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The postnatal mammary gland undergoes repeated cycles of proliferation and cell death, most notably when the fully differentiated (lactating) gland dedifferentiates to a prelactation state. Accumulation of milk proteins in the secretory epithelium creates the stress signal that triggers this process (involution). How this stress is perceived, and the cellular processes that are subsequently activated, remain unclear. We now report that Unfolded Protein Response (UPR), autophagy, and apoptosis related genes cluster separately during lactation and involution in the mouse mammary gland. Time-course experiments in rodents show that autophagy and UPR signaling are tightly co-regulated at the transition from reversible to irreversible involution. Inhibition of autophagy by chloroquine or genetic deletion of one ATG7 allele enhanced progression of mammary involution into the irreversible phase, as characterized by an early/ precocious induction of apoptosis. These are the ﬁrst preclinical in vivo data in support of a clinical trial testing an autophagy inhibitor for prevention of intraductal breast malignancy progression to invasive breast cancer. In marked contrast, stimulation of autophagy by low dose tunicamycin treatment reduced apoptosis and extended the reversible phase of involution by sustaining the secretory epithelium. Autophagy stimulators could be used short-term to promote lactation in women experiencing difﬁculties or irregularities in nursing. Taken together, these data indicate that UPR and autophagy play a key role in regulating the balance between cell survival and apoptosis during normal mammary gland regression. Introduction by which the terminally differentiated gland transitions, The mammary gland is a unique tissue in its ability to through de-differentiation, to a quiescent state similar to 1,2 undergo repeated cycles of cell proliferation, differentia- the prepregnancy gland . This process is of particular tion, death, and tissue remodeling during and after pub- interest, since mammary involution has been implicated erty, and during the process of involution that occurs after as a key contributor to pregnancy-associated breast can- 3,4 5 cessation of lactation. Involution is a normal process cer , and a higher metastatic potential . Involution can be divided into two distinct phases: the ﬁrst phase, 0–48 h (in mouse), is initiated by local factors triggered by milk Correspondence: Robert Clarke (firstname.lastname@example.org) 6,7 accumulation in the gland due to cessation of suckling . Department of Oncology and Lombardi Comprehensive Cancer Center, Cell and tissue architecture are maintained, involution Georgetown University Medical Center, Washington, DC 20057, USA Institute of Biomedicine, University of Turku Medical Faculty, Turku 20014, is reversible, and lactation can resume with suckling. Finland The second phase (48–144 h in mouse) is regulated by Full list of author information is available at the end of the article. These authors contributed equally: Anni Wärri, Katherine L. Cook Edited by N. Barlev © 2018 The Author(s) Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Ofﬁcial journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Wärri et al. Cell Death Discovery (2019) 5:40 Page 2 of 13 systemic hormones and is irreversible; tissue architecture dimerizes, autophosphorylates, and its unique endonu- is destroyed and a robust remodeling is accompanied clease becomes active and, among other functions, by adipogenesis . Apoptosis occurs during both phases, removes a 26 nucleotide base pair fragment from XBP1. triggered by detachment of luminal epithelial cells from This unconventional splicing action forms trancription- the basement membrane . ally active XBP1-Spliced (XBP1-S). XBP1-S then pro- IPK Autophagy has gained increasing attention in cancer motes the transcription of p58 (negative feedback research, including breast cancer, as a key mechanism loop to inhibit PERK signaling), lipid biogenesis proteins, cancer cells use to cope with cellular stress and produce and EnR-associated protein degradation components. energy . In contrast, in normal mammary gland phy- The UPR signaling cascade can also occur ﬁrst with siology, the role of autophagy as a cell survival or cell PERK activation, followed by ATF6 cleavage, and lastly 11,12 death process has remained controversial . Autophagy XBP1-S formation. In the lactating mammary gland, 15 20 has been implicated in the formation of growing ducts UPR pathways PERK-eIF2α-ATF4-CHOP and XBP1 during early mammary gland development, and in the have been implicated in prosurvival and lipogenic func- mature gland during the formation of epithelial acini tions, respectively. 