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This study identifies macroautophagy as a key mechanism of cell survival in estrogen receptor–positive (ER+) breast cancer cells undergoing treatment with 4-hydroxytamoxifen (4-OHT). This selective ER modifier is an active metabolite of tamoxifen commonly used for the treatment of breast cancer. Our study provides the following key findings: (a) only 20% to 25% of breast cancer cells treated with 4-OHT in vitro die via caspase-dependent cell death; more typically, the antiestrogen-treated ER+ breast cancer cells express increased levels of macroautophagy and are viable; (b) 4-OHT–induced cell death, but not 4-OHT–induced macroautophagy, can be blocked by the pan-caspase inhibitor z-VAD-fmk, providing strong evidence that these two outcomes of antiestrogen treatment are not linked in an obligatory manner; (c) 4-OHT–resistant cells selected from ER+ breast cancer cells show an increased ability to undergo antiestrogen-induced macroautophagy without induction of caspase-dependent cell death; and (d) 4-OHT, when used in combination with inhibitors of autophagosome function, induces robust, caspase-dependent apoptosis of ER+, 4-OHT–resistant breast cancer cells. To our knowledge, these studies provide the first evidence that macroautophagy plays a critical role in the development of antiestrogen resistance. We propose that targeting autophagosome function will improve the efficacy of hormonal treatment of ER+ breast cancer. [Mol Cancer Ther 2008;7(9):2977–87]

Supplementary Fig. S1 from A role for macroautophagy in protection against 4-hydroxytamoxifen–induced cell death and the development of antiestrogen resistance

EGFR is upregulated commonly in multiple cancer types, including breast cancer. However, the multiple role(s) of EGFR in breast cancer are not fully elucidated. In this study, we characterized a unique antiestrogen resistant breast cancer subline and provide compelling data that upregulation of the EGFR/MEK1/MAPK1/2 signaling axis during antiestrogen treatment facilitated escape from senescence, autophagy, and BimEL-dependent apoptosis. Our studies were conducted with the antiestrogen resistant TR5 cell line established by our laboratory, using a step-wise drug selection with the antiestrogen 4-hydroxytamoxifen (4-OHT) of the estrogen receptor α (ER+) MCF-7 breast cancer cells. A preliminary microarray comparing mRNA expression between 4-OHT-selected TR5 cells and MCF-7 parent cells identified upregulation of the epidermal growth factor receptor (EGFR). Western blot showed a greater than 5-fold upregulation of EGFR expression and downregulation of estrogen receptor alpha (ERα) compared to the levels in MCF-7 and T47-D (antiestrogen sensitive). Selective inhibition of EGFR, phosphorylated at Tyr 1068, was achieved with erlotinib. Inhibition blocked MEK1/MAPK1/2 signaling, which was also upregulated in 4-OHT selected TR5 cells, with detectable increases in the pro-apoptotic BH3 protein BimEL, and a fifty percent reduction in cell viability. However, a high number of cells survived erlotinib therapy due to autophagy induction determined to be cytoprotective. Autophagy induction was identified by analyzing the expression/turnover of autophagy proteins LC3-II and p62, a standard approach to ascertain autophagy levels in cells. Further, erlotinib treatment induced a 2-fold increase in the number of senescent cells in the surviving population. Senescent cells were identified by β-galactosidase staining of erlotinib-treated cells utilizing confocal microscopy. Interestingly, blockade of autophagy with the lysosomotrophic compound chloroquine enhanced the senescence cell population, suggesting that either autophagy blocked senescence induction or lysosome impairment increased senescence. Mechanistically, we have identified phosphorylated AMP-activated protein kinase (pAMPK) as being elevated under conditions of erlotinib treatment, and reduced in 4-OHT-selected TR5 cells relative to MCF-7 parent cells. This correlation supports the hypothesis that pAMPK was downregulated during 4-OHT selection of TR5 cells by elevated EGFR/MEK/MAPK1/2 signaling to allow surviving cells escape senescence induction and/or anti-proliferative autophagy. Overall we propose that EGFR mediated downregulation of pAMPK is a major regulatory pathway in breast cancer cells surviving endocrine treatment. Our current studies are dissecting the role of EGFR-mediated AMPK regulation with somatic cell-based studies and pharmacological intervention. Citation Format: Mackenzie L. Hagan, Michael McGrath, Carol Joseph, Allison Lewis, John R. Barrett, Muthusamy Thangaraju, David Gewirtz, Patricia V. Schoenlein. EGFR upregulation in breast cancer cells: A key mechanism of escape from antiestrogen induced death, autophagy, and senescence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB133.

