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PDF file - 28K, Supplementary Figure 2. A, the effects of the six most potent siRNA screen hits on IL-11 secretion in MDA-MB-231(SA) cells (n=3). B, the effects of heparan sulfate modifying enzymes on IL-11 secretion in MDA-MB-231(SA) cells (n=4). *, P < 0.05; **, P

Supplementary Figure 1 PDF file - 3511K, BCL2 is elevated in antiestrogen-resistant breast cancer

XLS file - 49K, Supplementary Table 2. Effects of 196 siRNAs (including 2 siRNAs targeting IL11) on TGF-�-induced IL-11 production in MDA-MB-231(SA) cells. The relative IL-11 concentration values were obtained by dividing the IL-11 concentration by the amount of viable cells in the well (as determined by the CellTiter-Blue assay). The values are shown relative to the average values for the negative control siRNA.

Supplementary Figure 2 PDF file - 56K, GX induces apoptosis in breast cancer cells

Supplementary Figure 5 PDF file - 51K, Cathepsin mRNA expression is unaffected by GX exposure

TGF-β regulates several steps in cancer metastasis, including the establishment of bone metastatic lesions. TGF-β is released from bone during osteoclastic bone resorption and it stimulates breast cancer cells to produce osteolytic factors such as interleukin 11 (IL-11). We conducted a cell-based siRNA screen and identified heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) as a critical gene for TGF-β–induced IL-11 production in highly bone metastatic MDA-MB-231(SA) breast cancer cells. HS6ST2 attaches sulfate groups to glucosamine residues in heparan sulfate glycosaminoglycans. We subsequently showed how heparin and a high-molecular-weight Escherichia coli K5-derived heparin-like polysaccharide (K5-NSOS) inhibited TGF-β–induced IL-11 production in MDA-MB-231(SA) cells. In addition, K5-NSOS inhibited bone resorption activity of human osteoclasts in vitro. We evaluated the therapeutic potential of K5-NSOS and fragmin in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) cells were inoculated into the left cardiac ventricle of athymic nude mice which were treated with fragmin, K5-NSOS, or vehicle once a day for four weeks. Both heparin-like glycosaminoglycans inhibited weight reduction, decreased osteolytic lesion area, and reduced tumor burden in bone. In conclusion, our data imply novel mechanisms involved in TGF-β induction and support the critical role of heparan sulfate glycosaminoglycans in cancer metastasis as well as indicate that K5-NSOS is a potential antimetastatic and antiresorptive agent for cancer therapy. This study illustrates the potential to translate in vitro siRNA screening results toward in vivo therapeutic concepts. Mol Cancer Res; 10(5); 597–604. ©2012 AACR.

PDF file - 6K, Supplementary Table 1. Primers and probes used for quantitative RT-PCR.

Supplementary Figure 4 PDF file - 29K, Knockdown of BECN1 by siRNA does not prevent GX-mediated inhibition of cell density

PDF file - 38K, Supplementary Figure 3. A, expression of HS6ST2 mRNA in MDA-MB-231(SA) versus parental MDA-MB-231 cells. B, validation of the knockdown efficacies of siRNAs by quantitative RT-PCR.

PDF file - 25K, Supplementary Figure 1. Basal and TGF-b-induced IL-11 production in parental MDA-MB-231 and highly metastatic MDA-MB-231(SA) cells. ***, P < 0.001 (t test).

Supplementary Figure 3 PDF file - 89K, Caspase inhibition fails to abrogate GX-mediated apoptosis

Supplementary Figure Legends PDF file - 65K, Legends for supplemental figures

Circulating mouse cytokine and chemokine involvement. A. ELISA analysis of IL-1β, IL-3, IL-4, IL-5, IL-6, IL-12p70, and IL-17 from mice treated with control, CQ, ICI, ICI+CQ, TAM, and TAM+CQ. n=3-6

Circulating mouse cytokine and chemokine involvement. A. Circulated levels of MCP-1, RANTES, Eotaxin, KC, MDC, TARC, and TCA3 were measured by ELISA from mouse plasma. n=3-6

Effect of antiestrogen therapy in vivo. A. LCC9 control, TAM, and ICI treated tumors sections were stained with an antibody against ERα or the progesterone receptor to show antiestrogen effectiveness.