13 9 in both the bovine and murine mammary glands . In this study, we used published gene expression In Beclin (Becn)1 null mouse embryos, widespread cell microarray datasets from different phases of mouse death occurs and is embryonic lethal , suggesting mammary gland development, including lactating and a prosurvival role for autophagy during early develop- involuting samples, and analyzed the expression of +/− ment. Becn1 heterozygote mice develop spontaneous apoptosis, autophagy, and UPR genes. We obtained tumors and mammary hyperplasias, consistent with a involution time-course samples from wild-type mice haploinsufﬁcient tumor suppressor role . In contrast, using forced weaning. Mice also were treated with and increased Becn1 expression has been described at the without drug interventions: autophagy stimulating (tuni- end of lactation in the adult mammary gland and inter- camycin), inhibiting (chloroquine) and control treat- preted to imply a cellular prodeath role . During invo- ments. We also used a genetic mouse model of ATG7 9,15 +/− +/+ lution, autophagy may exhibit either a prosurvival or haploinsufﬁciency (Atg7 vs. Atg7 ). We show here, prodeath function . to our knowledge for the ﬁrst time, that UPR and A complex interaction exists among autophagy, the autophagy are essential for survival of the terminally unfolded protein response (UPR), and cell fate outcomes differentiated mammary epithelium in the initial (rever- 17,18 in breast cancer sible) phase of involution by restraining apoptosis-driven . UPR, which is induced by the accumulation of unfolded/misfolded proteins in the irreversible changes and regression of the epithelium. endoplasmic reticulum (EnR), can regulate autophagy. Accumulation of milk proteins in the secretory mammary Results epithelium may create the stress signal that triggers PCA revealed separation of different time points in involution, perhaps through the integration of UPR involution process signaling, autophagy, and apoptosis in mammary gland To study the expression of UPR, autophagy, and apop- involution. An increase in unfolded/misfolded proteins tosis genes in the mammary gland, ﬁrst we searched in the EnR lumen activates the UPR by causing the dis- published gene expression microarray datasets obtained sociation of glucose regulated protein 78 (GRP78) from at different stages of (mouse) mammary development; the three UPR signaling arms: PKR-like endoplasmic only studies containing involution with several data 21,22 reticulum kinase (PERK), activating transcription factor points (no pooled samples) were selected and ana- 6 (ATF6), and inositol-requiring enzyme 1 (IRE1) . lyzed as presented in Materials and methods. We assigned Once released from GRP78, PERK dimerizes and autop- these genes to different groups based on their pathway hosphorylates to become active, resulting in phosphor- membership as annotated in Gene Ontology (GO; ylation of eIF2α and a halt in cap-dependent protein Autophagy Gene Ontology GO:0006914, Apoptosis Pro- translation, favoring ATF4 synthesis. Activated ATF4 cess GO:0006915 and Response to unfolded protein regulates the transcription of several genes including GO:0006986; Table 1; Supplementary Table S2). Principal GRP78, autophagy-related gene 12 (ATG12), and the component analysis (PCA) shows that UPR (internal), proapoptotic protein DNA-damage-inducible transcript UPR (downstream), autophagy, and apoptosis genes, 3 (DDIT3, also known as GADD153 or CHOP). ATF6 especially the ﬁrst three, clustered within their own translocates from the EnR to the Golgi complex where functional group in both datasets during lactation- site 1 and site 2 proteases cleave ATF6 to produce its involution phase (Fig. 1a, b). While the listing of some transcriptionally active form (cleaved ATF6) that pro- individual genes in more than one pathway (Table 1) motes transcription of X-box binding protein 1 (XBP1) likely reﬂects areas of signaling/pathway cross talk and GRP78. When activated by release from GRP78, IRE1 (as these pathways are known to cross talk) and makes Ofﬁcial journal of the Cell Death Differentiation Association Wärri et al. Cell Death Discovery (2019) 5:40 Page 3 of 13 Table 1 List of genes assigned to different groups based epithelial/adipose tissue area in the late involution glands on their pathway membership as annotated in Gene (96 h, 7 d). Concurrently, the appearance of apoptotic Ontology cells was visualized by terminal deoxynucleotidyl trans- ferase dUTP nick end labeling immunohistochemistry Gene Apoptosis Autophagy UPR (TUNEL IHC) (Fig. 2b). Adult virgin mammary glands (GO:0006915) (GO:0006914) (GO:0006986) and those from estradiol (E2) treated mice were used as negative controls. E2 treatment was used to inhibit ATF6 ATF6 ATF6 apoptosis, which occurs at low levels in the normal DDIT3 DDIT3 DDIT3 mammary glands of estrus-cycling mice. As shown in EIF2AK3 EIF2AK3 EIF2AK3 Fig. 2b, signiﬁcantly increased TUNEL staining was ATF4 ATF4 ATF4 detected at 48–72 h of involution, indicating a potent induction of apoptosis. An increase in CD68 staining EIF2A EIF2A (Fig. 2c) coincides with increased TUNEL staining, HSP90B1 HSP90B1 HSP90B1 showing that macrophages accompany the increasing HSPA5 HSPA5 HSPA5 number of dying cells in the alveolar lumens. Expression XBP1 XBP1 XBP1 XBP1 of proteins known to regulate and execute apoptosis (BCL-W, BCL-XL, Cleaved caspase-7, cleaved PARP; AMBRA1 AMBRA1 AMBRA1 Fig. 2d) also showed a temporal pattern. Expression of ATG12 ATG12 BCL-family members peaks early (at 24 h), whereas that ATG5 ATG5 ATG5 of caspases peak after onset of the irreversible phase of ATG7 ATG7 ATG7 involution (at 48–72 h; Fig. 2d). BCL2L1 BCL2L1 Autophagy, UPR, and apoptosis signaling components BCL2L2 BCL2L2 have a distinct temporal pattern during involution BECN1 BECN1 BECN1 We next investigated the expression of autophagy reg- MCL1 MCL1 MCL1 ulators relative to the progress of involution and apop- tosis. As shown by quantitative reverse transcription MTOR MTOR MTOR polymerase chain reaction (qRT-PCR) (Fig. 3a) and/or TSC2 TSC2 TSC2 Western (Fig. 3b) analysis, during the ﬁrst phase of BCL2 BCL2 BCL2 involution autophagy markers (low p62; high LC3-II) SQSTM1 SQSTM1 SQSTM1 coincide with peak expression of the known autophagy regulators Beclin-1, Atg7, Atg12, and pAMPK. The TSC1 TSC1 expression of Autophagy/Beclin-1 Regulator Ambra peaks 21, 22 Genes