Our long-term goal is to elucidate the role of JNK/Jun signaling in breast cancer cell autophagy induced by anti-estrogen treatment and/or chemotherapy in ERα expressing and TNBC cells, respectively. In this study, we analyzed the role of JNK/Jun signaling in regulating the high basal autophagy levels in TNBC. To do so, we utilized the highly metastatic murine 4T1 and the human MDA-MB-231 TNBC models. In addition, we performed autophagic flux assays after treating breast cancer cells with JNK-IN8, a non-reversible small molecule JNK1/2 inhibitor. Treatments were performed for various times in the presence and absence of chloroquine (CQ) to allow analysis of steady state levels of atg8 (LC3-II) and sequestosome-1 (p62). These LC3-II and p62 proteins are involved in autophagosome formation and function, and are catabolized during autolysosomal turnover. To impair this process, CQ is a lysosomotropic agent that raises the pH of the lysosome and blocks such turnover. We also determined the effect of JNK-IN-8 on 4T1 and MDA-MB-231 cell number (proliferation). These studies determined that JNK-IN8 was an effective inhibitor of autophagic flux in the TNBC cells when used at concentrations that effectively blocked cJun phosphorylation, the downstream readout of JNK-IN8 efficacy used for our experiments. Blockade of autophagic flux by JNK-1/2 inhibition in TNBC was in stark contrast to results obtained with JNK-IN-8 treatment of ERα expressing MCF-7 and T47-D breast cancer cells, in which JNK-IN-8 induced a robust autophagy response with elevations in the steady state levels and flux of LC3-II and p62. Induction of autophagy by JNK-IN-8 in ERα expressing breast cancer cells occurred concomitantly with a substantial reduction in cell proliferation but minimal induction of apoptosis. Overall, these pre-clinical in vitro studies support the following conclusions: (1) JNK1/2 targeting in TNBC effectively blocks autophagic flux and may be particularly effective when used in combination with chemotherapies that induce pro-survival autophagy which is a current focus of our studies; and (2) JNK1/2 targeting in ERα expressing breast cancers has the potential to mediate a pro-survival autophagic activity in response to endocrine therapy or chemotherapy and should be cautiously approached. Ongoing studies are aimed at understanding the underlying mechanisms of the differential effects of JNK/Jun signaling on autophagy in TNBC versus ERα expressing breast cancer. Such an understanding should provide important information to aid in the ongoing interest in utilizing JNK1/2 as a molecular target for improved treatment of TNBC. Citation Format: Michael K. McGrath, McKenzie L. Hagan, Timmothy Summers, Jesse Wayson, Carol Joseph, Amanda C. Barrett, John T. Barrett, Patricia V. Schoenlein. Effects of JNK/Jun blockade on breast cancer cell autophagy are dependent on breast cancer subtype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3006.

