential target for cancer therapy. 23,24 Autophagy is constitutive in all eukaryotic cell types containing a lysosomal compartment and contributes to organelle turnover as well as long-lived protein degradation. Autophagy occurs in parallel with the ubiquitin-proteasome system (UPS) that is specific to short-lived protein turnover. 25 Induction of autophagy involves the de novo formation of an autophagosome that nonspecifically engulfs portions of the cytoplasm and cytoplasmic organelles. The mature autophagosome subsequently fuses with a lysosome, forming the autolysosome within which the intra-autophagosomal components are degraded by lysosomal hydrolytic enzymes. Together, autophagy and the UPS prevent the accumulation of polyubiquitinated and aggregated proteins that can perturb cellular homeostasis and induce cell death. 23,26-30 Crosstalk between autophagy and the UPS has been reported. 26,31,32 In the present study, we specifically examined how proteasome inhibition affects autophagy function and cell survival in ER + breast cancer cells exposed to hormonal therapy. Our study shows that clinically achievable doses of bortezomib induced a significant death response in hormonally treated breast cancer cells with blockade of autophagy function as a major target. We further demonstrate that bortezomib attenuated cathepsin L and B activity and that cathepsin activity is required for autolysosomal turnover of proteins in ER + breast cancer cells. Thus, bortezomib may be a useful agent to target the prosurvival role of autophagy in breast cancer cells, and enhance the cytotoxic outcome of hormonal therapy. Results Proteasome inhibition induces a death response in antiestrogen-sensitive, ER + breast cancer cells undergoing hormonal therapy. To determine how proteasome inhibition influenced prosurvival autophagy in ER + breast cancer cells, MCF-7 cells were treated with E2 or E2 plus 4-OHT in the presence and absence of bortezomib at varying concentrations ranging from 1 50 nm. Examination of the cells by light microscopy showed that bortezomib-treated cells were highly stressed, with extensive cytosolic vacuolization (Fig. 1A) and an increase in cell detachment from the culture dish, a characteristic typical of MCF-7 cells undergoing cell death. 39,40 Cell counts determined that bortezomib treatment significantly reduced MCF-7 cell number under conditions of growth in E2 in the presence or absence of 4-OHT (Fig. 1B). The IC 50 s for bortezomib for MCF-7 growing in E2 - or E2 plus 4-OHT-supplemented medium was 4.0 nm and 2.9 nm, respectively. Clonogenic assays further demonstrated the sustained ability of bortezomib or bortezomib plus 4-OHT, to suppress MCF-7 cell growth (Fig. 1C). In this assay, one hit of drug was delivered to the cells and individual cells were given 4 weeks to form visible clones (Suppl. Fig. 1). In the clonogenic assay, IC 50 s for bortezomib were 1.9 nm for E2 and 2.0 nm for the E2 plus 4-OHT-treated cells. In contrast to bortezomib, 4-OHT treatment for this period of time was unable to effectively suppress MCF-7 clonogenic growth, mimicking the escape of breast cancer cells from the tumor suppressor effects of tamoxifen therapy (Fig. 1C). Based on the morphological indications of stress and the significant reduction in MCF-7 clonogenic survival by bortezomib treatment, the levels of intact and cleaved PARP and lamin A were determined. Cleavage of PARP and lamin A are established markers of MCF-7 cell death. 39,40 Elevated levels of both cleaved PARP and cleaved lamin A were detected in cells treated with bortezomib in the presence and absence of 4-OHT (Fig. 1D). Cleavage of lamin A showed a 8 10 fold increase in the E2 plus bortezomib-treated cells compared to the E2-treated cells (Fig. 1D, lanes 4 & 5 versus lane 1). In the 4-OHT-treated populations where lamin A cleavage occurs in response to 4-OHT treatment (Fig. 1D and compare lanes 6 to lane 1), lower concentrations of bortezomib were needed to generate a similar 10-fold increase in cleaved lamin A levels; i.e., 5 nm bortezomib in combination with 1 μm 4-OHT induced a 12-fold increase in lamin-a levels, whereas, E2-treated cells required 25 nm Bortezomib (Fig. 1D, lanes 8 and 5 compared to 20 Autophagy Volume 6 Issue 1

Figure 1. For figure legend, see page 22. lane 1). In all bortezomib-treated cells showing lamin A and PARP cleavage, the level of intact lamin A and PARP was reduced in a complimentary manner (Fig. 1D). Cleavage of lamin A and PARP were effectively blocked by the pan-caspase inhibitor zvad-fmk (Fig. 1E). Treatment with zvad-fmk also significantly reduced www.landesbioscience.com the number of trypan blue-positive cells in the bortezomib-treated cell populations (Fig. 1F). These data and the fact that mitochondrial integrity was severely compromised by bortezomib treatment (Fig. 1G), indicated that bortezomib treatment was primarily inducing an apoptotic cell death in breast cancer cells undergoing Autophagy 21