from two independent gene expression microarray data sets were later, at 72 h (Fig. 3a). IHC data for p62 speciﬁc staining acquired and analyzed as described in the Materials and methods and Supplementary Table S2, and a gene signature of 21 genes was selected. The (Fig. 3c) and autophagy speciﬁc LC3-GFP punctate for- GO terms for Autophagy Gene Ontology GO:0006914, Apoptosis Process mation in identical involution samples from LC3-GFP GO:0006915 and Response to unfolded protein GO:0006986. transgenic mice (Fig. 3d) are in accordance with p62 gene and LC3-II protein-expression quantiﬁcations. it more difﬁcult to fully separate the processes in only To verify further the temporal regulation of involution 3D, additional visual angles (Supplementary Fig. S2) we measured expression of the known upstream reg- show clustering of the apoptosis genes. ulators of autophagy, the UPR genes Grp78, Atf4, and Atf6, Xbp1 (unspliced form), phospho-eIF2a, and Chop/ Involution progress is deﬁned by changes in tissue Ddit3. As shown by qRT-PCR analysis (Fig. 4a), expres- architecture, apoptosis, and macrophage inﬁltration sion of the Grp78, Atf4, Atf6, and Xbp1 mRNAs peak To explore the extent of apoptosis and the possible during the ﬁrst phase of involution (24–48 h), while Chop/ contribution of UPR and autophagy during involution, Ddit3 mRNA expression peaks during the second phase we conducted a time-course experiment where involution (96 h). Protein expression by Western analysis (Fig. 4b) was initiated by forced weaning after 10 days (peak) shows the highest protein level of GRP78 and phospho- of lactation. Samples were collected at 0–96 h, and 7 d. eIF2a at 24 h, while ATF4 and CHOP/DDIT3 expression We ﬁrst established a timeframe of involution progression increase later (at 72 h and 72 h–7 d, respectively). Analysis 21,22 for our mouse model (Fig. 2a and Supplementary Fig. S1). of the published data sets showed both Xbp1 and We then established a quantitative grading for the Rela mRNA expression to be elevated during involution, early involution samples to accurately stage progression compared with virgin, pregnant, and lactating mammary (24–72 h; Fig. S1), and used ImageJ quantiﬁcation of the glands (Supplementary Fig. S3). IHC analyses show an Ofﬁcial journal of the Cell Death Differentiation Association Wärri et al. Cell Death Discovery (2019) 5:40 Page 4 of 13 21, 22 Fig. 1 Principal component analysis (PCA) of gene expression microarray data sets. Two published gene expression array data sets (A, B ) were used. Data from these studies were derived from nonpooled samples. Multiple time-points of mouse mammary gland developmental stages, including lactation and involution, were analyzed. Datasets were acquired and analyzed as explained in Materials and methods. PCA analysis was performed on involution samples for a 21 gene proﬁle representing apoptosis, autophagy, and UPR (Table 1; Supplementary Table S2) on each data set (a, b). A three-dimensional biplot shows the separation of apoptosis, autophagy, and UPR/downstream of UPR genes expressed at the different time points during mammary gland involution. Additional visual angles to show clustering of apoptosis genes better are presented in Supplementary Figure S2 early expression of GRP78 (Fig. 4c), while strong CHOP/ administration produced an enhanced onset of apoptosis and advanced initiation of the irreversible phase of invo- DDIT3-speciﬁc staining is apparent at later time points (Fig. 4d). Taken together, the time-course analyses of UPR lution (Fig. 5a, c, d middle panels). Quantiﬁcation mRNA and protein expression during mammary gland of changes in mammary gland histology in the Tm and involution show that their expression is tightly orche- CQ treatment groups is shown as fold-change relative strated, occurring concurrent with the expression of to control (vehicle treated mice; Fig. 5b). Taken together, autophagy genes and prior to expression of caspases the results show that autophagy is critical for regulating and cleaved PARP, hallmarks of the execution of apop- the progress of involution during its transition from the totic cell death. reversible to the irreversible phase. We also created an involution time-course, using an +/− 23 Autophagy and apoptosis are co-regulated during ATG7-deﬁcient (Atg7 ) mouse model , and measured involution changes in autophagy (p62 IHC, Fig. 6a, b, middle panels) To conﬁrm a mechanistic relationship for the link and apoptosis (TUNEL IHC, Fig. 6a, b, bottom panels) between autophagy and apoptosis during involution, we in mammary glands during their transition from the +/− performed drug intervention studies to either inhibit ﬁrst to the second phase of involution (24–72 h). Atg7 +/+ (daily chloroquine treatment) or stimulate autophagy mice were compared with their Atg7 littermate mice, (daily tunicamycin treatment). We recorded changes in and the experiment was performed as described for the the progress of involution relative to vehicle treated mice, involution models above. Progress of involution was based on grading of mammary gland histology (Fig. 5a) measured by quantiﬁcation of epithelium/fat pad area and quantiﬁcation of epithelium/adipose tissue area in the mammary glands at 96 h and 7 d of involution (Fig. 5b), autophagy (p62 IHC, Fig. 5c), and apoptosis (Fig. 6c, d). Strikingly, the progress of involution and +/− (TUNEL IHC, Fig. 5d). Results establish that low dose apoptosis in the mammary glands of Atg7 mice, +/+ tunicamycin (Tm), a commonly used pharmacologic when compared with their Atg7 control mice, were inducer of