// Feiyan Liu 1 , Xia Li 1 , Chunwan Lu 2,3 , Aiping Bai 4 , Jacek Bielawski 4 , Alicja Bielawska 4 , Brendan Marshall 5 , Patricia V. Schoenlein 5 , Iryna O. Lebedyeva 6 and Kebin Liu 2,3,7 1 College of Life Sciences, Zhejiang University, Hangzhou, China 2 Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA, USA 3 Charlie Norwood VA Medical Center, Augusta, GA, USA 4 Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA 5 Department of Cell Biology and Anatomy, Medical College of Georgia, Augusta, GA, USA 6 Department of Chemistry and Physics, Augusta University, Augusta, GA, USA 7 Georgia Cancer Center, Augusta University, Augusta, GA, USA Correspondence to: Feiyan Liu, email: // Keywords : MDSCs, cathepsin, ceramide, autophagy flux, Lysosomal cell death, Immunology and Microbiology Section, Immune response, Immunity Received : October 10, 2016 Accepted : November 07, 2016 Published : November 17, 2016 Abstract Myeloid-derived suppressor cells (MDSCs) are immune suppressive cells that are hallmarks of human cancer. MDSCs inhibit cytotoxic T lymphocytes (CTLs) and NK cell functions to promote tumor immune escape and progression, and therefore are considered key targets in cancer immunotherapy. Recent studies determined a key role of the apoptosis pathways in tumor-induced MDSC homeostasis and it is known that ceramide plays a key role in regulation of mammalian cell apoptosis. In this study, we aimed to determine the efficacy and underlying molecular mechanism of ceramide in suppression of MDSCs. Treatment of tumor-bearing mice with LCL521, a lysosomotropic inhibitor of acid ceramidase, significantly decreased MDSC accumulation in vivo . Using a MDSC-like myeloid cell model, we determined that LCL521 targets lysosomes and increases total cellular C16 ceramide level. Although MDSC-like cells have functional apoptosis pathways, LCL521-induced MDSC death occurs in an apoptosis- and necroptosis-independent mechanism. LCL521 treatment resulted in an increase in the number of autophagic vesicles, heterolysosomes and swollen ERs. Finally, concomitant inhibition of cathepsin B and cathepsin D was required to significantly decrease LCL521-induced cell death. Our observations indicate that LCL521 targets lysosomes to activate cathepsin B and cathepsin D, resulting in interrupted autophagy and ER stress that culminates in MDSC death. Therefore, a ceramidase inhibitor is potentially an effective adjunct therapeutic agent for suppression of MDSCs to enhance the efficacy of CTL-based cancer immunotherapy.

The c-Jun N-terminal kinases, JNK1/2 are members of the mitogen activated protein kinase (MAPK) family that can mediate inflammatory responses, cell differentiation, cell proliferation, and/or cell death. In breast cancer, JNK signaling has been associated with increased proliferation, angiogenesis, and tumor metastasis. Also, inhibition of JNK has been shown to promote tumor cytostasis, but this inhibition also attenuates the cytotoxic effects of certain chemotherapeutics, calling into question the therapeutic potential of JNK inhibitors. In this study, we hypothesized that JNK1/2 inhibition induced autophagy, as a mechanism of protection against chemotherapy- and endocrine-induced apoptosis. Autophagy is a normal physiologic process required for the elimination of damaged mitochondria and misfolded proteins that can be induced in cancer cells under certain stresses such as hypoxia and drug insult to provide protection from death. To characterize the role of JNK1/2 in autophagy, we utilized two ERα expressing human breast cancer cell lines, T47-D and MCF-7. Autophagy flux experiments were performed in which the steady state levels of the autophagy protein LC3 II were analyzed in control cells versus cells undergoing JNK1/2 inhibition with the non-reversible JNK-IN8 inhibitor. JNK-IN8 was used as a single agent or in combination with estradiol and/or antiestrogen treatment. These studies reproducibly identified JNK1/2 inhibition as a mechanism of autophagy activation. The induction of autophagy correlated with sustained inhibition of proliferation as determined by cell counts, MTT, and clonogenic assays, and induction of senescence (in T47-D cells) as determined by the expression of β-galactosidase. In contrast, induction of apoptosis was not a major outcome of JNK1/2 inhibition. These pre-clinical studies provide strong evidence that JNK1/2 is an attractive molecular target to improve the treatment of endocrine responsive breast cancer, but they also emphasize that cells are surviving by autophagy and/or senescence. Because cell survival by autophagy and/or senescence could ultimately lead to breast cancer relapse, ongoing studies are addressing approaches to effectively kill these surviving cell populations. Citation Format: Kyler Herrington, Carol Joseph, Samar Abdulmoneim, Allison Lewis, Jingwen Cai, Hannah Youngblood, Yutao Liu, Patricia Schoenlein. Targeting JNK1/2 induces a potent cytostatic response in estrogen receptor expressing breast cancer cells, with induction of autophagy and senescence as measurable outcomes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1231.