Figure 1. Bortezomib induces caspase-dependent MCF-7 cell death. (A) Light microscopy showed that bortezomib treatment induced cell detachment and vacuolization in cells treated with E2 or E2 plus 4-OHT (1 mm) that was clearly visible 144 h after treatment with 25 nm bortezomib (Parts c & d). The insets (boxed areas) shows vacuolated cells under higher magnification. (B) Cell counts showed a dose-dependent decrease in MCF-7 cell number by bortezomib treatment. Adherent and detached cells were collected and counted using a hemocytometer 144 h following treatment with E2 or E2 plus 4-OHT and 0, 1, 2.5, 5, 10, 25 and 50 nm bortezomib. (C) Bortezomib significantly reduced the clonogenic survival of MCF-7. Cells were treated with E2 or E2 plus 4-OHT and 0, 1, 2, 3, 4 and 5 nm bortezomib. (D) Immunoblotting detected increased levels of cleaved PARP and cleaved lamin A in cells treated with E2 or E2 plus 4-OHT and 0, 2.5, 5, 12.5 and 25 nm bortezomib for 96 h, while reduced levels of the full-length lamin A and PARP were noted. Fold diff. refers to the difference in signal intensity of cleaved lamin A and cleaved PARP relative to the E2-treated control which was arbitrarily assigned a value of 1. (E and F) Treatment with the pan-caspase inhibitor z-vad-fmk blocked bortezomib-induced cleavage of lamin A and PARP (E) and reduced the number of dead (trypan blue-positive) cells in the population (F). Cells were pretreated for 1 h with 50 μm z-vad-fmk prior to addition of hormonal treatments and/or bortezomib. Subsequent to treatment cells were harvested for SDS/PAGE and immunoblotting analysis 72 h after treatment (E) or harvested for cell counts after a 144 h treatment period (F), with a 2 nd hit of 50 um z-vad-fmk added after 72 h of the initial treatment. (G) Mitochondrial membrane potential was severely compromised by bortezomib treatment. After 96 h of bortezomib plus hormonal therapy, JC-1 was added to cells for 15 min and then cells were processed for flow cytometry. Bortezomib plus 4-OHT treatment reduced mitochondrial membrane potential of MCF-7 cells as compared to E2-treated cells. hormonal therapy and that caspase blockade in the bortezomib treated cells did not lead to a necrotic-type death. Proteasome inhibition blocks autolysosome catabolism in ER + breast cancer cells. The observed vacuolization in cells treated with bortezomib is a morphological feature observed in our previous study when bafilomycin was used to block autophagosome function, 20 suggesting that bortezomib was blocking autophagosome function in MCF-7 cells. To determine if this was the case, the levels of LC3 and p62 were determined by immunoblotting. The levels of p62 were consistently increased in MCF-7 cells following treatment with 10 and 25 nm bortezomib by 24 and 48 h (Fig. 2A and B, lanes 3 & 4, and 7 & 8, respectively). LC3 II levels also were consistently increased with 25 nm bortezomib by 24 and 48 h (Fig. 2A and B, respectively, compare lane 4 to 8 and lane 1 to 5). By 72 h of treatment, LC3 II levels were consistently increased with 5 and 10 nm Bortezomib treatment (data not shown). The fact that p62 levels were increased suggested that autolysosomal turnover of proteins was not occurring. 41-43 To confirm that p62 and LC3 do accumulate in MCF-7 cells when autolyosomal protein degradation is blocked, cells were treated with 10 mm chloroquine, a compound known to elevate the ph of the autolysosome and blocks lysosome function. 44 Chloroquine-treated cells, like bortezomib-treated cells, showed high levels of LC3 and p62 (Fig. 2C). Thus, we performed long-lived protein turnover studies to better understand how bortezomib was affecting autophagy function in MCF-7 cells. At 24 h (data not shown) and 48 h of treatment using various concentrations (1, 5, 10, 25 and 50 nm), bortezomib treatment blocked long-lived protein turnover in E2-treated and E2 plus 4-OHT-treated MCF-7 cells (Fig. 2D and E, respectively). With this assay, we were able to reproducibly demonstrate 4-OHT-mediated induction of long-lived protein turnover in MCF-7 cells; compare the ~43% proteolysis for E2-treated cells to the ~80% proteolysis of E2 plus 4-OHT-treated cells (Fig. 2E). Further, as a positive control we utilized chloroquine to block autolysosomal turnover and were able to demonstrate that a significant percentage of the 4-OHT-induced proteolysis was due to turnover of long-lived protein (Fig. 2F). Chloroquine was unable to enhance bortezomib-mediated blockade of proteolysis in E2-treated and E2 plus 4-OHT-treated cells (Fig. 2F). Our interpretation of these data is that bortezomib is as effective as chloroquine in blocking autolysosomal turnover of long-lived protein. We did note, however, that chloroquine was less effective than bortezomib in blocking proteolysis in the E2 plus 4-OHT treated cells and did not appear to block proteolysis in the E2 treated cells. We also utilized the more commonly used inhibitors 3-MA 45,46 and bafilomycin 47 to block lysosome function. These controls and the use of rapamycin 48 or serum starvation 49,50 to induce autophagy in MCF-7 cells confirmed the reproducibility and accuracy of our proteolysis studies. As seen in Figure 3A, approximately 15% of basal protein catabolism in E2-treated cells and greater-than 30% of long-lived protein catabolism in 4-OHT-treated, rapamycin-treated, and serum-starved cells was inhibited by 48 h of 3-MA treatment and, therefore, attributable to macroautophagy. Bafilomycin, like chloroquine, did not block macroautophagy in E2-treated cells, but did block ~20% of longlived protein catabolism in 4-OHT-treated, rapamycin-treated, and serum-starved cells (Fig. 3B). Data for the 24 h and 72 h time points at which proteolysis was analyzed showed similar results and can be seen in Supplement Figure 2. Importantly, bortezomib also blocked long-lived protein turnover induced by serum starvation (Fig. 3C) and rapamycin treatment (Fig. 3D), so bortezomib blockade of long-lived protein turnover and inhibition of macroautophagy appeared not to depend