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. Author manuscript; available in PMC: 2021 Oct 1.
Published in final edited form as: Biochim Biophys Acta Mol Cell Res. 2020 Jun 2;1867(10):118765. doi: 10.1016/j.bbamcr.2020.118765

Src Couples Estrogen Receptor to the Anticipatory Unfolded Protein Response and Regulates Cancer Cell Fate Under Stress

Liqun Yu 1, Lawrence Wang 1, Ji Eun Kim 1, Chengjian Mao 1, David J Shapiro 1,2
PMCID: PMC7653967  NIHMSID: NIHMS1600465  PMID: 32502618

Abstract

Accumulation of unfolded protein, or other stresses, activates the classical reactive unfolded protein response (UPR). In the recently characterized anticipatory UPR, receptor-bound estrogen, progesterone and other mitogenic hormones rapidly elicit phosphorylation of phospholipase C γ (PLCγ), activating the anticipatory UPR. How estrogen and progesterone activating their receptors couples to PLCγ phosphorylation and anticipatory UPR activation was unknown. We show that the oncogene c-Src is a rate-limiting regulator whose tyrosine kinase activity links estrogen and progesterone activating their receptors to anticipatory UPR activation. Supporting Src coupling estrogen and progesterone to anticipatory UPR activation, we identified extranuclear complexes of estrogen receptor α (ERα):Src:PLCγ and progesterone receptor:Src:PLCγ. Moreover, Src inhibition protected cancer cells against cell death. To probe Src’s role, we used the preclinical ERα biomodulator, BHPI, which kills cancer cells by inducing lethal anticipatory UPR hyperactivation. Notably, Src inhibition blocked BHPI-mediated anticipatory UPR activation and the resulting rapid increase in intracellular calcium. After unbiased long-term selection for BHPI-resistant human breast cancer cells, 4/11 BHPI-resistant T47D clones, and nearly all MCF-7 clones, exhibited reduced levels of normally growth-stimulating Src. Notably, Src overexpression by virus transduction restored sensitivity to BHPI. Furthermore, in wild type cells, several-fold knockdown of Src, but not of ERα, strongly blocked BHPI-mediated UPR activation and subsequent HMGB1 release and necrotic cell death. Thus, Src plays a previously undescribed pivotal role in activation of the tumor-protective anticipatory UPR, thereby increasing the resilience of breast cancer cells. This is a new role for Src and the anticipatory UPR in breast cancer.

Keywords: Src, Unfolded Protein Response, Estrogen receptor, Cancer cell death, Calcium, Dasatinib

Graphical Abstract

graphic file with name nihms-1600465-f0001.jpg

1. Introduction

The endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR), maintains protein folding quality control and homeostasis. In the well-studied reactive UPR, cancer cells respond to unfolded or misfolded proteins, hypoxia, nutritional deprivation and therapy-induced stress by activating the three arms of the UPR, PERK, IRE1α and ATF6α, reducing protein production and increasing protein folding capacity1. Building on initial work in B cells2, we, and others, described a protective anticipatory UPR pathway in which estrogen (17β-estradiol, E2), progesterone (P4), and the mitogenic hormones, epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) act through their respective receptors to pre-activate the UPR and anticipate future needs for increased protein folding capacity required to support hormone-stimulated cell proliferation3–7. Notably, mild and transient E2 and EGF activation of the anticipatory UPR elicits an adaptive response that protects cancer cells against subsequent UPR-mediated apoptosis3,4. Moreover, analysis of data from ~1,000 breast cancer patients showed that E2-estrogen receptor α (ERα)-related activation of a UPR gene index at diagnosis was tightly correlated with subsequent tamoxifen resistance, tumor recurrence and a poor prognosis3. While a role for ERα in activation of the anticipatory UPR was clearly established, still unknown was the identity of the key regulator that initiates the anticipatory UPR pathway (Graphical abstract) by coupling E2-ERα to tyrosine phosphorylation and activation of phospholipase C γ (PLCγ)3–5. We demonstrate that the well-studied oncogene cellular Src (c-Src) is the rate-limiting tyrosine kinase that couples E2-ERα and P4-PR to activation of the anticipatory UPR.

The proto-oncogene c-Src is a non-receptor tyrosine kinase with critical roles in signaling pathways involved in cancer cell proliferation, survival, angiogenesis and metastasis8–10. Although Src was known to interact with ERα and progesterone receptor (PR)11–14, a role for Src in coupling ERα and PR action to PLCγ activation and the anticipatory UPR had not been reported.

While moderate and transient activation of the reactive and anticipatory UPR pathways promotes cancer progression and therapy resistance3,15, severe unresolvable EnR stress elicits strong and sustained activation of the reactive UPR, activating multiple pro-apoptotic pathways16–18. In contrast, we recently showed that strong and sustained activation of the anticipatory UPR results in ATP depletion and necrotic cell death (Graphical abstract)19. We targeted the anticipatory UPR pathway with the promising preclinical breast cancer drug, BHPI, which acts through ERα to induce lethal anticipatory UPR hyperactivation19–22. We explored the role of Src in both protective and cytotoxic anticipatory UPR activation. Our studies describe an often rate-limiting role for Src in coupling steroid receptor action to life-death decisions based on activation of the protective and cytotoxic anticipatory UPR.

2. Material and Methods

2.1. Cell Culture and Reagents

T47D and MCF-7 cells are available from ATCC. MCF7-ERαHA cells were provided by E. Alarid. T47D-ERαY537S (TYS) cells were made as described21. BHPI-resistant clones were derived from T47D and MCF-7 cells. Cells were grown in the following conditions: T47D (MEM, 10% FBS), MCF-7 (MEM, 5% FBS), MCF7-ERαHA (DMEM, 10% FBS), TYS (MEM, 10% CD-FBS), BHPI-resistant clones (MEM, 10% FBS + 1 μM BHPI for T47D clones; MEM, 5% FBS + 5 μM BHPI for MCF-7 clones). Reagents used were: Dasatinib, Saracatinib, MG132 (Selleck Chemicals, TX), E2-BSA, Tunicamycin (Sigma-Aldrich, MO), 35S-methionine (Perkin Elmer, MA).