SLC5A8 is a putative tumor suppressor that is inactivated in more than 10 different types of cancer, but neither the oncogenic signaling responsible for SLC5A8 inactivation nor the functional relevance of SLC5A8 loss to tumor growth has been elucidated. Here, we identify oncogenic HRAS (HRAS(G12V)) as a potent mediator of SLC5A8 silencing in human nontransformed normal mammary epithelial cell lines and in mouse mammary tumors through DNMT1. Further, we demonstrate that loss of Slc5a8 increases cancer-initiating stem cell formation and promotes mammary tumorigenesis and lung metastasis in an HRAS-driven murine model of mammary tumors. Mammary-gland-specific overexpression of Slc5a8 (mouse mammary tumor virus-Slc5a8 transgenic mice), as well as induction of endogenous Slc5a8 in mice with inhibitors of DNA methylation, protects against HRAS-driven mammary tumors. Collectively, our results provide the tumor-suppressive role of SLC5A8 and identify the oncogenic HRAS as a mediator of tumor-associated silencing of this tumor suppressor in mammary glands. These findings suggest that pharmacological approaches to reactivate SLC5A8 expression in tumor cells have potential as a novel therapeutic strategy for breast cancer treatment.

The NAD-dependent histone deacetylase silent information regulator 1 (SIRT1) is overexpressed and catalytically activated in a number of human cancers, but recent studies have actually suggested that it may function as a tumor suppressor and metastasis inhibitor in vivo. In breast cancer, SIRT1 stabilization has been suggested to contribute to the oncogenic potential of the estrogen receptor α (ERα), but SIRT1 activity has also been associated with ERα deacetylation and inactivation. In this study, we show that SIRT1 is critical for estrogen to promote breast cancer. ERα physically interacted and functionally cooperated with SIRT1 in breast cancer cells. ERα also bound to the promoter for SIRT1 and increased its transcription. SIRT1 expression induced by ERα was sufficient to activate antioxidant and prosurvival genes in breast cancer cells, such as catalase and glutathione peroxidase, and to inactivate tumor suppressor genes such as cyclin G2 (CCNG2) and p53. Moreover, SIRT1 inactivation eliminated estrogen/ERα-induced cell growth and tumor development, triggering apoptosis. Taken together, these results indicated that SIRT1 is required for estrogen-induced breast cancer growth. Our findings imply that the combination of SIRT1 inhibitors and antiestrogen compounds may offer more effective treatment strategies for breast cancer. Cancer Res; 71(21); 6654–64. ©2011 AACR.

SLC6A14, also known as ATB(0,+), is an amino acid transporter with unique characteristics. It transports 18 of the 20 proteinogenic amino acids. However, this transporter is expressed only at low levels in normal tissues. Here, we show that the transporter is up-regulated specifically in estrogen receptor (ER)-positive breast cancer, demonstrable with primary human breast cancer tissues and human breast cancer cell lines. SLC6A14 is an estrogen/ER target. The transport features of SLC6A14 include concentrative transport of leucine (an activator of mTOR), glutamine (an essential amino acid for nucleotide biosynthesis and substrate for glutaminolysis), and arginine (an essential amino acid for tumor cells), suggesting that ER-positive breast cancer cells up-regulate SLC6A14 to meet their increased demand for these amino acids. Consequently, treatment of ER-positive breast cancer cells in vitro with α-methyl-DL-tryptophan (α-MT), a selective blocker of SLC6A14, induces amino acid deprivation, inhibits mTOR, and activates autophagy. Prolongation of the treatment with α-MT causes apoptosis. Addition of an autophagy inhibitor (3-methyladenine) during α-MT treatment also induces apoptosis. These effects of α-MT are specific to ER-positive breast cancer cells, which express the transporter. The ability of α-MT to cause amino acid deprivation is significantly attenuated in MCF-7 cells, an ER-positive breast cancer cell line, when SLC6A14 is silenced with shRNA. In mouse xenograft studies, α-MT by itself is able to reduce the growth of the ER-positive ZR-75-1 breast cancer cells. These studies identify SLC6A14 as a novel and effective drug target for the treatment of ER-positive breast cancer.