the canonical EnR stress response, signiﬁcantly enhanced as also seen when chloroquine treated mice delayed the appearance of, and decreased the number where compared with their vehicle treated controls. of, apoptotic cells (Fig. 5a, c, d bottom panels). In con- These data further highlight a critical role for autophagy trast, inhibiting autophagy with chloroquine (CQ) in the regulation of mammary gland involution. Ofﬁcial journal of the Cell Death Differentiation Association Wärri et al. Cell Death Discovery (2019) 5:40 Page 5 of 13 Fig. 2 Progression of mammary gland involution based on changes in tissue architecture, apoptosis, inﬁltrating magrophages, and the expression of apoptosis proteins. Involution time course was created and samples collected as described in the Materials and methods. Mammary glands of virgin mice ± 17β-estradiol (E2) were used as negative controls; E2 treatment was used to block apoptosis. a Progression of involution was assessed based on tissue histology in the H&E stained slides and quantiﬁed in early involution (24–72 h) by grading of the H&E slides (shown in Supplementary Fig. S1) and in late involution (96 h, 7 d) by using ImageJ analysis of epithelial/adipose tissue area of the H&E stained slides (see Materials and methods); all slides were photographed using Olympus BX 61 microscope and 10× magniﬁcation (A–C). b Detection of apoptotic cells by TUNEL IHC. c Macrophage inﬁltration visualized by CD68 staining (IHC). d Expression of the known apoptosis markers and regulators BCL-W, BCL- XL, Cleaved caspase-7, Cleaved PARP proteins were measured by Western analysis. Results at each time point show average ± SD, n = 3 Discussion differentiated mammary gland returns to a prepregnancy Pathways that control common house-keeping func- state resembling the virgin gland. The process involves tions in normal cells are often activated in cancer cells, a complex and robust period of programmed cell death including breast cancer, a phenomenon termed “non- and changes in tissue architecture that include extra- 24 1,6,7 oncogenic addiction” . Several of these processes con- cellular matrix (ECM) remodeling and adipogenesis . tribute to normal mammary gland involution including Reﬂecting the ability of the normal adult mammary gland 2,8,22,25 apoptosis, inﬂammation, and wound healing . to undergo robust de-differentiation and tissue remodel- Involution is a normal process initiated after pregnancy, ing, mammary involution has been implicated as a driver without or following an intervening period of lactation of ductal carcinoma in situ (DCIS), and may be a key 3,4 (breast feeding), during which the terminally contributor to pregnancy-associated breast cancer . Ofﬁcial journal of the Cell Death Differentiation Association Wärri et al. Cell Death Discovery (2019) 5:40 Page 6 of 13 Fig. 3 Evaluation of autophagy in the mammary gland involution. a Expression of the autophagy genes Atg7, Atg12, Ambra-1, Beclin-1, and p62 was analyzed by qRT-PCR. Lactating mammary glands (involution 0 h) were used as controls; the expression of each gene at each time point is presented as fold-change relative to control. b Western analysis of the autophagy proteins BECLIN-1, ATG7, LC3-II, and pAMPK are shown. The horizontal black bars/arrows show the involution switch from reversible to irreversible stage, which has occurred by 72 h after forced weaning (A, B). The vertical bar indicates the involution 0 h (B, Western blot inset). Results at each time point show average ± SD, n = 3. c Autophagy marker, (downregulation of) p62 speciﬁc staining (IHC) at different time points during involution and in virgin control mammary glands. d H&E staining (upper panel) and autophagy speciﬁc LC3-GFP punctate formation (lower panel) in identical involution samples from LC3-GFP transgenic mice. All slides were photographed using Olympus BX 61 microscope and 10× magniﬁcation (C, D) In rodent models, multiple studies have shown involution haploinsufﬁciency results in enhanced/precocious invo- to enhance tumor cell growth, local invasion, and lution through enhanced apoptosis. 5,26 metastasis . To date, studies have focused mostly on Comprehensive global gene expression analyses of genes the role of apoptosis and classical/nonclassical pathways differentially expressed during mammary development 27,28 of cell death in mammary involution . We now show have previously been used to identify involution-associated that the UPR regulates autophagy (macroautophagy), genes that may contribute to breast tumorigen- 8,21,22,29,30 and that both activities are co-regulated with apoptosis esis . Here, we used only data sets that included during involution. These integrated activities provide a several involution time points and without pooling tissue 21 22 key survival component that regulates transition from the samples (C57Bl/6 mice ; BALB-c mice ). Since involu- reversible to irreversible phases (“involution switch”) early tion is triggered by the increased cellular stress that in the initiation of mammary gland involution. Using arises from milk accumulation in the secretory epithelium normal mouse mammary tissue, we establish that autop- after cessation of nursing , we focused on UPR genes and hagy stimulation with tunicamycin treatment leads to its downstream signals. The UPR is activated by an inap- prolonged persistence of secretory mammary epithelium, propriate accumulation of proteins in the endoplasmic while inhibition of autophagy with chloroquine or ATG7 reticulum, and we have previously shown how the UPR Ofﬁcial journal of the Cell