on the agent used to induce macroautophagy. Further, similar results were obtained with the proteasome inhibitor MG132 (Suppl. Fig. 3A and B) and lactacystin (Suppl. Fig. 3C), providing strong evidence that proteasome inhibition in general results in blockade of autolysomal catabolism of long-lived proteins. To determine if bortezomib inhibits autolysosomal-mediated catabolism in ER + breast cancer cells other than MCF-7 cells, we performed similar studies with T-47D cells, another commonly used ER-positive breast cancer cell line. 51 Bortezomib effectively reduced T-47D cell number with an IC 50 of 3.7 nm for E2-treated cells and 4.0 nm for E2 plus 4-OHT-treated cells (Fig. 4A). Bortezomib also effectively induced T-47D cell death as determined by trypan blue uptake (Fig. 4B) and increased levels of cleaved lamin A and cleaved PARP in bortezomib-treated cells (Fig. 4C). Electron microscopic examination confirmed the presence of dead cells in the bortezomib-treated cell populations, with greater than 25% of the cells having an apoptotic-appearing nucleus by 48 h of treatment. Supplement Figure 4 shows representative T-47D cells following 48 h of treatment with 4-OHT and 4-OHT plus 10 nm bortezomib, respectively. T-47D cells 22 Autophagy Volume 6 Issue 1

Figure 2. Bortezomib blocks autolysosomal-mediated turnover of long-lived proteins in MCF-7 cells undergoing hormonal treatment. (A and B) Immunoblotting demonstrates increased LC3 (16 kd plus 18 kd forms) and p62 levels in bortezomib-treated cells. Cells were incubated with E2 or E2 plus 4-OHT in the absence or presence of bortezomib (1, 10 and 25 nm) for 24 h and 48 h at which time cell lysates were prepared and protein was subjected to SDS/PAGE, followed by immunoblotting using antibodies to LC3, p62 and b-actin. Data are representative of at least four independent experiments. Fold diff. refers to the difference in signal intensity of LC3 (16 plus 18 kd form) and p62 relative to the E2-treated control which was arbitrarily assigned a value of 1. (C) Immunoblotting demonstrates increased LC3 (16 kd plus 18 kd forms) and p62 levels in cells treated with 10 mm chloroquine. (D and E) Bortezomib treatment decreases the rate of autophagic degradation of long-lived proteins in E2 - treated (D) or E2 plus 4-OHTtreated MCF-7 cells (E). Proteolysis of long-lived proteins in E2 - treated cells in the absence or presence of bortezomib (1, 5, 10, 25 and 50 nm) was determined after 48 h of treatment as described in Materials and Methods. The results show that bortezomib attenuates the autophagic degradation. (F) Chloroquine (10 mm) treatment decreases the rate of autophagic degradation of long-lived protein in E2 plus 4-OHT treated MCF-7 cells, but does not affect bortezomib-blockade of long-lived protein turnover. Values are expressed as mean ± S.D. (n = 3). Comparisons are made between the following treatment groups: a, E2 versus E2 plus bortezomib; b, E2 versus E2 plus 4-OHT; c, E2 plus 4-OHT versus E2 plus 4-OHT plus bortezomib, d, E2 plus 4-OHT versus E2 plus 4-OHT plus chloroquine; e, E2 plus 4-OHT plus bortezomib versus E2 plus 4-OHT plus bortezomib plus chloroquine. The symbols *, and ** represent statistical significance: at p < 0.01 and p < 0.001, respectively. treated with 4-OHT showed visible autolysosomes in their cytosol, but the nucleus of the cells often appeared healthy (S4A- C). In contrast, bortezomib treatment of T-47D cells by 48 h typically showed high levels of vacuolization in the cytoplasm of the majority of cells (S4D) and compacted chromatin in the nuclei of the dying cells indicative of an apoptotic process (S4D). Prior to cell death, bortezomib blocked long-lived protein turnover in T-47D cells treated with E2, E2 plus 4-OHT, or serum www.landesbioscience.com Autophagy 23

Figure 3. 3-MA, bafilomycin, and bortezomib block autolysosomal-mediated turnover of long-lived proteins in serum-starved and rapamycin-treated MCF-7 cells. Long-lived protein degradation was measured after 48 h of treatment. (A) 3-MA decreased autophagic proteolysis in 4-OHT, rapamycin, and serum-starved cells. (B) Bafilomycin decreased autophagic proteolysis in 4-OHT, rapamycin, and serum-starved cells. (C) Bortezomib decreased autophagic proteolysis in a dose-dependent manner during serum deprivation. (D) The induction of autophagic proteolysis by rapamycin was attenuated by bortezomib treatment. Serum starvation was performed by removing serum from attached cells with PBS washes 24 h after seeding and in the absence or presence of 10 mm 3-MA (A), 50 nm bafilomycin (B) or bortezomib at 1, 5, 10, 25 and 50 nm (C). Rapamycin was used at 50 ng/ml in the absence or presence of 10 mm 3-MA (A), 50 nm bafilomycin (B) or 1 or 10 nm Bortezomib (D). Values are expressed as mean ± S.D. (n = 3). Comparisons are made between the following treatment groups as follows: a, designated treatment compared to E2; b, E2 plus 4-OHT versus E2 plus 4-OHT plus 3-MA; c, E2 plus rapamycin versus E2 plus rapamycin plus 3-MA; d, serum-free versus serum-free plus 3-MA; e, E2 plus 4-OHT versus E2 plus 4-OHT plus bafilomycin; f, E2 plus rapamycin versus E2 plus rapamycin plus bafilomycin; g, serum-free versus serum-free plus bafilomycin; h, serum-free versus serum-free plus bortezomib; i, rapamycin-versus rapamycin plus bortezomib. The symbols *, and ** represent statistical significance: at p < 0.01 and p < 0.001, respectively. starvation (Fig. 4D) or E2 (Fig. 4E). Increased levels of the LC3 and p62 proteins were also detected in bortezomib-treated T47-D cells (data not shown). 