Antiestrogen resistance is still a major clinical challenge preventing the eradication of estrogen receptor positive (ER+) breast cancer. A major goal of our laboratory is to identify targeted therapies that can be combined with antiestrogen treatment to block the emergence of antiestrogen resistant breast cancer. Toward this goal, we previously determined that the catabolic process of autophagy facilitates the emergence of antiestrogen resistance (Sammadar et al., Molecular Cancer Ther. 9, 2008) and identified cathepsin L as a key lysosomal protease required for antiestrogen induced pro-survival autophagy ER+ MCF-7cells (Periyasamy-Thandavan et al., Autophagy. 6:19-35, 2010). Thus, we hypothesize that blocking cathepsin L expression / activity is one approach to targeting pro-survival autophagy during antiestrogen treatment of breast cancer. In support of this hypothesis, we now show that antiestrogen resistant TR5 cells show increased levels of active cathepsin L compared to the levels in the parent antiestrogen sensitive MCF-7 cells. The upregulation of cathepsin L expression is consistent with the fact that TR5 cells utilized autophagy to survive long-term 4-hydroxytamoxifen (4-OHT) selection. Withdrawal of 4-OHT selection during TR5 passage does not lead to a reduction in cathepsin L levels, suggesting that the increased expression of cathepsin L is a stable genetic or epigenetic alteration. Elevated expression of active (phosphorylated) MAPK1/2 is also present in TR5 cells and targeting MEK1/MAPK1/2 with the selective inhibitor U0126 consistently reduced the levels of active cathepsin L. We further established a key role for MEK1/MAPK1/2 in the regulation of cathepsin L in the parent MCF-7 cells by performing cathepsin L activity assays. Cells treated with 4-OHT showed increased cathepsin L activity compared to E2-treated control cells; whereas, cathepsin L activity in cells treated with 4-OHT + UO126 was significantly reduced compared to the levels in 4-OHT-treated cells. This reduction in cathepsin L by U0126-mediated blockade of MEK1/MAPK1/2 correlated directly to “impaired” autophagic flux and increased BimEL-dependent apoptosis in the antiestrogen-treated cell populations and was mediated, at least in part, via transcriptional regulation of cathepsin L as determined by quantitative PCR. Further, MEK1/MAPK1/2 up-regulation of cathepsin L is selective, as cathepsin B activity is not reduced by MEK1 targeting. These studies provide evidence that the targeting MEK1/MAPK1/2 in combination with antiestrogen treatment has the potential to reduce cathepsin-L mediated pro-survival autophagy in ER+ breast cancer. Citation Format: Annie Liu, Carol Joseph, Jesse Wayson, Timothy Summers, Haifeng Cai, Patricia V. Schoenlein. MEK1:MAPK1/2 targeting attenuates pro-survival autophagy and enhances antiestrogen-induced apoptosis in breast cancer cells via a cathepsin L-dependent mechanism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1337.