Death Differentiation Association Wärri et al. Cell Death Discovery (2019) 5:40 Page 7 of 13 Fig. 4 Evaluation of UPR in the mammary gland involution. a Expression of UPR genes Grp78, Atf4, Atf6, Xbp1 (unspliced form), and Chop/Ddit3 was analyzed by qRT-PCR. Lactating mammary gland (involution 0 h) was used as a control, and the expression of each gene at each time point is presented as fold-change relative to control. The horizontal black bar/arrow shows the involution switch from reversible to irreversible stage, which has occurred by 72 h after forced weaning (A, B). b Western analysis of UPR proteins GRP78, ATF4, ATF6, XBP1 (unspliced form), phospho-eIF2a, and CHOP/DDIT3. The vertical bar indicates the involution 0 h (B, Western blot inset). Results at each time point show average ± SD, n = 3. c GRP78 and d CHOP/DDIT3-speciﬁc staining (IHC) at different time points during involution and in virgin control mammary glands. All slides were photographed using Olympus BX 61 microscope and 10× magniﬁcation (C, D) 19,31 can activate autophagy . In both data sets, we found PARP (Fig. 2d). Increased expression of anti-apoptotic differential expression of UPR, autophagy, and apoptosis BCL2 family members was detected early, prior to genes when compared with nonpregnant (virgin) mam- increased TUNEL staining and expression of the proa- mary glands (Supplementary Table S2). Principal compo- poptotic proteins. Our results are consistent with obser- nent analysis (PCA) of the differentially expressed genes vations from Bcl-2 overexpressing and Bcl-XL knockout clearly shows that apoptosis, autophagy, and UPR and its mice indicating a key protective role for BCL-2 family 35–37 downstream genes were present in separable clusters members in the regulation of involution . deﬁned by the top three principal components (Fig. 1a, b; Controlled inﬂux of macrophages and other immune Supplementary Fig. S2). Thus, these genes are expressed in cell types, to eliminate the dead and dying mammary cells, 38,39 a temporally orchestrated fashion relative to the progress is a central component of involution . Depletion of of involution. To validate these observations indepen- macrophages leads to delayed postpartum involution dently, we applied an established mouse model of forced (measured at day 3 ). We show that increased inﬁltration involution and created a time course of mammary gland of macrophages coincided with the peak in cell death samples. Consistent with previous studies, tissue archi- (highest TUNEL positivity) after 48–72 h of involution, tecture (assessed in histological sections; Fig. 2a, Supple- reﬂecting initiation of the irreversible phase of involution mentary Fig. S1) and the extent of apoptosis (Fig. 2b) (Fig. 2c). 32–34 conﬁrmed the two distinct phases of involution .The We hypothesized that, through UPR signaling, autop- number of apoptotic cells peaked at the beginning of the hagy could provide a key survival component in the early irreversible phase of involution (48–72 h), concurrent phase of mammary gland involution, during which tissue with increased levels of cleaved caspase-7, and cleaved architecture is preserved and involution is still reversible. Ofﬁcial journal of the Cell Death Differentiation Association Wärri et al. Cell Death Discovery (2019) 5:40 Page 8 of 13 Fig. 5 Drug interventions inhibiting and stimulating autophagy enhanced and delayed involution, respectively. At the time of forced weaning (involution 0 h) drug treatments were started to inhibit (with low dose chloroquine [CQ], middle panels) and stimulate (with low dose tunicamycin [Tm], bottom panels) autophagy, as described in the Materials and methods. Time-course involution samples were collected as in Figs. 2–4. a, b Histology and quantiﬁcation of involution progress by ImageJ analysis of epithelial/fat pad area of the H&E stained mammary gland slides, as described in Materials and methods. Average ± SD are shown, n = 3. Tm vs. vehicle control: Student’s t test, P ≤ 0.001 (96 h); Mann–Whitney rank sum test P ≤ 0.001 (7d). CQ vs. vehicle control: Mann–Whitney rank sum test P ≤ 0.001 (72 h). c Representative slides of (c) autophagy marker (downregulation of) p62 speciﬁc staining (IHC), and d apoptosis marker TUNEL IHC are shown A physiologically critical window would thereby be protein expression (IHC) in the mammary epithelium maintained during which lactation can resume if suckling was increased up to 96 h after initiation of involution, restarts. Thus, any temporary interruption in lactation suggesting a decrease in autophagy during the irreversible would not lead to the end (failure) of nursing and pos- phase of mammary gland involution (Fig. 3c). These sible death of the offspring. We focused on the signaling results appear consistent with earlier observations events around the “involution switch”, when reversible showing induction of autophagy by the detachment of involution becomes irreversible (after 48 h in mouse), and luminal cells from their extracellular matrix ,and 9,15 40,41 show an orchestrated regulation of autophagy and UPR autophagy protecting mammary epithelial ,DCIS , 10,42 genes after 24–96 h of mammary involution initiation. and breast cancer cells from anoikis. We observed a decline in p62 expression levels (Fig. 3a), Other observations suggest that autophagy can con- upregulation of LC3-II protein expression (Fig. 3b), and tribute to both prosurvival and prodeath outcomes during 9,15 16 increased LC3-GFP punctate formation in IHC (Fig. 3d) mammary involution (refs. vs. ref. ). Here, we show at 24–48 h compared with