3-MA treatment of E2-treated, E2 plus 4-OHT-treated, and serum-starved cells showed remarkable similarity to the effects of bortezomib (Suppl. Fig. 5A F) further demonstrating that blockade of the 4-OHT-induced macroautophagy in T-47D cells can be detected with our proteolysis assays. Thus, bortezomib action includes blockade of autolysosomal-mediated protein catabolism in antiestrogen-sensitive ER + breast cancer cells and this blockade occurs prior to induction of a robust cell death response. Proteasome inhibition blocks autophagosome function in antiestrogen-resistant ER + breast cancer cells. To determine if bortezomib was able to block prosurvival autophagy in antiestrogen-resistant cells, we utilized the TR5 cell line recently described. 20 In contrast to the parent MCF-7 cells, TR5 cells are not growth-stimulated by E2 and not growth-inhibited by 1.0 mm 4-OHT (Fig. 5A). In addition, the cells have a significantly slower growth rate than the parental MCF-7 cells as seen in Figure 5A. By 24 h, the number of MCF-7 cells had doubled from the number seeded and by 168 h, the total number of cells was increased by ~7-fold. In contrast, TR5 cells showed only a ~4-fold overall increase in the total cell number. This hormone independence does not appear to be due to ER alpha loss because TR5 cells express significant levels of ER alpha (Fig. 5B). Nonetheless, bortezomib treatment of TR5 cells growing in the presence of E2 or E2 plus 4-OHT reduced cell number in a dose-dependent manner, with moderately higher IC 50 s than previously seen for MCF-7 and T-47D cells; (7.7 nm and 6.8 nm for E2-treated and E2 plus 4-OHT-treated cells, respectively) (Fig. 5C). A dose-dependent, bortezomibinduced blockade of autolysosomal catabolism of proteins also was observed in TR5 cells treated with E2 or E2 plus 4-OHT (Fig. 5D), with 10 nm bortezomib inhibiting ~50% long-lived protein turnover. Both cleavage of lamin A and PARP were detected by 72 h of bortezomib treatment, and the increase in the cleaved forms of these proteins occurred concomitantly with a decrease in total lamin A and PARP, respectively (Fig. 5E). Bortezomib-treated cells also showed increased levels of LC3 and p62 levels and this increase was sustained up to 144 h, which is a longer time interval than observed for MCF-7 or T-47D cells (Fig. 5F). Chloroquine treatment showed a similar increase in the levels of LC3 and p62, thus confirming that these proteins increase concomitantly with functional blockade of the autolysosome (Fig. 5G). Overall, these data show that bortezomib effectively blocked autolysosomal turnover of protein and induced cell death in the antiestrogen resistant TR5 cells. When bortezomib-treated TR5 cells were examined with fluorescence microscopy following immunocytochemistry with LC3 antibody, the typical punctate staining for LC3-localized to autophagosomes was observed at 24, 48 and 72 h following treatment with no apparent decrease in staining intensity within cells (Fig. 6A and data not shown). Also, assays with 24 Autophagy Volume 6 Issue 1

Figure 4. Bortezomib treatment of antiestrogen-sensitive T-47D breast cancer cells induced cell death and attenuates autophagic catabolism. Cells were treated with E2 with or without 4-OHT (1 mm) in the presence or absence of bortezomib (1, 2.5, 5, 10 and 25 nm). (A) Bortezomib treatment reduced T-47D cell number in a dose-dependent manner. Cells were treated for 144 h, harvested and counted with a hemocytometer. (B) Bortezomib treatment induced cell death in E2-treated and E2 plus 4-OHT-treated T-47D cells as evidenced by an increase in the number of trypan blue-positive cells and a decrease in the number of trypan blue-negative cells. Cells were treated for 144 h and harvested for cell counts; trypan blue to a final concentration of 0.1% was added to the cell suspension 5 min prior to counting. (C) Bortezomib induces cell death with cleavage of lamin A and PARP by 72 h of treatment. Fold diff. refers to the difference in signal intensity of cleaved PARP relative to the E2-treated control which was arbitrarily assigned a value of 1. (D and E) Autophagic proteolysis by 4-OHT and serum starvation in T-47D cells was attenuated by bortezomib treatment. Cells were harvested after 48 h of bortezomib treatment. Values are expressed as mean ± S.D. (n = 3). Comparisons are made between the following treatment groups as follows: a, E2 versus E2 plus 4-OHT; b, E2 versus serum-starved cells; c, E2 plus 4-OHT versus E2 plus 4-OHT plus bortezomib; d, serumstarved versus serum-starved plus bortezomib; e, E2 versus E2 plus bortezomib. The symbols *, and ** represent statistical significance: at p < 0.01 and p < 0.001, respectively. monodansylcadavarine (MDC) showed that the autolysosomes and lysosomes within the cells were functionally intact in their ability to sequester the MDC at 24, 48 and 72 h (Fig. 6B and data not shown). MDC sequestration analysis conducted in MCF-7 cells showed similar results (Fig. 6C). EM analysis of TR5 cells (Suppl. Fig. 6) also demonstrated the presence of intact autophagosomes and autolysosomes in at least 50% of the cell population; thus it did not appear that bortezomib was blocking the fusion of the autophagosome with lysosomes. EM anlaysis in MCF-7 cells also showed intact autophagosomes and autolysosomes (Suppl. Fig. 7). However, in at least 25% of the MCF-7 and TR5 cell populations treated with 10 nm bortezomib, dilation of the endoplasmic reticulum (ER) and protein aggregates was evident. These characteristics of ER stress were apparent by 48 h of bortezomib treatment in MCF-7 cells, but were not evident in TR5 cells until 72 h after bortezomib treatment. These combined data show that bortezomib-treated cells show characteristics of ER stress concomitant with their inability to catabolize long-lived proteins, but are still capable of LC3 integration into the autophagosome membrane and autolysosomal sequestration of MDC. Bortezomib induces an ER stress response with CHOP and BNIP3 as effectors of cell death. It is well established that inhibition of the proteasome induces an ER stress and the unfolded www.landesbioscience.com Autophagy 25