In recent studies, we and others showed that autophagy is critical to estrogen receptor positive (ER+) breast cancer cell survival and the development of antiestrogen resistance. Consequently, new approaches are warranted for targeting autophagy in breast cancer cells undergoing antiestrogen therapy. Because crosstalk has been demonstrated between the autophagy- and proteasome-mediated pathways of protein degradation, this study investigated how the proteasome inhibitor bortezomib affects autophagy and cell survival in antiestrogen-treated ER+ breast cancer cells. Bortezomib, at clinically achievable doses, induced a robust death response in ER+, antiestrogen-sensitive and antiestrogen-resistant breast cancer cells undergoing hormonal therapy. Cleavage of PARP and lamin A was detectable as a read-out of cell death, following bortezomib-induced mitochondrial dysfunction. Prior to induction of cell death, bortezomib-treated cells showed high levels of light chain 3 (LC3) and p62, two protein markers for autophagy. The accumulation of these proteins was due to bortezomib-mediated blockade of long-lived protein turnover during macroautophagy. This novel action of bortezomib was linked to its blockade of cathepsin-L activity, which is required for autolysosomal-mediated protein turnover in ER+ breast cancer cells. Further, bortezomib-treated breast cancer cells showed induction of the unfolded protein response, with upregulation of CH OP and GRP78. Bortezomib also induced high levels of the pro-apoptotic protein BNIP3. Knockdown of CH OP and/or BNIP3 expression via RNAi targeting significantly attenuated the death-promoting effects of bortezomib. Thus, bortezomib inhibits prosurvival autophagy, in addition to its known function in blocking the proteasome, and is cytotoxic to hormonally treated ER+ breast cancer cells. These findings indicate that combining a proteasome inhibitor like bortezomib with antiestrogen therapy may have therapeutic advantage in the management of early-stage breast cancer.

Autophagy is a cellular process of “self-eating.” During autophagy, a portion of cytoplasm is enveloped in double membrane-bound structures called autophagosomes, which undergo maturation and fusion with lysosomes for degradation. At the core of the molecular machinery of autophagy is a specific family of genes or proteins called Atg. Originally identified in yeast, Atg orthologs are now being discovered in mammalian cells and have been shown to play critical roles in autophagy. Traditionally, autophagy is recognized as a cellular response to nutrient deprivation or starvation whereby cells digest cytoplasmic organelles and macromolecules to recycle nutrients for self-support. However, studies during the last few years have indicated that autophagy is a general cellular response to stress. Interestingly, depending on experimental conditions, especially stress levels, autophagy can directly induce cell death or act as a mechanism of cell survival. In this review, we discuss the molecular machinery, regulation, and function of autophagy. In addition, we analyze the recent findings of autophagy in renal systems and its possible role in renal pathophysiology.

A major impediment to the successful treatment of estrogen receptor alpha (ERalpha)-positive breast cancer is the development of antiestrogen resistance. Tamoxifen, the most commonly used antiestrogen, exerts its pharmacological action by binding to ERalpha and blocking the growth-promoting action of estrogen-bound ERalpha in breast cancer cells. Tamoxifen treatment primarily induces cytostasis (growth arrest) and the surviving breast cancer cells commonly acquire tamoxifen resistance. Numerous clinically-relevant mechanisms of acquired antiestrogen resistance have been identified by in vitro studies. Our recent studies (Mol Cancer Ther 2008; 7:2977-87) now demonstrate that autophagy (also referred to as macroautophagy) is critical to the development of antiestrogen resistance. Under conditions of compromised autophagy, including treatments with pharmacological inhibitors and RNAi targeting of the beclin 1 gene, the cytotoxicity (death-inducing effects) of the antiestrogen 4-hydroxytamoxifen (4-OHT) was significantly increased. 4-OHT is an active metabolite of tamoxifen commonly used for in vitro studies. A step-wise drug selection protocol, using 4-OHT as the selecting drug, established antiestrogen-resistant breast cancer cell lines. Analysis of a representative resistant cell line showed an increased ability of the cells to sustain high levels of antiestrogen-induced autophagy without progression to death. Importantly, blockade of autophagosome function in the 4-OHT-treated, antiestrogen-resistant cells induced a robust death response. These data provide strong evidence that autophagy is a key mechanism of cell survival during antiestrogen challenge and progression to antiestrogen resistance. We discuss the potential benefit of blocking autophagosome function to significantly reduce the emergence of antiestrogen-resistant breast cancer cells.