Purpose: Only few predictive factors for the clinical activity of anti–epidermal growth factor receptor (EGFR) therapy are available. Mammary-derived growth inhibitor (MDGI) is a small cytosolic protein suggested to play a role in the differentiation of epithelial cells. Here, we have investigated the effect of MDGI expression on the EGFR signaling and cetuximab responsiveness of cancer cells.Experimental Design: MDGI mRNA expression was investigated in clinical breast and lung cancer samples and in nontransformed and malignant cell lines. The effect of ectopic expression of MDGI on EGFR, ErbB2, and integrin function and traffic was investigated in breast and lung cancer cell lines using multiple methods. The effect of anti-EGFR agents on these cells were tested by cell proliferation measurements and by assessing tumor growth of breast cancer cells in cetuximab treated and control athymic nude mice.Results: Here, we show that although MDGI is absent in cultured cell lines because of epigenetic silencing, MDGI mRNA is expressed in 40% of clinical breast carcinomas and 85% of lung cancers. Ectopic expression of MDGI rendered breast and lung cancer cells resistant to the anti-EGFR antibody cetuximab in vitro and in an orthotopic breast cancer xenograft model in vivo. When expressed in cancer cells, MDGI induces intracellular accumulation of EGFR, but not ErbB2, and the internalized receptor is phosphorylated and not degraded.Conclusions: MDGI-driven inherent desensitization of cancer cells is a novel molecular mechanism for resistance to the anti-EGFR antibody therapy, and MDGI may be a biomarker for responsiveness to anti-EGFR antibody therapy.(Clin Cancer Res 2009;15(21):6570–81)

Circulating mouse cytokine and chemokine involvement. A. Interferon-γ and TNFÎ{plus minus} were measured by ELISA in plasma from mice treated with control, CQ, ICI, ICI+CQ, TAM, and TAM+CQ. n=3-6

Supplementary Data from Mammary-Derived Growth Inhibitor Alters Traffic of EGFR and Induces a Novel Form of Cetuximab Resistance

Antiestrogens effect angiogenesis. A. LCC9 cells were treated with vehicle, 1 Î1/4M CQ, 100 nM ICI, or 100 nM TAM for 72 hours and protein homogenates were collected. Western blot hybridization was used to confirm levels of pVEGFR2 and total VEGFR2. Gel loaded was confirmed by β-actin. n=3, *p

Autophagic gene expression in human breast cancer. A. Autophagy genes significantly regulated in early versus never reoccurring breast cancers. B. Breast cancer survival in patients with high/low ATG7 and BECN1 expression.

Purpose: Estrogen receptor-α (ERα)-targeted therapies including tamoxifen (TAM) or Faslodex (ICI) are used to treat ER+ breast cancers. Up to 50% of tumors will acquire resistance to these interventions. Autophagy has been implicated as a major driver of antiestrogen resistance. We have explored the ability of chloroquine (CQ), which inhibits autophagy, to affect antiestrogen responsiveness.Experimental Design: TAM-resistant MCF7-RR and ICI-resistant/TAM cross-resistant LCC9 ER+ breast cancer cells were injected into mammary fat pads of female athymic mice and treated with TAM and/or ICI in combination with oral low-dose CQ.Results: We show that CQ can increase antiestrogen responsiveness in MCF7-RR and LCC9 cells and tumors, likely through the inhibition of autophagy. However, the combination of ICI+CQ was less effective than CQ alone in vivo, unlike the TAM+CQ combination. Antiestrogen treatment stimulated angiogenesis in tumors but did not prevent CQ effectiveness. The lower efficacy of ICI+CQ was associated with ICI effects on cell-mediated immunity within the tumor microenvironment. The mouse chemokine KC (CXCL1) and IFNγ were differentially regulated by both TAM and ICI treatments, suggesting a possible effect on macrophage development/activity. Consistent with these observations, TAM+CQ treatment increased tumor CD68+ cells infiltration, whereas ICI and ICI+CQ reduced peripheral tumor macrophage content. Moreover, macrophage elimination of breast cancer target cells in vitro was reduced following exposure to ICI.Conclusion: CQ restores antiestrogen sensitivity to resistant tumors. Moreover, the beneficial combination of TAM+CQ suggests a positive outcome for ongoing neoadjuvant clinical trials using this combination for the treatment of ER+ ductal carcinoma in situ lesions. Clin Cancer Res; 20(12); 3222–32. ©2014 AACR.