Figure 5. Bortezomib blocks autophagic catabolism and induces cell death in antiestrogen-resistant TR5 breast cancer cells. (A) TR5 cells are antiestrogen-resistant and proliferated in an E2-independent manner. Cell count was determined 168 h after seeding in medium without E2 (E2 - ), with E2 (E2 + ), 4-OHT, or E2 plus 4-OHT. (B) TR5 cells express ERα. SDS/PAGE, followed by immunoblotting identified ERα expression in TR5 cells. (C) Bortezomib reduced TR5 cell number in a dose dependent manner. Cells were treated with E2 with or without 4-OHT and bortezomib (0, 1, 2.5, 5, 10 and 25 nm). After 144 h of treatment; cells were counted using a hemocytometer. (D) Bortezomib blocks long-lived protein turnover in a dose-dependent manner. Cells were harvested at 48 h after treatment and long-lived protein catabolism in TR5 cells was determined as described in materials and methods. (E and F) Bortezomib treatment results in increased levels of cleaved lamin A and cleaved PARP by 72 h of treatment (E) and the accumulation of LC3 and p62, which persisted for up to 144 h after treatment (F). (G) Chloroquine treatment leads to the accumulation of LC3 and p62 in TR5 cells. For the studies in (B) and (E G), SDS/PAGE, followed by immunoblotting was performed as described in Materials and Methods. Fold diff. refers to the difference in signal intensity of ERα, cleaved lamin A, cleaved PARP, LC3 (16 plus 18 kd form), and p62 relative to the E2-treated control which was arbitrarily assigned a value of 1. Data are representative of at least four separate experiments. protein response (UPR) in cancer cells. 26,52-54 Downstream effectors of the UPR include activation of BiP/GRP78 to maintain ER integrity, 54 and the C/EBP homologous protein (CHOP) to mediate death when ER stress is beyond a cell s tolerance. 55 We observed a dose-dependent increase in CHOP and GRP78 after 48 h (Fig. 7A) in all three cell lines, confirming induction of ER stress. The fact that both CHOP and GRP78 were upregulated in bortezomib-treated cells suggested that cells were trying to survive (GRP78 upregulation), but this was ultimately a failed attempt with CHOP upregulation mediating death. We also analyzed levels of the pro-apoptotic protein BNIP3 in MCF-7 and TR5 cells treated with bortezomib (Fig. 7B). The rationale for analyzing the levels of this protein stemmed from previous unpublished data from our laboratory showing induction of the 26 Autophagy Volume 6 Issue 1

Figure 6. Bortezomib-treated breast cancer cells exhibit punctate fluorescent LC3 staining and retained the ability to sequester MDC in acidic compartments. (A) LC3 staining is punctate in TR5 cells after 48 hour treatment with E2, E2 plus 4-OHT, or E2 plus 4-OHT plus 10 nm bortezomib. (B and C) MDC is sequestered in TR5 (B) and MCF-7 cells (C) treated with 4-OHT and/or bortezomib, indicating structural integrity of autolysosomes in cells. TR5 and MCF-7 cells were treated with E2 and E2 plus 4-OHT in the absence or presence of 1, 5 or 10 nm bortezomb. TR5 and MCF-7 cells were treated for 48 h and 24 h, respectively, and then subjected to MDC sequestration assays as outlined in Materials and Methods. E2, E2 plus 4-OHT, Values are expressed as mean ± S.D. (n = 3). Comparisons are made between the following treatment groups as follows: a, E2 versus E2 plus bortezomib; b, E2 versus E2 plus 4-OHT; c, E2 plus 4-OHT versus E2 plus 4-OHT plus bortezomib. The symbols *, and ** represent statistical significance: at p < 0.01 and p < 0.001, respectively. pro-apoptotic protein BNIP-3 (Schoenlein PV, unpublished) concomitant with the bafilomycin-induced blockade of autophagy and induction of vacuolization and death recently described in MCF-7 and TR5 cells. 20 Figure 7B shows that in a relatively dose-dependent fashion, BNIP3 levels increase in bortezomibtreated MCF-7 and TR5 cells. An increase in BNIP3 was also observed in T-47D cells but BNIP3 levels in T-47 D cells peak by 24 h (data not shown) and is nondetectable by immunoblotting approaches at later time points. In bortezomib-treated MCF-7 cells, CHOP expression peaked at 48 h and was difficult to detect by 96 h. BNIP3 expression peaked at 48 72 h of expression, but BNIP3 protein appeared more stable because detectable levels persisted longer in the cells (Fig. 7C and data not shown). A prodeath role for BNIP3 and CHOP was confirmed with RNAi targeting experiments (Fig. 8). Control experiments determined that RNAi could downregulate CHOP and BNIP3 (Fig. 8A). In these experiments, RNAi or scrambled RNAi was administered to MCF-7 and TR5 cells for 48 h, after which E2 plus or minus bortezomib was provided to the cells for 24 and 48 h (Fig. 8A). CHOP protein levels were reduced by 24 h of RNAi treatment (Fig. 8A), but this reduction was transient and not detected by 48 h of