2.2. Western Blotting

Western blotting was carried out as previously described23. 300,000 cells were harvested from a 6 well plate and 50 μg of total protein was used for western blots. Primary antibodies are listed in Supplementary Table 1. Antibodies were probed with HRP-conjugated secondary antibodies (ThermoFisher, MA) and imaged with the ECL2 detection kit (ThermoFisher) using a PhosphorImager (GE Healthcare, IL). All western blot experiments have been repeated at least 3 times. Western blot bands were quantified using ImageJ. In brief, a rectangular area was selected to include the band of interest and intensity of each band was calculated using the analysis tool. A blank area with the same rectangular size was selected and quantified and served as the background. After subtracting the background, band intensity was then normalized by dividing the intensity with the first lane in the blot.

2.3. Co-immunoprecipitation

10 million cells were lysed with non-denaturing lysis buffer (20 mM Tris HCl, pH 8; 137 mM NaCl; 1% NP-40; 2 mM EDTA) with proteinase inhibitor cocktail. Whole cell lysates obtained by centrifugation were incubated with 1 μg of ERα or PR antibody and cross-linked protein A Dynabeads (ThermoFisher) overnight at 4°C. The immunocomplexes were then washed with washing buffer (10 mM Tris, pH 7.4; 1 mM EDTA; 1 mM EGTA, pH 8.0; 150 mM NaCl; 1% Triton X-100; 0.2 mM sodium orthovanadate; protease inhibitor cocktail) three times and separated for immunoblot analysis.

2.4. siRNA Knockdown

siRNA knockdowns were performed using DharmaFECT1 and 100 nM ON-TARGET plus non-targeting pool or SMARTpools for ERα, PLCγ and Src (Dharmacon, CO). Transfection conditions were as described23.

2.5. Real-time PCR

300,000 cells were plated and cultured as described23. Total RNA from each of three biological replicates was collected using a Qiagen RNAeasy kit. 1 μg of total RNA was used for cDNA library preparation using a ProtoScript® First Strand cDNA Synthesis Kit (NEB, MA). PCR was performed in MicroAmp™ Optical 96-Well Reaction Plate (Applied Biosystems) on cDNA equivalent to 10 ng RNA. Primers are listed in Supplementary Table 2.

2.6. Cell Proliferation Assay

The indicated number of cells were plated into 96-well plates. The next day, the medium was changed to the indicated treatment. The treatment medium was changed after 48 hr and cell numbers were measured with MTS reagent (Promega, WI) after 96 hr (72 hr for siRNA KD experiments) with indicated treatments.

2.7. ATP Measurement

10,000 cells were plated and cultured, and ATP depletion assays were performed as previously described19 and ATP levels were measured using the ATPlite Luminescence Assay System (Perkin Elmer, MA). Luminescence was measured using a PHERAStar plate reader (BMG Labtech, Germany).

2.8. Cell Viability Assay

300,000 cells were cultured and plated as previously described19. Cell viability and cell volume after the indicated treatment were measured using a Countess II cell counter (ThermoFisher).

2.9. Protein Synthesis Assay

Protein synthesis was analyzed by measuring incorporation of 35S-methionine into newly synthesized protein. Cells were incubated with the indicated treatments and 3 μCi 35S-methionine (Perkin Elmer) was added for the last 30 min of treatment. Cells were washed, lysed and clarified by centrifugation. Supernatants were spotted onto Whatman 540 filter paper discs (Fisher Scientific, PA) and subsequently washed with 10% trichloroacetic acid (TCA) and 5% TCA. Trapped protein was solubilized, and radioactivity was measured in a scintillation counter.

2.10. Lentivirus Production

Src cDNA was amplified and inserted into pCDH-CMV-MCS-EF1-PURO vector using an In-Fusion HD cloning kit (Clontech, CA). GCaMP3 was amplified from pcDNA3-Cyto-GCaMP3 (Addgene #64853) and inserted into pCDH-CMV-MCS-EF1-PURO by restriction enzymes XbaI (NEB, R0145S) and NotI (NEB, R0189S) digestion and T4 DNA ligase (NEB, M0202S) ligation. Lentivirus was produced by cotransfecting pCDH-Src-PURO, pCDH-GCaMP3-PURO or pHIV-Luciferase vector (Addgene #21375) with packaging vectors pCI-VSVG (Addgene #1733) and psPAX2 (Addgene #12260) into HEK293 cells using Lipofectamine 3000 (ThermoFisher).

2.11. Calcium Imaging

10,000 T47D cells were plated in 35 mm-fluorodish cell culture plates (World Precision Instruments, #FD35–100). The next day, cells were transduced with GCaMP3 expressing lentivirus for 24 hours and incubated for an additional 48 hours. To remove extracellular calcium, 30 minutes prior to imaging, cells were washed three times with calcium free HEPE-based buffer (140 mM NaCl, 4.7 mM KCl, 1.13 mM MgCl2, 10 mM HEPES, 10 mM Glucose, pH =7.4). The indicated treatment was added and GFP fluorescence was measured at 10X magnification using a Leica DMI8 microscope for >200 seconds. Images were acquired and analyzed with LAS X (Leica) and ImageJ.

2.12. Statistical Analysis

RNAseq data from normal tissues and primary tumors was obtained from the TCGA Research Network. Samples were grouped by hormone receptors status determined by IHC. The Pearson correlation test was used to determine gene expression correlation coefficients. A two-tailed Student’s t-test was used for comparisons between groups. One-way ANOVA followed by Tukey’s post hoc test was used for multiple comparisons.