PDF file - 388K, GRP78 promotes survival signaling schematic

Patient/tumor characteristics of human breast cancer tissue microarrays.

PDF file - 78K, Cell surface GRP78 localization does not mediate the cells anti-estrogen responsiveness

LCC9 (A) or ZR-75-1 (B) cells were transfected with control siRNA, GRP78 siRNA, or GRP78 cDNA for 24 hours and then plated in an ACEA E-plate in the presence of vehicle or 100 nM 4-OHT; cell index was measured by electrical impedance every 12 hours for 72 hours. n=3, *p

Relationship between IRF1 and ATG7 in human tissue microarrays.

PDF file - 46K, Elevated LC3-II protein levels are observed in anti-estrogen resistant cell lines

Efficient knockdown of IRF1 using siRNA.

A. Heat map of lipid metabolites in LCC1 and LCC9 cells transfected with control or GRP78 siRNA and treated with or without TAM. n=6. B. Relative levels of oleic ethanolamide and palmitoyl ethanolamide in LCC1 and LCC9 cells treated with vehicle, TAM, GRP78 siRNA or GRP78 siRNA+TAM. n=6. C. LCC9 cells were treated with 10 nM -1 microM oleic ethanolamide (OEA), and/or various doses (vehicle, 10 nM, 100 nM, 1000 nM) TAM for 6 days. Relative cell density was determined by crystal violet assay. n=4, *p

Interferon regulatory factor-1 (IRF1) is a tumor suppressor that regulates cell fate in several cell types. Here, we report an inverse correlation in expression of nuclear IRF1 and the autophagy regulator ATG7 in human breast cancer cells that directly affects their cell fate. In mice harboring mutant Atg7, nuclear IRF1 was increased in mammary tumors, spleen, and kidney. Mechanistic investigations identified ATG7 and the cell death modulator beclin-1 (BECN1) as negative regulators of IRF1. Silencing ATG7 or BECN1 caused estrogen receptor-α to exit the nucleus at the time when IRF1 nuclear localization occurred. Conversely, silencing IRF1 promoted autophagy by increasing BECN1 and blunting IGF1 receptor and mTOR survival signaling. Loss of IRF1 promoted resistance to antiestrogens, whereas combined silencing of ATG7 and IRF1 restored sensitivity to these agents. Using a mathematical model to prompt signaling hypotheses, we developed evidence that ATG7 silencing could resensitize IRF1-attenuated cells to apoptosis through mechanisms that involve other estrogen-regulated genes. Overall, our work shows how inhibiting the autophagy proteins ATG7 and BECN1 can regulate IRF1-dependent and -independent signaling pathways in ways that engender a new therapeutic strategy to attack breast cancer. Cancer Res; 75(6); 1046–55. ©2015 AACR.

Silencing ATG7 and BECN1 induces IRF1 expression independently of ROS.