RNAi targeting (data not shown). BNIP3 knockdown was detected by 24 h (data not shown) and still detectable 48 h following RNAi transfection (Fig. 8A). Knockdown of BNIP3 and CHOP resulted in a reduction in levels of cleaved PARP and lamin A in cells treated with E2 plus bortezomib (10 nm) for 72 h (Fig. 8B). A clear reduction in cleaved levels of lamin A was detected in cells treated for 72 h with E2 plus bortezomib plus 4-OHT only if both BNIP3 and CHOP were knocked down (Fig. 8C). At later time points, a reduction in levels of cleaved lamin A was noted with RNAi to CHOP or BNIP3 (data not shown). To confirm our sirna studies, we measured mitochondrial membrane permeability in cells treated with E2 plus 4-OHT plus bortezomib following RNAi targeting of CHOP or BNIP3 or neither. In these experiments, bortezomib induced mitochondrial membrane permeability and RNAi to CHOP significantly reduced this effect (Fig. 8D). RNAi to BNIP3 also showed a reduction in the level of bortezomib-induced mitochondrial membrane permeability (Fig. 8D), but the effect was more modest than that mediated by CHOP downregulation. These studies www.landesbioscience.com Autophagy 27

Figure 7. Bortezomib induced ER stress in ER + breast cancer cells and BNIP3 expression. ER + breast cancer cells MCF-7, T-47D and TR5 cells were incubated with E2 or E2 plus 4-OHT with or without bortezomib (1, 10, 25 and 50 nm for MCF-7 and TR5; 1, 5, 10 and 25 nm for T-47D) for 48 h. Total protein was isolated, subjected to SDS/PAGE, and transferred to PVDF membrane. Immunoblotting was conducted to determine GRP78 and CHOP levels. b-actin immunoblotting served as a loading control. (A) Bortezomib treatment induces ER stress markers GRP78 and CHOP in a dose-dependent manner. (B) Bortezomib treatment increased BNIP3 expression in MCF-7 and TR5 in a dose dependant fashion. Fold diff. refers to the difference in signal intensity of GRP78 and CHOP (A) or BNIP3 (B) relative to the E2-treated control which was arbitrarily assigned a value of 1. Data are representative of at least four separate experiments. (C) Bortezomib induced CHOP and BNIP3 in a time-dependent fashion with CHOP expression induced early (24 h) and BNIP3 expression induced at later (72 h). The time course was performed on cells that were treated with E2 plus 4-OHT in the presence of bortezomib (10 nm) for 144 h, with protein harvests performed at 6, 12, 24, 48, 72, 96, 120 and 144 h. L blockade provides an explanation for the buildup in LC3 II and p62 levels seen in bortezomib-treated cells. The fact that cathepsin B is also blocked is consistent with bortezomib s ability to block autolysosomal turnover of long-lived proteins in general. 37 Thus, minimally bortezomib is blocking cathepsin L and B, two cathepsins recently demonstrated to contribute equally to autophagy-mediated turnover in mammalian cells. 37 We further determined that blockade of cathepsin L with the cathepsin L inhibitor 1-Naphthalenesulfonyl-Ile-Trp-Aldehyde 58 led to complete attenuation of 4-OHT-induced macroautophagy in MCF-7 cells (Fig. 10A and B). These data and the fact that 4-OHT treatment induced cathepsin L activity (Fig. 9D F) provides strong evidence that 4-OHT treatment induces autophagy in MCF-7 cells via a cathepsin L-dependent mechanism. Inhibition of cathepsin L also induced CHOP expression and cleavage of lamin A and PARP in hormonally-treated MCF-7 breast cancer cells (Fig. 10C). Thus, cathepsin L-mediated autophagy appears to be prosurvival in MCF-7 cells. Discussion provide strong evidence that both BNIP3 and CHOP are contributing to the death of bortezomib-treated breast cancer cells. Bortezomib blocks cathepsin activity which is required for autolysosomal-mediated protein turnover. In order to understand how bortezomib was blocking autolysosomal turnover of proteins, we focused on cathepsin activities as potential targets. Several cathepsins have been shown to be involved in autophagy, including cathepsin L 37,56,57 and cathepsin B. 37 Cathepsin-L and -B activities were assayed in cells treated with E2 or E2 plus 4-OHT in the presence and absence of 10 nm bortezomib. In three independent experiments, bortezomib treatment reduced cathepsin B and L activity at 24, 48 and 72 h (Fig. 9A F). Blockade of cathepsin L has been shown to be involved in the autolysosomal degradation of autophagosomal membrane markers, 37 so cathepsin In this study, we have investigated the potential crosstalk between the catabolic process of autophagy and the proteasome-mediated protein degradation pathway. This study was executed due to several recent reports of an observed crosstalk between these two protein degradation pathways. 23,31,42,59,60 Based on these studies, we reasoned that if the blockade of the proteasomal pathway leads to induction of autophagy, then this may be one approach to kill ER + breast cancer cells, i.e., pushing prosurvival autophagy to prodeath autophagy, followed by autophagocytic cell death or apoptosis. Thus, we examined bortezomib s effects on autophagy using a variety of approaches including determination of the levels of the LC3 and p62 proteins by immunoblotting and immunocytochemistry, MDC sequestration, and visualization of cell morphology using electron microscopy. Finally, and most importantly, we directly determined longlived protein turnover in the breast cancer cells in response to bortezomib. This functional assay of the autolysosome has been previously described by Cogdono and colleagues 35,36 and was key 28 Autophagy Volume 6 Issue 1