3. Results

3.1. Steroid hormones activate phospholipase C γ through Src

To identify the tyrosine kinase that couples E2-ERα to the UPR, we used the finding that Src is a major tyrosine kinase in cancer cells9. Since we previously observed that UPR markers are elevated in ERα+ mammary carcinoma3, we tested whether c-Src expression follows a similar pattern. c-Src expression is significantly elevated in ERα+ and in PR+ mammary carcinomas (Figure 1a). To explore whether Src mediates E2-ERα activation of PLCγ, we evaluated the effect of Src inhibition or knockdown on E2-ERα stimulation of PLCγ phosphorylation. In ERα+ T47D and MCF-7 human breast cancer cells, E2 increased phosphorylation and activation of Src and PLCγ with a maximum at 20 min. The Src inhibitor, dasatinib (Das), abolished phosphorylation and activation of Src and PLCγ (Figure 1b and Supplementary Figure 1a). PR interacts with Src14, suggesting progesterone (P4) might also activate PLCγ and the anticipatory UPR through Src. Progesterone treatment stimulated Src and PLCγ phosphorylation in T47D cells and in TYS (T47D-ERαY537S) cells, which express the ERαY537S mutation that is associated with reduced survival in metastatic breast cancer21,23. In both T47D and TYS cells, dasatinib pretreatment blocked the P4-mediated increase in Src and PLCγ phosphorylation (Figure 1c and Supplementary Figure 1b). Notably, Src knockdown by two sets of Src siRNAs blocked E2- and P4-stimulated PLCγ phosphorylation (Figure 1d and Supplementary Figure 1c). Since two sets of Src siRNA blocked PLCγ phosphorylation these effects are unlikely to be due to off-target effects of the siRNA. Src knockdown slightly reduced PLCγ levels (Figure 1d and Supplementary Figure 1c,d). Since the decline in PLCγ phosphorylation was much larger than the decline in total PLCγ (Figure 1d and Supplementary Figure 1c), the effect of Src knockdown is not due to a reduction in PLCγ level. PLCγ knockdown did not alter Src levels (Supplementary Figure 1e).

Figure 1.

Figure 1.

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Src mediates steroid hormone-stimulated PLCγ phosphorylation. (a) c-Src gene expression in normal tissues (NT), ERα+ primary breast cancer (ER+ TP) and PR+ primary breast cancer (PR+ TP). * indicates a significant difference among groups using one-way ANOVA followed by Tukey’s post hoc test. *** P < 0.001. (b,c) Western blot analysis of phosphorylated PLCγ (p-PLCγ, tyrosine 783), total PLCγ, phosphorylated Src (p-Src), total Src and β-actin in ERα+ T47D cells treated with vehicle control or dasatinib (Das) for 5 min, followed by treatment with 10 nM E2 (b) or 10 nM progesterone (P4) (c). (d) Western blot analysis of Src, p-PLCγ, PLCγ and β-actin protein levels following treatment of T47D cells with either 100 nM non-coding (NC) or Src siRNA SMARTpool, followed by treatment with vehicle, E2 or P4 for 30 min. (e) Co-immunoprecipitation and western blot analysis of ERα, Src and PLCγ interactions in MCF7-ERαHA cells. Using magnetic beads, cell lysates were immunoprecipitated with ERα or mouse IgG antibody. The immunoprecipitates were blotted with PLCγ, Src and ERα antibodies. (f) Co-immunoprecipitation and western blot analyses of PR, Src and PLCγ interactions in T47D-ERαY537S (TYS) cells. Cell lysates were immunoprecipitated with PR or mouse IgG antibody. The immunoprecipitates were blotted with PLCγ, Src and PR antibodies.

3.2. Identification of multiprotein complexes containing ERα, Src and PLCγ and PR, Src and PLCγ

Rapid PLCγ phosphorylation stimulated by E2 and P4 suggested direct interactions between ERα, Src and PLCγ and between PR, Src and PLCγ. We therefore tested for the existence of ERα:Src:PLCγ and PR:Src:PLCγ complexes using co-immunoprecipitation (co-IP). Since extranuclear ERα is only ~5% of the total cellular pool24, MCF7-ERαHA cells were used to increase ERα expression20. In MCF7-ERαHA cells, ERα is doxycycline inducible. Although levels of ERα in doxycycline-treated MCF7-ERαHA cells are several-fold higher than in commonly used breast cancer cells like MCF-7 and T47D, and are higher than ERα levels in most human breast cancers, they are in the range seen in some human breast cancers25,26. Compared to control lysates immunoprecipitated using non-specific rabbit IgG, immunoprecipitation with ERα antibody resulted in an E2-dependent increase in co-immunoprecipitated Src and PLCγ (Figure 1e). In TYS cells where PR expression is high23, Src and PLCγ co-immunoprecipitated with anti-PR but not with control rabbit IgG (Figure 1f). Unlike the ERα:Src:PLCγ complex, association of PR, Src and PLCγ was not further enhanced by P4. This is consistent with earlier work showing that ERα and PR interact differently with Src14. The presence of E2-ERα:Src:PLCγ, and P4-PR:Src:PLCγ complexes supports the view that E2-ERα and P4-PR activate Src kinase, which then phosphorylates and activates PLCγ, initiating the anticipatory UPR.