PDF file - 132K, Representative Western blot images of data quantified in Table 1

RT-PCR gene expression of SCD1 (A), FASN (B), ACC (C), and CPT1A (D) was measured in LCC1 and LCC9 breast cancer cells transfected with control or GRP78 siRNA for 72 hours and normalized with HRPT housekeeping gene expression. n=3; *p

The unfolded protein response is an endoplasmic reticulum stress pathway mediated by the protein chaperone glucose regulated-protein 78 (GRP78). Metabolic analysis of breast cancer cells shows that GRP78 silencing increases the intracellular concentrations of essential polyunsaturated fats, including linoleic acid. Accumulation of fatty acids is due to an inhibition of mitochondrial fatty acid transport, resulting in a reduction of fatty acid oxidation. These data suggest a novel role of GRP78-mediating cellular metabolism. We validated the effect of GRP78-regulated metabolite changes by treating tumor-bearing mice with tamoxifen and/or linoleic acid. Tumors treated with linoleic acid plus tamoxifen exhibited reduced tumor area and tumor weight. Inhibition of either GRP78 or linoleic acid treatment increased MCP-1 serum levels, decreased CD47 expression, and increased macrophage infiltration, suggesting a novel role for GRP78 in regulating innate immunity. GRP78 control of fatty acid oxidation may represent a new homeostatic function for GRP78. Cancer Res; 76(19); 5657–70. ©2016 AACR.

Nuclear IRF1 is induced following ATG7 and BECN1 knockdown.

dnIRF1 blocks ICI sensitivity in LCC1 cells.

Model ranges, their range, and description.

Knockdown of IRF1 alters BECN1 and IGF1R/mTOR expression.

A. LCC9 breast cancer cells were transfected with control siRNA, GRP78 siRNA, control pcDNA, or GRP78 cDNA for 72 hours and CD47, calreticulin, and HMGB1 protein expression measured by Western blot hybridization. B. LCC9 xenografts from control mice or mice treated with TAM, GRP78M, or GRP78M+TAM were stained for CD47 and visualized at 20X. n=6; *p

Supplementary figure legends

We previously showed that environmentally-induced epigenetic inheritance of cancer occurs in rodent models. For instance, we reported that paternal consumption of an obesity-inducing diet (OID) increased breast cancer susceptibility in the offspring (F1). Nevertheless, it is still unclear whether programming of breast cancer in daughters is due to systemic alterations or mammary epithelium-specific factors and whether the breast cancer predisposition in F1 progeny can be transmitted to subsequent generations. In this study, we show that mammary glands from F1 control (CO) female offspring exhibit enhanced growth when transplanted into OID females compared to CO mammary glands transplanted into CO females. Similarly, carcinogen-induced mammary tumors from F1 CO female offspring transplanted into OID females has a higher proliferation/apoptosis rate. Further, we show that granddaughters (F2) from the OID grand-paternal germline have accelerated tumor growth compared to CO granddaughters. This between-generation transmission of cancer predisposition is associated with changes in sperm tRNA fragments in OID males. Our findings indicate that systemic and mammary stromal alterations are significant contributors to programming of mammary development and likely cancer predisposition in OID daughters. Our data also show that breast cancer predisposition is transmitted to subsequent generations and may explain some familial cancers, if confirmed in humans.