those obtained 72 h post- that signaling through UPR regulates both functions of weaning, indicative of activated autophagic ﬂux. Upre- autophagy (prosurvival; prodeath), but in a sequential and gulation of phosphorylated AMPK and decreased TORC1 orchestrated fashion. Results of time course experiments complex (preliminary data) expression show that the showed elevated Grp78 (Fig. 4a, b), Xbp1 (unspliced form; promotion of autophagy in the reversible phase of invo- Fig. 4a), and phosphorylated eIF2α (Fig. 4b) expression lution might be regulated by mTOR inhibition. p62 24–48 h postweaning, the time during which involution Ofﬁcial journal of the Cell Death Differentiation Association Wärri et al. Cell Death Discovery (2019) 5:40 Page 9 of 13 +/+ Fig. 6 Enhanced involution in autophagy gene deﬁcient mice. An involution time course was created using wild type (Atg7 ) and Atg7 +/− heterozygous (Atg7 ) mice. Samples were collected as in Figs. 2–4 and as described in the Materials and methods. a, b H&E staining (top panels), +/+ +/− p62 IHC (middle panels), and TUNEL IHC (bottom panels) of mammary glands of Atg7 and Atg7 mice after 24 h, 48 h, and 72 h of involution. c, d Quantiﬁcation of epithelial/adipose tissue area of the H&E stained slides as in Fig. 2, and as described in the Materials and methods, and in late involution (96 h, 7 d) by using ImageJ analysis, as described in the Materials and methods. Average ± SD are shown, n = 3–5. Student’s t test, P = 0.026 (96 h), P = 0.001 (7 d) is reversed if suckling resumes. However, stimulation time of forced weaning accelerated involution (as deter- of Atf4 and Chop/Ddit3 occurred later, at 72–168 h mined by H&E, Fig. 5a middle panel, Fig. 5b) and apop- of involution (Fig. 4a, b). Overexpression of ATF4 can tosis (Fig. 5d middle panel), implying that autophagy lead to impaired lactation and accelerated involution promotes survival and is a vital signaling component marked by increased apoptosis, suggesting that increased of the reversible phase of involution. The increase in ATF4 promotes cell death in the mammary gland . p62 expression conﬁrms the inhibition of autophagy by ATF4 stimulates the proapoptotic protein, CHOP/ chloroquine (Fig. 5c middle panel). The opposing inter- DDIT3 , and we found that CHOP/DDIT3 expression vention, where lactating mice are treated with tunicamy- increased 48–168h after forced weaning and correlated cin starting at the time of forced weaning, reduced, and/or with increased cell death (Fig. 4a, b, d). Our results delayed apoptosis (Fig. 5d, bottom panel) and sustained highlight the timing and duration of UPR-autophagy autophagy, seen as low expression of p62 (Fig. 5c bottom signaling molecules/pathways contributing both to pro- panel). Thus, prolonged autophagy can extend the survival and prodeath outcomes in response to cellular reversible phase of mammary gland involution. These stress in normal mammary tissue. data imply a possible translational application of autop- To verify a causal relationship between autophagy and hagy stimulation to promote and prolong lactation in apoptosis in the progression of involution, we performed women with difﬁculties or irregularities in breast feeding, drug intervention experiments to both block (with especially in developing countries where mother’s milk chloroquine) and enhance (with tunicamycin) autophagy. production provides the primary source of babies’ nutri- Dosing lactating mice with chloroquine starting at the tion. In contrast, chloroquine administration likely has an Ofﬁcial journal of the Cell Death Differentiation Association Wärri et al. Cell Death Discovery (2019) 5:40 Page 10 of 13 adverse effect on some nursing women in those countries Materials and methods where this drug is used for malaria prevention/treatment. Materials We also performed an involution time course study Antibodies were purchased from the following vendors: +/− +/+ 23 using Atg7 heterozygous ( ) and wild-type ( ) mice GRP78 (Western blots), CHOP/DDIT3 (Western blot), to conﬁrm further the causal relationship between intact phospho-eIF2α, Beclin-1, Atg7, LC3B, Bcl-W, Bcl-XL, autophagy signaling and apoptosis in the progression of Cleaved caspase-7 and PARP (Cell Signaling Technology), +/− involution. As with chloroquine treated mice, Atg7 GRP78 (IHC), ATF4, β-Actin and horseradish peroxidase female mice exhibited an enhanced mammary involution (HRP)-secondary antibodies (Santa Cruz Biotechnology), compared with wild-type mice (Fig. 6a, b; quantiﬁcation CHOP/DDIT3 (IHC) (Abcam). Apoptosis was measured Fig. 6c, d), characterized by an early/precocious induction by TUNEL staining using a ﬂuorescein-based in situ cell of apoptosis (Fig. 6a, b, bottom panels). Since ATG7 is death detection kit (Roche). 