3.3. E2 and P4 activate the Src-mediated anticipatory UPR, protecting breast cancer cells against drug-induced stress

We previously reported that E2-ERα moderately induces the UPR marker, transcription factor spliced XBP1 (sp-XBP1) mRNA and the oncogenic UPR-induced chaperone HSPA5/BiP/GRP78, and that increased expression of UPR-related genes at diagnosis is strongly correlated with a poor prognosis in ERα+ breast cancer3. If Src both couples E2-ERα to anticipatory UPR activation and is overexpressed in mammary carcinomas (Figure 1a), Src expression should correlate with overexpression of HSPA5 and other UPR marker genes in tumors and with induction of sp-XBP1 mRNA27. In vivo data from The Cancer Genome Atlas (TCGA) demonstrated that in ERα+ and PR+ mammary carcinomas in which c-Src was also upregulated, there was up-regulation of UPR marker genes, especially the IRE1α pathway targets XBP1 and HSPA5. Pearson correlation analysis further confirmed that in ER+ breast cancer patients there is a significant positive association between expression of c-Src and expression of XBP1 and HSPA5 (Figure 2a). Thus, Src-mediated PLCγ activation may be responsible for anticipatory UPR activation in these ERα and PR positive breast cancers. To test our hypothesis, we evaluated the effect of Src inhibition or knockdown on levels of sp-XBP1 mRNA and the sp-XBP1-induced oncogenic chaperone HSPA5/BiP/GRP7828. Both E2 and P4 increased sp-XBP1 mRNA in breast cancer cells. Inhibiting Src with dasatinib or saracatinib significantly reduced sp-XBP1 mRNA (Figure 2b,c and Supplementary Figure 2a,b). Dasatinib alone did not significantly increase sp-XBP1 mRNA (Supplementary Figure 2c). Consistent with their induction of sp-XBP1 mRNA, E2 and P4 elevated BiP expression in T47D cells and Src knockdown blocked BiP induction (Figure 2d). To further test the role of an extranuclear pathway in estrogen activation of the anticipatory UPR, we studied the effects of E2-BSA on Src and UPR activation. E2-BSA is a cell membrane impermeable conjugate. Similar to what we observe in experiments using E2, E2-BSA rapidly induces Src phosphorylation (Supplementary Figure 3a), followed by production of sp-XPB1 and induction of BiP (Supplementary Figure 3b,c). Notably, E2-BSA stimulates BiP expression as effectively as E2 (Supplementary Figure 3c). This suggests that extranuclear estrogen signaling at or near the plasma membrane is the major pathway regulating activation of the anticipatory UPR.

Figure 2.

Figure 2.

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Estrogen and progesterone activate the protective anticipatory unfolded protein response (UPR) through Src. (a) (Left) UPR marker gene expression in normal breast tissues (NT), ERα positive primary breast cancer (ER+ TP) and PR positive primary breast cancer (PR+ TP). * indicates a significant difference among groups using one-way ANOVA followed by Tukey’s post hoc test. (Right) Pearson correlation analysis of gene expression data in ER+ breast cancer patients. The Pearson correlation test was performed to analyze co-expression of Src mRNA and UPR marker mRNA expression using data from the TCGA database. * P < 0.05, *** P < 0.001. (b,c) qRT-PCR analysis of spliced XBP1 (sp-XBP1) mRNA levels in T47D cells treated for 15 min with vehicle, or with 200 nM dasatinib, followed by treatment with vehicle, 10 nM E2 (b) or P4 (c) for 4 hr. Different letters indicate a significant difference among groups (P<0.05) using one-way ANOVA followed by Tukey’s post hoc test (mean ± s.e.m., n = 3). (d) Western blot analysis of BiP and β-actin protein levels following treatment of T47D cells with either 100 nM non-coding (NC), or Src siRNA SMARTpool, followed by treatment with vehicle, E2 or P4 for 4 hr. (e) Western blot analysis of PR, phosphorylated ERK (p-ERK), total ERK and β-actin protein levels in T47D cells and TYS cells treated with vehicle, 10 nM E2 or 10 nM P4 for 24 hr. (f,g) Weak dasatinib-sensitive anticipatory activation of the UPR with progesterone protects cells against subsequent tunicamycin-induced cell death. T47D (f) and TYS (g) cells were maintained in medium with ethanol-vehicle (Untreated), 10 nM P4 or P4 plus 200 nM dasatinib for 48 hr Vehicle control, P4 and dasatinib were removed from medium and cells were then treated with the indicated concentrations of tunicamycin (TUN) for 96 hr. Different letters indicate a significant difference among groups (P<0.05) using one-way ANOVA followed by Tukey’s post hoc test (mean ± s.e.m., n = 6).

E2 pretreatment induces chaperones and renders cells resistant to lethal concentrations of tunicamycin (TUN)3. If Src plays a central role in activation of the protective anticipatory UPR, Src inhibition might reduce protection of the cancer cells. Unlike E2, which activates the pro-proliferation ERK pathway, increasing cell proliferation and complicating analysis of experimental data, P4 did not increase cell number or activate the ERK pathway (Figure 2e and Supplementary Figure 3d,e). Notably, pretreating cells with P4 elicited 4-fold and 2-fold increases in the concentration of TUN required to induce apoptosis in T47D and TYS cells, respectively; blocking UPR activation with dasatinib re-sensitized cells to TUN (Figure 2f,g). Thus, Src-mediated anticipatory activation of the UPR results in increased chaperone production and protects tumor cells against apoptotic cell death induced by prolonged EnR stress. Since these data provided strong evidence that Src couples E2-ERα and P4-PR to moderate and tumor-protective activation of the anticipatory UPR, we next explored whether Src also plays a central role in strong and sustained lethal hyperactivation of the anticipatory UPR.

3.4. Src is required for strong and sustained activation of the UPR

BHPI, acting through ERα, elicits strong and sustained activation of the anticipatory UPR19,20. The opening of IP3R calcium channels induces sustained Ca2+ release from the lumen of the EnR into the cytosol. To restore Ca2+ homeostasis, sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) pumps use ATP to pump Ca2+ back into the EnR lumen20, creating a futile cycle of pumping and release that depletes intracellular ATP (Graphical abstract). To obtain a synchronized increase in intracellular calcium in a large fraction of the cells in a time frame measured in seconds, a higher concentration of BHPI was used. BHPI induced a rapid increase in cytosol calcium, which was completely blocked by inhibiting Src activation using dasatinib (Figure 3a,b and Supplementary Movie 1). Moreover, BHPI stimulated a ~40% decline in ATP levels, which was partially blocked by Src knockdown (Figure 3c). We next explored whether Src was critical for strong UPR activation. BHPI robustly induced sp-XBP1 mRNA and dasatinib largely inhibited sp-XBP1 induction (Figure 3d). BHPI strongly stimulates the PERK arm of the UPR, leading to inhibition of protein synthesis20. Pretreatment with dasatinib blocked BHPI-induced PERK activation, reducing downstream phosphorylation of eIF2α (Supplementary Figure 4a).