BackgroundWhile genetics explains some familial breast cancer cases, we showed that environmentally-induced epigenetic inheritance of breast cancer can also occur in rodent models. We previously reported that paternal consumption of a high-fat diet and ensuing obesity increased breast cancer susceptibility in the offspring (F1). Nevertheless, it is still unclear whether paternal-induced programming of breast cancer in daughters is associated with systemic alterations or mammary epithelium-specific factors. It also remains to be determined whether the ancestrally programmed breast cancer predisposition in F1 progeny can be transmitted to subsequent generations.MethodsMale mice (F0) were fed either a control (CO) diet or an obesity-inducing diet (OID) for seven weeks and then mated with female mice (F0) reared on a CO diet. The resulting offspring (F1), also exclusively fed CO diet, were either used for mammary gland and tumor transplantation surgeries or to generate the F2 generation. To induce the mammary tumors, female mice were treated with 7,12 dimethylbenz[a]anthracene (DMBA). Total RNA extracted from F0 or F1 males sperm was used for small RNA-Seq analysis.ResultsMammary glands from F1 CO female offspring exhibited enhanced development when transplanted into OID females [OID(CO-MG)], as shown by higher mammary gland area, epithelial branching and elongation, compared to CO females that received a CO mammary gland [CO(CO-MG)]. Similarly, mammary tumors from F1 CO female offspring transplanted into OID females [OID(CO.T)] displayed improved growth with a higher proliferation/apoptosis rate. We also found that granddaughters (F2) from the OID grand-paternal germline showed accelerated tumor growth compared to COxCO granddaughters (F2). Transmission of breast cancer predisposition to the F2 generation through OID male germline was associated with alterations in specific sperm tRNA fragments (tRF) in both F0 and F1 males.ConclusionsOur findings indicate that systemic metabolic and mammary stromal alterations are the most significant contributors to paternal programming of mammary gland development and cancer predisposition in female offspring rather than mammary epithelium confined factors. Our data also show breast cancer predisposition in OID daughters can be transmitted to subsequent generations and could explain some familial cancers, if confirmed in humans.

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.

Purpose: Whether it is safe for estrogen receptor–positive (ER+) patients with breast cancer to consume soy isoflavone genistein remains controversial. We compared the effects of genistein intake mimicking either Asian (lifetime) or Caucasian (adulthood) intake patterns to that of starting its intake during tamoxifen therapy using a preclinical model. Experimental Design: Female Sprague-Dawley rats were fed an AIN93G diet supplemented with 0 (control diet) or 500 ppm genistein from postnatal day 15 onward (lifetime genistein). Mammary tumors were induced with 7,12-dimethylbenz(a)anthracene (DMBA), after which a group of control diet–fed rats were switched to genistein diet (adult genistein). When the first tumor in a rat reached 1.4 cm in diameter, tamoxifen was added to the diet and a subset of previously only control diet–fed rats also started genistein intake (post-diagnosis genistein). Results: Lifetime genistein intake reduced de novo resistance to tamoxifen, compared with post-diagnosis genistein groups. Risk of recurrence was lower both in the lifetime and in the adult genistein groups than in the post-diagnosis genistein group. We observed downregulation of unfolded protein response (UPR) and autophagy-related genes (GRP78, IRE1α, ATF4, and Beclin-1) and genes linked to immunosuppression (TGFβ and Foxp3) and upregulation of cytotoxic T-cell marker CD8a in the tumors of the lifetime genistein group, compared with controls, post-diagnosis, and/or adult genistein groups. Conclusions: Genistein intake mimicking Asian consumption patterns improved response of mammary tumors to tamoxifen therapy, and this effect was linked to reduced activity of UPR and prosurvival autophagy signaling and increased antitumor immunity. Clin Cancer Res; 23(3); 814–24. ©2017 AACR.

In the mammary gland, vimentin intermediate filaments are expressed in stromal cells and in basal epithelial cell populations, including gland-reconstituting mammary stem cells, with largely undefined functions. Here, we have studied how vimentin deficiency affects mouse mammary gland development. We find that, in adult vimentin knockout mice (Vim−/−), mammary ductal outgrowth is delayed. The adult Vim−/− glands display dilated ducts and a reduced basal-to-luminal mouse mammary epithelial cell (MMEC) ratio indicative of altered progenitor cell activity. Accordingly, isolated Vim−/− MMECs form fewer mammospheres and basal-like organoids in vitro than their wild-type counterparts. Importantly, reduced basal MMEC number translates into defects in Vim−/− mammary gland regeneration in vivo. Global gene expression profiling of basal MMECs reveals that lack of vimentin alters multiple pathways, including adhesion, cancer and Wnt signalling. Furthermore, vimentin contributes to stem-like cell properties in MDA-MB-231 breast cancer cells, wherein vimentin depletion reduces tumoursphere formation and attenuates expression of breast cancer stem cell-associated surface markers. Together, our findings identify vimentin as a positive regulator of stemness in the developing mouse mammary gland and in breast cancer cells.