9,16,45 also required for efferocytosis , the engulfment and phagocytosis of dead cells and apoptotic bodies by viable Murine mammary gland involution model 45,46 mammary epithelium , delayed involution was repor- All animal procedures were approved by the George- ted in epithelial Atg7-deﬁcient mammary glands .In town University Animal Care and Use Committee and +/− contrast, we found accelerated involution in Atg7 performed following the National Institutes of Health mouse mammary glands. However, Teplova et al. used guidelines for the proper and humane use of vertebrate a mouse model where most mammary epithelial cells, animals in biomedical research. Mice were housed in a but not the mammary stromal cells, were ATG7 deﬁcient. temperature- and humidity-controlled room under a 12-h +/+ Both cell compartments are ATG7 deﬁcient in the mouse light–dark cycle. Female ATG7 wild type ( ), hetero- +/− model used in the present study. As indicated by Castello- zygous ( ; Tokyo Metropolitan Institute of Medical 47 10 23 Cros et al. . and Sanchez et al. , stromal cells are of Sciences, Japan ,) and LC3-GFP mice (obtained through major importance because they use autophagy to support NIH from RIKEN BioResource Center ) and C57Bl/6 the survival of epithelial cells. Consistent with our results, mice (Harlan, USA) were mated to produce and nurse Debnath et al. . reported that depletion of either ATG5, pups for approximately 10 days before forced weaning to ATG6, or ATG7 inhibited autophagy and enhanced induce involution (Inv. 0 h); litters were harmonized to luminal apoptosis, concluding that autophagy promotes contain six to eight pups. At the end of each experiment, mammary epithelial cell survival during anoikis. ECM animals were euthanized and mammary glands were detachment-induced autophagy occurs even in the collected at necropsy 24, 48, 72, 96 h, and 7 d after forced absence of apoptosis in Bcl2 overexpressing cells. Thus, weaning. Abdominal #4 glands (from the same animal) the contributions of autophagy to cell survival during were snap frozen and used later for gene expression ECM detachment are independent of the cells’ apoptosis analyses by qRT-PCR. Thoracic #2–3 glands were snap competency . frozen for protein analyses by Western and ﬁxed in for- In conclusion, we have established an integral role for malin for immunohistochemical analyses. 3-5 mice were the temporally orchestrated expression of key UPR and used at each time point. PCR primers for murine ATG7 autophagy signaling molecules in mammary gland invo- and LC3 are given in Supplementary Table S1A. lution during the critical transition from a reversible to irreversible phase of tissue regression/remodeling. Intervention studies Conﬁrming the causal relationship, inhibition of autop- As with the above involution model, C57Bl/6 mouse hagy by either drug treatment or ATG7 haploinsufﬁciency dams were allowed to nurse litters of six to eight pups for enhances epithelial cell death and advances involution. 10 days before forced weaning (Inv. 0 h), at which time Conversely, stimulation of autophagy delays robust cell point either chloroquine (CQ; Sigma-Aldrich, USA), death and prolongs the reversible phase of mammary tunicamycin (Tm; Calbiochem EMB Bioscience Inc.) or gland involution. To our knowledge, our results are vehicle treatments were initiated. CQ was given in the ﬁrst preclinical in vivo data supporting the hypothesis drinking water (0.24 mg/ml, resulting in ~1 mg daily dose that autophagy may promote the survival of breast cells per mouse ). Tunicamycin was injected i.p. (63 ng per lacking an appropriate matrix contact in DCIS lesions mouse ) once daily until the mice were euthanized. and/or disseminating tumor cells. The clinical value Mammary glands were collected after 0, 24, 48, and 72 h of this hypothesis is currently being tested in the PINC as above. At each time point, three to ﬁve mice were trial (“Preventing Invasive Breast Neoplasia with Chlor- included and the glands were collected as indicated above. oquine” [clinicaltrials.gov/show/NCT01023477] ), which is assessing the efﬁcacy of neoadjuvant anti-autophagy Quantiﬁcation of involution progression therapy in inhibiting the progression of DCIS to invasive Ten ﬁelds in each hematoxylin and eosin (H&E) stained disease such as invasive ductal carcinoma. slide of a thoracic #3 mammary gland were systematically Ofﬁcial journal of the Cell Death Differentiation Association Wärri et al. Cell Death Discovery (2019) 5:40 Page 11 of 13 photographed using an Olympus BX 61 microscope and each 20 μl qRT-PCR reaction to detect mRNA levels. The 10× magniﬁcation at each involution time point, treat- reference gene used for probe normalization in the qRT- ment, and for each genotype. Ten ﬁelds (H&E stained PCR was β-Actin (Actβ). Primers used in the reaction slides, 10× magniﬁcation) in each mammary gland of 24, were summarized in Supplementary Table S1B. Primers 48, and 72 h involution time points were graded, adapted for murine Ambra1, Atf4, Atf6, Atg12, Atg7, Becn1, p62, with permission from Pai and Horseman , in stages 1–8 Chop/Ddit3, Grp78, Grp94, and Xbp1 (unspliced) were designed to give <150-base pair products. Primer speci- as shown in Supplementary Fig. S1. Results are given as fold-change relative to the appropriate vehicle control ﬁcity was conﬁrmed by gel electrophoresis (produce a gland at each time point and treatment. For the 96 h and single band). Reactions were performed using Biorad 7 d involution time points, the percentage of epithelium/ SYBR Green Fast qRT-PCR mix and the ABI real-time adipose tissue per total mammary gland area in the H&E PCR detection system. Relative mRNA levels were cal- stained slides (ten ﬁelds of each slide, 10× magniﬁcation) culated using the comparative Ct method (ΔCt ). was measured using ImageJ software [http://rsbweb.nih. +/− gov/ij/] according to the IJ 1.46r user guide. Atg7 Western blot hybridization +/+ mouse mammary glands were compared with Atg7 Mammary glands were solubilized by sonication in glands at each time point. Statistical analyses (t test; RIPA lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, Mann–Whitney rank sum test) were performed using 1% NP40, 0.25% Na-deoxycholate, 1 mM PMSF, 1 mM Sigma Stat 3.0. sodium orthovanadate, 1× Roche complete mini protease inhibitor cocktail). Protein concentration was determined PCA and correlation analysis