Figure 3.

Figure 3.

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BHPI kills ERα positive breast cancer cells by hyperactivating the UPR through Src. (a) Microscopic visualization of intracellular calcium using the GCaMP3 calcium sensor. Note that because this sensor exhibits a kd for Ca2+ of ~300 nM, a small increase in Ca2+ that is not blocked by dasatinib will be below the limit of detection. Color scale from basal Ca2+ to highest Ca2+: blue, green, red, white. T47D cells were treated with 1 μM dasatinib 15 min prior imaging and 50 μM BHPI was added at 15 sec. (b) Graph depicts quantitation of cytosolic calcium levels in T47D cells pre-treated with either vehicle-control or dasatinib, followed by BHPI (BHPI addition indicated by the black arrow). Total fluorescence intensity of a single cell was divided by the area of each cell to get the average fluorescence intensity (mean ± s.e.m, n = 10). (c) Src knockdown partially blocks cytotoxic BHPI-mediated ATP depletion. ATP level analysis following treatment of T47D cells with either 100 nM NC siRNA, or Src siRNA SMARTpool, followed by treatment for 24 hr with vehicle or the indicated concentration of BHPI. * indicates a significant difference among groups using Student’s t-test. * P< 0.05, ** P < 0.005 (mean ± s.e.m., n = 6). (d) qRT-PCR analysis of sp-XBP1 gene expression in T47D, TYS and MCF-7 cells treated with vehicle or 200 nM dasatinib for 15 min, followed by treatment with vehicle or 100 nM BHPI. Different letters indicate a significant difference among groups (P<0.05) using one-way ANOVA followed by Tukey’s post hoc test (mean ± s.e.m., n = 3). (e) Western blot analysis of BHPI-stimulated HMGB1 release into the cell culture medium at 50 or 100 nM BHPI following treatment of T47D cells with either 100 nM NC or Src siRNA SMARTpool. (f) Src knockdown blocks BHPI-induced cell death. T47D and TYS cells were treated with 100 nM NC or Src siRNA followed by treatment with 100 nM BHPI for 24 (T47D) or 1 hr (TYS). (g) Inhibiting Src activity blocks BHPI-induced cell death. MCF-7 cells were treated for 2 hr with vehicle, 100 nM BHPI, 200 nM dasatinib or dasatinib + BHPI. (f,g) Cell death was monitored by an automated trypan blue assay. Different letters indicate a significant difference among groups (P<0.05) using one-way ANOVA followed by Tukey’s post hoc test (mean ± s.e.m., n = 3). (h) MTS assay following treatment of T47D cells with either 100 nM NC or Src siRNA SMARTpool, followed by treatment with vehicle or the indicated concentration of BHPI for 72 hr. * indicates a significant difference among groups using Student’s t-test. ** P < 0.005, ns not significant (mean ± s.e.m., n = 6).

Sustained UPR activation induced by BHPI results in classical necrosis phenotypes, including plasma membrane disruption and leakage of intracellular contents19. We assessed loss of membrane integrity by monitoring leakage into the medium of the marker, high mobility group box 1 (HMGB1). 24h BHPI treatment increased HMGB1 release and Src knockdown reduced HMGB1 leakage (Figure 3e). To evaluate cell viability, we quantitated cell death using an automated trypan blue exclusion assay19. In T47D, TYS and MCF-7 cells, ablating Src signaling by knockdown, or with dasatinib, nearly abolished BHPI’s ability to induce cell death (Figure 3f,g). Moreover, a longer three-day MTS assay confirmed that Src knockdown reversed BHPI inhibition of cell proliferation (Figure 3h). Since Src knockdown only reduced Src levels ~3-fold (Figure 1d), these data suggested Src, not ERα, might be limiting for activation of the anticipatory UPR. To explore this, we evaluated the effect of ERα knockdown on BHPI inhibition of cell proliferation. Although the extent of ERα knockdown was at least as great as Src knockdown (Supplementary Figures 1c, 4b), ERα knockdown did not block BHPI inhibition of cell proliferation (Supplementary Figure 4c). These data suggest that Src, not ERα, is limiting in the complex that initiates activation of the anticipatory UPR. We therefore explored whether reduced expression of Src might be associated with the development of resistance to BHPI.