Antiestrogen therapy induces the unfolded protein response (UPR) in estrogen receptor-positive (ER(+)) breast cancer. X-box binding protein 1 (XBP1), which exists in the transcriptionally inactive unspliced form [XBP1(U)] and the spliced active form [XBP1(S)], is a key UPR component mediating antiestrogen resistance. We now show a direct link between the XBP1 and NF-κB survival pathways in driving the cell fate decisions in response to antiestrogens in ER(+) breast cancer cells, both in vitro and in a xenograft mouse model. Using novel spliced and nonspliceable forms of XBP1, we show that XBP1(U) functions beyond being a dominant negative of XBP1(S). Both isoforms regulate NF-κB activity via ERα; XBP1(S) is more potent because it also directly regulates p65/RelA expression. These findings provide new insights into the fundamental signaling activities of spliced and unspliced XBP1 in breast cancer, establish NF-κB to be a mediator of these activities, and identify XBP1 and its splicing to be novel therapeutic targets.

In the mammary gland, vimentin intermediate filaments are expressed in stromal cells and in basal epithelial cell populations including gland-reconstituting mammary stem cells (MaSC), with largely undefined functions. Here, we studied how vimentin deficiency affects mouse mammary gland development. Our results demonstrate that in adult vimentin knockout mice (Vim-/-) mammary ductal outgrowth is delayed. The adult Vim-/- glands are characterised by dilated ducts, an imbalance in the proportion of basal to luminal mammary epithelial cells and a reduction in cells expressing Slug (Snai2), an established MaSC regulator. All of these features are indicative of reduced progenitor cell activity. Accordingly, isolated Vim-/- mammary epithelial cells display reduced capacity to form mammospheres, and altered organoid structure, compared to wt counterparts, when plated in a 3D matrix in vitro. Importantly, altered basal epithelial cell number translates into defects in Vim-/- mammary gland regeneration in vivo in cleared fat pad transplantation studies. Furthermore, we show that vimentin contributes to stem-like cell properties in triple negative MDA-MB-231 breast cancer cells, wherein vimentin depletion reduces tumorsphere formation and alters expression of breast cancer stem cell-associated surface markers. Together, our findings identify vimentin as a positive regulator of stemness in the developing mouse mammary gland and in breast cancer cells.

SHARPIN is a widely expressed multifunctional protein implicated in cancer, inflammation, linear ubiquitination and integrin activity inhibition; however, its contribution to epithelial homeostasis remains poorly understood. Here, we examined the role of SHARPIN in mammary gland development, a process strongly regulated by epithelial–stromal interactions. Mice lacking SHARPIN expression in all cells ( Sharpin cpdm ), and mice with a stromal ( S100a4‐Cre ) deletion of Sharpin, have reduced mammary ductal outgrowth during puberty. In contrast, Sharpin cpdm mammary epithelial cells transplanted in vivo into wild‐type stroma, fully repopulate the mammary gland fat pad, undergo unperturbed ductal outgrowth and terminal differentiation. Thus, SHARPIN is required in mammary gland stroma during development. Accordingly, stroma adjacent to invading mammary ducts of Sharpin cpdm mice displayed reduced collagen arrangement and extracellular matrix (ECM) stiffness. Moreover, Sharpin cpdm mammary gland stromal fibroblasts demonstrated defects in collagen fibre assembly, collagen contraction and degradation in vitro . Together, these data imply that SHARPIN regulates the normal invasive mammary gland branching morphogenesis in an epithelial cell extrinsic manner by controlling the organisation of the stromal ECM.