using a standard bicinchoninic acid assay. Proteins were The data sets published by Clarkson et al. and Stein size fractionated by polyacrylamide gel electrophoresis et al. were acquired by following the data collection and then transferred to a nitrocellulose membrane. method in Zhao et al. . Data normalization was per- Nonspeciﬁc binding was blocked by incubation with formed using the Plier method in Affymetrix expression Blotto (tris-buffered saline with 5% powdered milk and 1% console; log2 transformation was made subsequently. Triton X-100) for 1 h at room temperature. Membranes A gene signature of 21 genes was selected (Supplementary were incubated overnight at 4 °C with primary antibodies, Table S2) from the QIAGEN PCR Array list to represent followed by incubation with polyclonal HRP-conjugated autophagy, UPR (downstream) UPR (internal), and secondary antibodies (1:2000) for 1 h at room tempera- apoptosis. Assignement of these genes to different groups ture. Immunoreactive products were visualized by che- was based on their pathway membership as annotated in miluminescence (SuperSignal Femto West, Pierce GO. In the human genes feature using the GO terms Biotechnology, Rockford, IL) and quantiﬁed by densito- for Autophagy Gene Ontology GO:0006914, Apoptosis metry using the ImageJ digital densitometry software Process GO:0006915 and Response to unfolded protein (http://rsbweb.nih.gov/ij/) with β-Actin and Ponceau-S GO:0006986, only 5/21 of the genes we detected belong staining as loading control. uniquely to a single GO term (Table 1). Thus, since many genes are shared between these pathways, we did PCA Tissue staining and IHC (Fig. 1a, b, Supplementary Fig. S2) on each pathway Mammary glands were ﬁxed in 10% formalin for 24 h respectively and made a one-to-one gene—pathway prior to embedding in parafﬁn. Embedded tissues were cut mapping based on the importance of a gene in their into 5 µm thick sections and stained with H&E to deter- corresponding pathway. mine morphology. Apoptosis was visualized by TUNEL In addition, in order to investigate the relationship staining. Brieﬂy, mammary gland tissue sections were between autophagy and UPR during the involution stage, examined for apoptosis using ﬂuorescein dUTP nick end we selected n = 4 genes/group (Table 1, Supplementary labeling. Positive- and negative-control slides provided Table S2) and calculated the correlation coefﬁcients of with the kit were used in each assay to ensure consistency. all possible permutation among the genes representing Immunostaining was performed with an antibody to CD68 autophagy and UPR (UPR was combined). In both data- (1:100), p62 (1:100), CHOP/DDIT3 (1:100), GRP78 (1:100) sets, the top ten combinations all has a correlation coef- using the streptavidin–biotin method. Stained sections ﬁcient >0.95. were visualized and photographed. GFP ﬂuorescence was detected from samples ﬁxed in 4% paraformaldehyde–PBS Quantitative real-time reverse transcription PCR overnight, followed by immersion in 20% sucrose/PBS at Total RNA was extracted from frozen mammary gland 4 °C for 24 h and cryoprotection in 30% sucrose/PBS samples by the trizol method. 1 µg RNA was used at 4 °C until they sunk. Samples were embedded in optimal for cDNA generation using the Biorad iScript cDNA cutting temperature compound, frozen, and cut in slides synthesis kit. 1/400 of the cDNA products were used in using routine procedures. Ofﬁcial journal of the Cell Death Differentiation Association Wärri et al. Cell Death Discovery (2019) 5:40 Page 12 of 13 Statistical analyses 14. Yue, Z., Jin, S., Yang, C., Levine, A. J. & Heintz, N. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufﬁcient tumor All statistical analyses (Student’s t test; Mann–Whitney suppressor. Proc. Natl Acad. Sci. USA 100, 15077–15082 (2003). rank sum test) were performed using Sigma Stat 3.0, and 15. Avivar-Valderas, A. et al. PERK integrates autophagy and oxidative stress detailed in the text where applicable. responses to promote survival during extracellular matrix detachment. Mol. Cell Biol. 31,3616–3629 (2011). 16. Teplova, I. et al. ATG proteins mediate efferocytosis and suppress inﬂammation Acknowledgments in mammary involution. Autophagy 9 459–475 (2013). The work was funded by NIH grants U01-CA184902, U54-CA149147, and P30- 17. Tyson, J. J. et al. Dynamic modelling of oestrogen signalling and cell fate CA51008 to R.C.; D.O.D. postdoc fellowship BC112023 to K.L.C. in breast cancer cells. Nat. Rev. Cancer 11,523–532 (2011). 18. Clarke, R. et al. Endoplasmic reticulum stress, the unfolded protein response, Author details 1 autophagy, and the integrated regulation of breast cancer cell fate. Cancer Res. Department of Oncology and Lombardi Comprehensive Cancer Center, 2 72,1321–1331 (2012). Georgetown University Medical Center, Washington, DC 20057, USA. Institute 19. Cook, K. L., Shajahan, A. N. & Clarke, R. Autophagy and endocrine resistance of Biomedicine, University of Turku Medical Faculty, Turku 20014, Finland. 3 in breast cancer. Expert Rev. Anticancer Ther. 11,1283–1294 (2011). Department of Surgery and Comprehensive Cancer Center, Wake Forest 4 20. Gregor, M. F. et al. The role of Adipocyte XBP1 in metabolic regulation University, Winston Salem, NC 27157, USA. National Heart Lung and Blood 5 during lactation. Cell Rep. 3,1430–1439 (2013). Institute, National Institutes of Health, Bethesda, MD 20892, USA. University of 21. Clarkson, R. 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Cell Death Discovery – Springer Journals
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