3.5. In ER+ breast cancer cells, Src downregulation confers resistance to BHPI

We performed long-term selections for breast cancer cells resistant to growth inhibition by BHPI. Although 50 nM BHPI induced death of most T47D and MCF-7 cells, some cells survived. Therefore, much higher concentrations of BHPI were needed to avoid outgrowth of false positive clones and enable identification of clonal lines of BHPI-resistant cells. Treatment of T47D and MCF-7 cells with 1 and 5 μM BHPI, respectively, led to rapid death of nearly all the cells, followed by slow outgrowth of BHPI-resistant colonies. BHPI resistance could arise from changes that diminish lethal BHPI hyperactivation of the anticipatory UPR, or from changes in growth or death pathways that counter UPR hyperactivation. We therefore evaluated the effects of the apoptosis inducer staurosporine (STS) and of BHPI in T47D and MCF-7 cells and in BHPI-resistant T47D clone 1 (TB1) and MCF-7 clone 5 (MB5) cells. Flow cytometry showed that while both BHPI and STS induced cell death in the wild-type T47D and MCF-7 cells, only STS induced death of the BHPI-resistant TB1 and MB5 cells (Supplementary Figure 5). Since this showed there was not a general defect in cell death in BHPI-resistant cells, we explored effects on proteins in the anticipatory UPR pathway, and on UPR activation. Since long-term treatment with aromatase inhibitors selects for tumors that contain constitutively active ERα mutations29 and BHPI acts through ERα, it seemed possible we would identify mutations in ERα with diminished interaction with BHPI. Interestingly, sequencing of all 11 BHPI-resistant T47D clones showed no mutations in ERα. Since BHPI acts through the ERα-Src-PLCγ complex to hyperactivate the anticipatory UPR pathway, we explored levels of these proteins in the BHPI-resistant clones. Consistent with a rate-limiting role for Src in the anticipatory UPR, 4 of 11 T47D clones (clone 1, 3, 4 and 11) and 7 of 8 MCF-7 clones showed reduced Src expression (Figure 4a and Supplementary Figure 6a). Several BHPI-resistant clones displayed reduced levels of ERα. We further characterized several BHPI-resistant clones. T47D clones: TB1 (moderately reduced Src, ERα and PLCγ), TB3 (reduced Src) and TB11 (reduced ERα, PLCγ and slightly reduced Src) and MCF-7 clone MB5 (reduced Src and ERα) and MB7 (reduced Src and ERα). T47D clone TB8 (Src not reduced) was a control. TB1, TB3 and TB11 exhibited reduced sensitivity to estrogen and showed slow growth in BHPI (Figure 4b,c). In contrast, TB8 lost E2 stimulation of cell proliferation, while BHPI slightly increased its proliferation (Figure 4b,c). This suggests TB8 cells with normal Src levels exhibit a different BHPI resistance mechanism than cells with reduced levels of Src. Compared to parental MCF-7 cells, MB5 and MB7 cells showed slightly reduced E2-stimulated cell proliferation and unlike T47D cells, 1 μM BHPI had minimal effects on their proliferation (Supplementary Figure 6b,c). Since T47D and MCF-7 cells display different sensitivity to growth inhibition and killing by BHPI (Figure 4c and Supplementary Figure 6c), data obtained from the two sets of BHPI-resistant cell lines should be broadly applicable.

Figure 4.

Figure 4.

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Identification and characterization of the BHPI-resistant clones. (a) Western blot of PLCγ, ERα, Src, phosphorylated eIF2α (p-eIF2α), eIF2α and β-actin in T47D parental cells and BHPI-resistant clones treated with vehicle or 1 μM BHPI. All BHPI-resistant clones were cultured in medium containing 1 μM BHPI. Proliferation of T47D and resistant clones treated with vehicle, 10 nM E2 (b) and indicated concentrations of BHPI (c) for 96 hr (b,c). Different letters indicate a significant difference among groups (P < 0.05) using one-way ANOVA followed by Tukey’s post hoc test (mean ± s.e.m., n = 6).

Src functions at the start of the anticipatory UPR pathway. If down-regulation of Src plays a key role in BHPI resistance, and BHPI works by strongly activating the anticipatory UPR pathway, resistant cells that down-regulated Src should exhibit impaired BHPI induction of the UPR marker sp-XBP1 and BiP chaperone, but activators of the reactive UPR should retain the ability to induce sp-XBP1 mRNA and BiP. We therefore compared sp-XBP1 mRNA and BiP protein expression in parental cells and in BHPI-resistant clones treated with BHPI and with the classic UPR activators TUN and thapsigargin (THG). Compared to parental cells, induction of sp-XBP1 mRNA was impaired in the TB1 and MB5 cells expressing reduced levels of Src (Supplementary Figure 6d, f). Because BHPI robustly activates the PERK-p-eIF2α arm of the UPR, eliciting near-quantitative inhibition of protein synthesis20, BiP protein is not induced by BHPI (Supplementary Figure 6h). In contrast, the induction of sp-XBP1 mRNA and BiP protein by the reactive UPR activators was nearly identical in parental and BHPI-resistant clones (Supplementary Figure 6e–h). These data indicate that the reactive UPR pathway is intact and are consistent with Src down-regulation resulting in reduced initial activation of the anticipatory UPR. Consistent with a defect in the early steps in the anticipatory UPR, in 100 nM BHPI, which blocks growth and kills wild type T47D cells (Figure 4a), PERK-p-eIF2α activation and inhibition of protein synthesis were nearly abolished in the resistant clones (Figure 5a, b). While increasing BHPI to 1 μM restored substantial BHPI inhibition of protein synthesis, these cells still grow slowly (Figures 5b, 4c). Notably, while 100 nM and 1 μM BHPI strongly reduced ATP levels in wild type T47D and MCF-7 cells, in the BHPI-resistant clones, BHPI lost the ability to reduce ATP levels (Figure 5c and Supplementary Figure 6i) and therefore failed to induce necrotic cell death (Figure 5d) and HMGB1 release (Figure 5e). If the reduced levels of Src in BHPI-resistant clones contributes to BHPI resistance, restoring the level of Src might re-sensitize resistant clones to BHPI. We therefore explored the effect of restoring Src levels on BHPI resistance.

Figure 5.

Figure 5.

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Src downregulation confers BHPI resistance on breast cancer cells. (a) Western blot of p-eIF2α, total eIF2α and β-actin protein levels in T47D and BHPI-resistant clones treated with vehicle or 100 nM BHPI for 1 hr. (b) Protein synthesis assay in T47D and BHPI-resistant clones treated with vehicle, 100 nM BHPI or 1 μM BHPI for 1 hr. Different letters indicate a significant difference among groups using one-way ANOVA followed by Tukey’s post hoc test (mean ± s.e.m., n=4). (c) ATP levels in T47D cells and BHPI-resistant clones treated with vehicle or the indicated concentrations of BHPI for 24 hr. * indicates a significant difference among groups using Student’s t-test (mean ± s.e.m., n = 6). (d) Trypan blue exclusion assay for cell death in T47D cells and BHPI-resistant clones after 24 hr treatment with vehicle, 100 nM BHPI or 1 μM BHPI. * indicates a significant difference among groups using one-way ANOVA followed by Tukey’s post hoc test (mean ± s.e.m., n = 3). (e) Western blot showing HMGB1 release into the cell culture medium in 100 nM and 1 μM of BHPI. (c,d) * P < 0.05, *** P < 0.001, ns not significant.