Figure 8. RNAi knockdown of BNIP3 and/or CHOP attenuates bortezomib-induced MCF-7 and TR5 death. (A) RNAi targeting results in knockdown of CHOP and BNIP3 within 24 and 48 h of delivery. (B and C) RNAi knockdown of BNIP3 and/or CHOP attenuates bortezomib-induced cell death in E2-treated (B) and E2 plus 4-OHT-treated breast cancer cells (C), with a measurable reduction in cleaved lamin A and cleaved PARP. Fold diff. refers to the difference in signal intensity of CHOP and BNIP3 (A) or cl PARP and cl lamin A (B and C) relative to the E2 plus Bortezomib or E2 plus 4-OHT plus bortezomib treatment groups which was arbitrarily assigned a value of 1. (D) RNAi knockdown of BNIP3 or CHOP attenuates bortezomib induced mitochondrial depolarization in E2 plus 4-OHT-treated. *, indicates statistical significance determined by using the student s t test. Statistical significance was defined as p value of <0.05 (compared to the % mitochondrial depolarization in the control group, i.e., treatment with E2 plus bortezomib plus 4-OHT plus scrambled RNAi). For RNAi studies, cells were transfected with BNIP3 and/or CHOP RNAi or non-targeting control RNAi for 48 h, and then treated with E2 in presence of bortezomib (10 nm) and/or 4-OHT. Total protein was isolated from cells at 24 h and 48 h (A), 72 h after E2 plus bortezomib treatment (B), and 72 h after E2 plus 4-OHT plus bortezomib treatment (C and D). Immunoblotting for CHOP, BNIP3, PARP, cleaved lamin A, and β-actin (A C) and the mitochondrial depolarization assay (D) were conducted as described in Materials and Methods. to deciphering results of this study and concluding that bortezomib blocked autophagy-mediated catabolism, in addition to the known inhibitory effects of bortezomib on the proteasomal pathway of degradation. This study clearly showed a robust death response induced by bortezomib at relatively early time points (24 h for T-47D, 48 h for MCF-7, and 72 h for TR5 cells). Following bortezomib treatment, we demonstrated a time-dependent induction of CHOP and BNIP3, with BNIP3 expression occurring later and being more sustained in the ER +, antiestrogen-sensitive MCF-7 and antiestrogen-resistant TR5 cells. Concomittant with bortezomib-induced CHOP induction, an increase in mitochondrial dysfunction was reproducibly detected. Using an RNAi targeting approach, we demonstrated that both CHOP and BNIP3 contributed to the death response. Importantly, knockdown of CHOP via sirna targeting completely blocked bortezomib-induced mitochondrial permeability. In contrast, BNIP3 downregulation did not effectively block bortezomib-induced mitochondrial permeability. Nor were BNIP3 levels affected by CHOP downregulation, so BNIP3 does not appear to be a downstream effector of CHOP. Also, BNIP3 expression consistently occurred ~24 h later than CHOP expression following bortezomib treatment. In unpublished studies, we have recently demonstrated that chloroquine treated MCF-7 cells show upregulated BNIP3 expression, littleto-no induction of CHOP expression, and cell death but significantly less than that seen with bortezomib treatment (Raney et al. manuscript in preparation). It is interesting to speculate that bortezomib induces two cell death pathways, a BNIP3-mediated death pathway that specifically kills autophagocytic cells, and a CHOP-mediated pathway that kills nonautophagocytic cells, but www.landesbioscience.com Autophagy 29

Figure 9. Bortezomib attenuates cathepsin B and L activity: MCF7 (8 x 10 5 cells) were seeded, treated, harvested at the indicated times, and assayed as described in Materials and Methods. (A C) Cathepsin B activity is blocked by bortezomib at 24 h, 48 h and 72 h respectively. (D F) Cathepsin L activity is increased by 4-OHT, but blocked by bortezomib treatment at 24 h, 48 h and 72 h respectively. Fold-increase in Cathepsin B and L activity was determined by comparing the relative fluorescence units (RFU) per μg protein with the level of the E2 treated samples. All experiments were performed in triplicates. Values are expressed as mean ± S.D. (n = 3). Statistical significance was determined by the student s t test: *p value of <0.05; **pvalue of <0.001. Comparisons are made between the following treatment groups as follows: a, designated treatment compared to E2; and b, designated treatment compared to E2 plus 4-OHT. this speculation will have to be rigorously tested, particularly considering that the mechanism of UPR induction by proteasome inhibition is complex, with multiple ER transmembrane signaling molecules impacting transcription and translation, including transcription factor 6 (ATR6), PERK and IRE1. 61,62 The significance and mechanism of BNIP3 upregulation is currently under investigation in our laboratory. As part of these ongoing studies, we will attempt to resolve discrepancies in the role of BNIP3 and address the role of BNIP3L since both of these proteins have recently been shown to be prosurvival. 63 One possibility is that BNIP3 will induce death, not autophagy, if autolysosomal function is blocked as occurs with bortezomib treatment. The bortezomib-induced death response was preceded by a discernable increase in the levels of LC3 and p62 in MCF-7, T-47D and TR5 breast cancer cells treated with bortezomib. The lipidated 16 kd LC3 is typically induced by effectors of autophagy such as starvation, 42,64 but is also turned over once the autophagosomes fuse with lysosomes. 23,60 Thus, LC3 accumulation fails to differentiate between induction of autophagy or a blockade in autolysosomal turnover that would prevent cellular constituents and proteins from being degraded within the autophagosomes. 23 The p62 protein, on the other hand, is not typically induced by autophagy but is turned over in the autolysosome. 41-43 Thus, the build up in the levels of both of these proteins for a prolonged period was suggestive of a defective autophagy pathway. Mechanistically, one possibility is that bortezomib is blocking fusion of the autophagosome with the lysosome; however, electron microscopic examination did not clearly demonstrate such an effect. Another possibility is that bortezomib has off-target effects on specific autophagy proteins. However, we also utilized MG-132 and lactacystin in this study and obtained similar results. These two proteasome inhibitors are quite different from bortezomib and would not necessarily have similar nonspecific, off-target effects. 26,28,65 Based on a previous study, which had determined that 30 Autophagy Volume 6 Issue 1

Figure 10. Inhibition of cathepsin L blocks macroautophagy in MCF-7 breast cancer cells. MCF-7 cells were seeded and treated with E2, E2 plus 4-OHT, serum-starvation or rapamycin in the absence or presence of cathepsin L inhibitor (20 μm). Proteolysis studies are shown in (A and B), for which cells were harvested 24 and 48 h after treatment, respectively. Values are expressed as mean ± S.D. (n = 3). (C) Immunoblotting determined levels of CHOP, cleaved lamin A and cleaved PARP. For these studies, cells were harvested 96 h after treatment. (A and B) Comparisons are made between the following treatment groups as follows: (a) E2 verus E2 plus 4-OHT; (b) E2 plus 4-OHT versus E2 plus 4-OHT plus cathepsin L inhibitor; (c) E2 plus rapamycin versus E2; (d) E2 plus rapamycin versus E2 plus rapamycin plus cathepsin L inhibitor; (e) serum-starved versus E2; (f) serum starved versus serum-starved plus cathepsin L inhibitor. The symbols *, and ** represent statistical significance: at p < 0.01 and p < 0.001, respectively. (C) Fold diff., refers to the difference in signal intensity of cl lamin A or cl PARP relative to the E2 treatment group which was arbitrarily assigned a value of 1. MG-132 blocks cathepsin activity, 66 we analyzed the effects of bortezomib on cathepsins. We focused on cathepsin L and B, which are now known to be involved in autophagy 37,56,57,67 and demonstrated that bortezomib blocked cathepsin L and B activity. Also, we utilized an inhibitor of cathepsin L, and demonstrated that inhibition of cathepsin L activity blocked macroautophagy function (long-lived protein turnover) and induced cell death in ER + breast cancer cells. Thus, our data show that bortezomib blocks cathepsin L activity and that cathepsin L activity may be key to the prosurvival autophagy in ER + breast cancer cells previously described by us 20 and others. 19,21 Such a prosurvival role for cathepsin L is being appreciated for other cancer cells 57 and cathepsin L activity has recently been shown to be key to cancer cell drug resistance. 68 In addition, it should be noted that both cathepsin L and cathepsin B have been shown to be upregulated in human breast cancer and to potentially serve as prognostic factors for tumor recurrence in human breast cancer. 69,70 Further, the vacuolization that we have shown to occur during bortezomib treatment may result from an imbalance in cathepsin activity 57 and/or dilation of the ER membrane. 71 We currently are determining the origin of the vacuolization and the contribution of cathepsin blockade to this characteristic of bortezomib-treated ER + breast cancer cells. The results in this study are in contrast to a recent study in which high-dose bortezomib treatment of MCF-7 cells showed no induction of cell death. 72 This apparent discrepancy can be explained by the different media used in our study. In the study by Xu et al. 72 the authors use DMEM-F12 medium supplemented with 10% FBS, insulin, and EGF. We have analyzed the effects of insulin, EGF and IGF1 in MCF-7 and T-47D breast cancer cells undergoing antiestrogen therapy and determined that these growth factors effectively block cell death and autophagy (Periyasamy-Thandavan S, et al. manuscript in preparation). Thus, media composition can affect outcome of treatments delivered in vitro and potentially mimic different environmental influences in vivo. The proposed potential of bortezomib plus SERM therapy versus SERM therapy alone will be better tested in our ongoing in vivo studies. In summary, this study underscores the need to execute proteolysis studies to determine the functionality of autolysosomes. Analysis of LC3 and p62 levels as well as induction of LC3 cytosolic punctate staining, MDC sequestration, and EM quantitation of autophagosomes were not adequate by themselves as measures of the catabolic functionality of autophagy during bortezomib treatment. In addition, this study provides strong data supporting a potential use of proteasome inhibitors like bortezomib as an approach to induce caspase-dependent apoptosis in breast cancer cells undergoing cathepsin-dependent, prosurvival autophagy during hormonal therapy. We are aware of clinical studies that have demonstrated the efficacy of bortezomib and other proteasome inhibitors against breast cancer. 73 However, in these clinical studies, the breast cancers were end-stage breast cancers that failed conventional therapies such as antiestrogen treatment and/ www.landesbioscience.com Autophagy 31