3.6. Overexpressing Src re-sensitizes resistant breast cancer cells to BHPI-stimulated UPR hyperactivation

To test whether down-regulation of Src was critical for acquisition of BHPI resistance and was rate-limiting for UPR hyperactivation, we transduced the cells with Src lentivirus or control luciferase lentivirus. Although the Src lentivirus increased Src levels in T47D cells, it did not alter levels of ERα and PLCγ (Supplementary Figure 7a, b). Consistent with a saturating and rate-limiting level of Src in wild type T47D cells, elevated Src levels only minimally increased BHPI-induced cell death, while Src knockdown significantly reduced cell death (Figure 6a). Notably, the higher Src level in T47D cells transduced with Src lentivirus greatly attenuated the effect of Src knockdown on BHPI-induced cell death (Figure 6a). Restoration of Src in BHPI-resistant cells was complicated by the surprising finding that BHPI treatment elicited rapid turnover of Src protein in the BHPI-resistant clones, but not in parental T47D cells. Treatment with 100 nM BHPI for 1 hour did not reduce Src levels in T47D cells, or in the TB8 clone (WT Src level), but reduced Src levels in TB1, TB3 and TB11 cells (Supplementary Figure 7c). Moreover, 1-hour treatment with 1 μM BHPI, but not 100 nM BHPI, significantly reduced Src expression in MB5 cells (Supplementary Figure 7d). Surprisingly, Src depletion was not blocked by the proteasome inhibitor MG132 (Supplementary Figure 7e). Thus, viral transduction may only partially restore Src levels and function in the BHPI-resistant clones. While restoring Src protein in BHPI-resistant TB1 cells did not significantly increase ATP depletion (Supplementary Figure 7f), it increased phosphorylation of eIF2α, and partially reversed BHPI inhibition of protein synthesis and cell proliferation (Figure 6b–d). To further validate our observation, we transduced TB11 BHPI resistant cells with Src expressing virus. Src restoration re-sensitized TB11 cells to BHPI induced ATP depletion and cell proliferation inhibition but had little effects in protein synthesis inhibition (Supplementary Figure 7g,i,k).

Figure 6.

Figure 6.

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In BHPI-resistant cells, Src overexpression partially restores sensitivity to BHPI. (a-h) For all virus transduction experiments, cells were cultured in medium containing virus for 24 hr, then kept in virus-free medium for 24 hr to allow protein overexpression. (a) Western blot of Src and β-actin and trypan blue exclusion viability assay of T47D cells transduced with luciferase-expressing or Src-expressing virus following knockdown with either 100 nM NC or Src siRNA SMARTpool. All cells were treated with 100 nM BHPI for 24 hours. Different letters indicate a significant difference among groups (P < 0.05) using one-way ANOVA followed by Tukey’s post hoc test (mean ± s.e.m., n = 3). (b) Western blot of Src, p-eIF2α, total eIF2α and β-actin protein levels in TB1 cells treated with vehicle or 100 nM BHPI for 1 hr after transduction with control or Src-expressing virus. (c) Protein synthesis in TB1 cells treated with vehicle or 100 nM BHPI for 1 hr after transduction with control or Src-expressing virus (mean ± s.e.m., n = 4). (d) MTS assay of TB1 cells treated with vehicle or 100 nM BHPI for 96 hr after transduction with control or Src-expressing virus (mean ± s.e.m., n = 6). (e) Western blot of Src and β-actin in MB5 cells treated with vehicle or 1 μM BHPI for 8 hr after transduction with luciferase-expressing or Src-expressing virus. (f) ATP level in MB5 cells treated with vehicle or 1 μM BHPI for 1 hr after transduction with control or Src-expressing virus (mean ± s.e.m., n = 4). (g) Protein synthesis in MB5 cells treated with vehicle or 1 μM BHPI for 1 hr after transduction with control or Src-expressing virus (mean ± s.e.m., n = 4). (h) MTS assay of MB5 cells treated with vehicle or 1 μM BHPI for 72 hr after transduction with control or Src-expressing virus (mean ± s.e.m., n = 6). (c,d,f,g,h) * indicates a significant difference among groups using Student’s t-test. * P< 0.05, ** P < 0.005.

Consistent with our observations in T47D cells, 1 μM BHPI greatly reduced Src protein in MCF-7-derived MB5 cells and viral transduction partially restored Src expression (Figure 6e). Restoring Src expression in MB5 and MB7 cells increased BHPI-mediated ATP depletion and inhibition of protein synthesis (Figure 6f,g, Supplementary Figure 7h,j). Notably, while 1 μM BHPI did not inhibit proliferation in MB5 and MB7 cells transduced with control luciferase virus, increasing Src levels by transduction with Src virus partially restored BHPI inhibition of cell proliferation (Figure 6h, Supplementary Figure 7l). These data show that restoring the level of Src oncogene in BHPI-resistant cells partially re-sensitizes them to BHPI-mediated UPR hyperactivation and cell death.

4. Discussion

How signals from hormones and biomodulators bound to steroid receptors elicit phosphorylation of PLCγ and activation of the anticipatory UPR was unknown. We show that a single activating kinase, Src, triggers both types of anticipatory UPR activation; the mild and transient tumor-protective mode and the strong and sustained cancer-targeting lethal mode (Graphical abstract). Although Src is abundant in ERα+ breast cancer cells, and has been reported to modulate estrogen-related reactive UPR activation and subsequent apoptotic cell death 30,31, its key role in activation of the hormone-mediated anticipatory UPR and in BHPI-mediated necrotic cell death unveils previously unexplored oncogenic and therapeutic mechanisms. Src’s role in rapid activation of the anticipatory UPR in cancer cells plays an enabling role in facilitating the diverse mitogenic activities of Src. In ERα+ breast cancer cells, acting through the anticipatory UPR, Src level and activity serve as a molecular rheostat integrating diverse signals. Activation of the UPR at diagnosis is strongly correlated with tumor recurrence, tamoxifen-resistance and a poor prognosis3. Consistent with our finding that constitutively active ERα mutations that are partially tamoxifen resistant elicit weak long-term activation of the anticipatory UPR21,23, in tamoxifen-resistant MCF-7 cells, sustained and gradually increasing Src activation is observed30.

While there have been several reports that Src activates PLCγ32–34, in the absence of a downstream PLCγ activated pathway, these studies did not attract a great deal of interest. Our finding that Src is a rate-limiting controller of the anticipatory UPR provides a framework and context that integrates these seemingly diverse observations33,35,36.

Usually resistance to an anticancer drug involves either mutation of its target, as is seen with aromatase inhibitors and ERα mutations in breast cancer29,37, or overexpression of the target protein as is seen with antiandrogens and androgen receptor (AR) in prostate cancer38. Unexpectedly we found that clonal cell lines identified in unbiased long-term selection for resistance to BHPI often exhibited down-regulation of pro-growth Src. Since UPR activation by classical UPR activators, tunicamycin and thapsigargin, was unaffected, it is likely that downregulation of Src was a key event in acquisition of BHPI resistance. Notably, Src overexpression partially re-sensitized resistant clones to BHPI-stimulated UPR hyperactivation. This is a partial effect because Src oncogene is up-regulated in breast cancers (Figure 1a) and plays important roles in cancer cell proliferation and survival39. Consequently, complete silencing of Src expression is likely to strongly reduce, or abolish, proliferation of the breast cancer cells. Thus, while Src is critical and rate-limiting for lethal BHPI hyperactivation of the anticipatory UPR, Src can only be down-regulated a few fold in BHPI-treated cells before the level of Src becomes too low to carry out its pro-proliferation and pro-survival functions. Therefore, in our selection, a moderate down-regulation of Src allows breast cancer cells to be minimally resistant to BHPI and still maintain other critical Src functions. These minimally resistant cells will proliferate very slowly and are under continued selection for additional changes that increase their resistance to BHPI and their rate of cell proliferation. Because, in addition to Src down-regulation, the mutant cells contain other changes that contribute to BHPI resistance, restoring the level of Src only partially restores sensitivity to BHPI.

Consistent with a regulatory role for degradation controlling Src level and ultimately activity, in several BHPI-resistant breast cancer cell lines, Src was rapidly degraded through a proteasome-independent mechanism (Supplementary Figure 7c, d, e). In normal cells, activated Src undergoes reversible protein dephosphorylation and ubiquitin-mediated protein degradation40,41. In cancer cells, therapeutic HSP90 inhibitors had been shown to enhance Src degradation42,43.

An important feature of the anticipatory UPR pathway is that the proteins in the early steps, ERα, Src and PLCγ have multiple functions in cells and only a fraction of each protein is likely localized in the multiprotein complex that initiates the anticipatory UPR. This has important consequences at multiple levels, including the observed phosphorylation of PLCγ and the partitioning of Src between multiple binding partners and pathways. For example, the tyrosine residues of PLCγ can be phosphorylated by both receptor and non-receptor tyrosine kinases, including EGFR, PDGFR and subunits of G protein. PLCγY783 phosphorylation shown in the western blots is due to both Src and other tyrosine kinases. While effects on PLCγ phosphorylation due to Src activation are significant, activation of Src is not the only pathway contributing to PLCγ phosphorylation. This is confirmed by our finding that blocking Src activation reduced PLCγ phosphorylation back to the basal level (0.99 in Fig. 1b and 0.98 in Fig. 1c), suggesting that basal PLCγ phosphorylation is independent of Src activity. Our findings that moderate down-regulation of Src leads to BHPI resistance in long-term culture and that 3–4 fold knockdown of Src is sufficient to strongly inhibit BHPI-induced cell death might seem surprising in light of the overexpression and abundance of Src in ERα+ breast cancer cells. Since only a small fraction of ERα is extranuclear near the plasma membrane, and much of the extranuclear ERα is devoted to activating ERK and other signaling pathways, total Src is in large excess over what is required to complex with ERα and PLCγ and activate the anticipatory UPR. Partitioning of limiting Src between the ERα- and PLCγ-regulated anticipatory UPR and other signal transduction pathways likely provides a novel mechanism for coordinating activity of the UPR and the growth-stimulating signaling pathways. Unlike Src, under normal conditions reducing the level ERα by siRNA knockdown does not reverse BHPI inhibition of cell proliferation (Supplementary Figure 4c). Under prolonged selection for BHPI resistance, the reduced level of the other components of the complex, Src and PLCγ can influence partitioning of limiting ERα into the complex; then reducing the level of ERα, seen in several of the clones, likely further reduces levels of the ERα:Src:PLCγ complex and makes a contribution to BHPI resistance. While this type of limited availability due to competition between different complexes is well studied in the context of transcription factors, such as CBP44, it has been less widely considered in the control of UPR activation.

Taken together, our findings that Src is down-regulated in BHPI-resistant clones, and that down-regulation of Src attenuates both the protective activation of the anticipatory UPR and BHPI-induced lethal hyperactivation of the anticipatory UPR strongly suggest elevated levels and activity of Src in breast cancer play a major role in key life-death decisions that surround activation of the anticipatory UPR in cancer cells (Supplementary Figure 8).

Supplementary Material

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Highlights.

Src regulates estrogen and progesterone activation of the anticipatory UPR

Inhibiting Src blocks the Ca2+ increase that triggers anticipatory UPR activation

Src levels control cancer cell death by the anticipatory UPR hyperactivator, BHPI

Breast cancer cell lines selected for BHPI resistance often down-regulate Src

Acknowledgements

This research was supported by grants NIH [RO1DK071909], DOD [BCRPW81XWH-13], the E. Howe Scholar Award to DS and a C.F. Kade fellowship to LY. We are grateful to Dr. Kai Zheng for use of his microscope in calcium imaging.

Footnotes

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Conflict of Interest

The authors declare no conflict of interest.

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Supplementary Materials

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