IFN regulatory factor 8 (IRF8) has been shown to suppress tumor development at least partly through regulating apoptosis of tumor cells; however, the molecular mechanisms underlying IRF8 regulation of apoptosis are still not fully understood. Here, we showed that disrupting IRF8 function resulted in inhibition of cytochrome c release, caspase-9 and caspase-3 activation, and poly(ADP-ribose) polymerase cleavage in soft tissue sarcoma (STS) cells. Inhibition of the mitochondrion-dependent apoptosis signaling cascade is apparently due to blockage of caspase-8 and Bid activation. Analysis of signaling events upstream of caspase-8 revealed that disrupting IRF8 function dramatically increases FLIP mRNA stability, resulting in increased IRF8 protein level. Furthermore, primary myeloid cells isolated from IRF8-null mice also exhibited increased FLIP protein level, suggesting that IRF8 might be a general repressor of FLIP. Nuclear IRF8 protein was absent in 92% (55 of 60) of human STS specimens, and 99% (59 of 60) of human STS specimens exhibited FLIP expression, suggesting that the nuclear IRF8 protein level is inversely correlated with FLIP level in vivo. Silencing FLIP expression significantly increased human sarcoma cells to both FasL-induced and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)–induced apoptosis, and ectopic expression of IRF8 also significantly increased the sensitivity of these human sarcoma cells to FasL- and TRAIL-induced apoptosis. Taken together, our data suggest that IRF8 mediates FLIP expression level to regulate apoptosis and targeting IRF8 expression is a potentially effective therapeutic strategy to sensitize apoptosis-resistant human STS to apoptosis, thereby possibly overcoming chemoresistance of STS, currently a major obstacle in human STS therapy. [Cancer Res 2009;69(3):1080–8]

This study identifies macroautophagy as a key mechanism of cell survival in estrogen receptor–positive (ER+) breast cancer cells undergoing treatment with 4-hydroxytamoxifen (4-OHT). This selective ER modifier is an active metabolite of tamoxifen commonly used for the treatment of breast cancer. Our study provides the following key findings: (a) only 20% to 25% of breast cancer cells treated with 4-OHT in vitro die via caspase-dependent cell death; more typically, the antiestrogen-treated ER+ breast cancer cells express increased levels of macroautophagy and are viable; (b) 4-OHT–induced cell death, but not 4-OHT–induced macroautophagy, can be blocked by the pan-caspase inhibitor z-VAD-fmk, providing strong evidence that these two outcomes of antiestrogen treatment are not linked in an obligatory manner; (c) 4-OHT–resistant cells selected from ER+ breast cancer cells show an increased ability to undergo antiestrogen-induced macroautophagy without induction of caspase-dependent cell death; and (d) 4-OHT, when used in combination with inhibitors of autophagosome function, induces robust, caspase-dependent apoptosis of ER+, 4-OHT–resistant breast cancer cells. To our knowledge, these studies provide the first evidence that macroautophagy plays a critical role in the development of antiestrogen resistance. We propose that targeting autophagosome function will improve the efficacy of hormonal treatment of ER+ breast cancer. [Mol Cancer Ther 2008;7(9):2977–87]

Apoptotic resistance is often associated with metastatic phenotype in tumor cells and is considered a hallmark of tumor progression. In this study, IFN regulatory factor 8 (IRF8) expression was found to be inversely correlated with an apoptotic-resistant and metastatic phenotype in human colon carcinoma cell lines in vitro. This inverse correlation was further extended to spontaneously arising primary mammary carcinoma and lung metastases in a mouse tumor model in vivo. Exogenous expression of IRF8 in the metastatic tumor cell line restored, at least partially, the sensitivity of the tumor cells to Fas-mediated apoptosis, and disruption of IRF8 function conferred the poorly metastatic tumors with enhanced apoptotic resistance and metastatic capability. DNA demethylation restored IRF8 expression and sensitized the metastatic tumor cells to Fas-mediated apoptosis. Analysis of genomic DNA isolated from both primary and metastatic tumor cells with methylation-sensitive PCR revealed hypermethylation of the IRF8 promoter in metastatic tumor cells but not in primary tumor cells. Taken together, our data suggest that IRF8 is both an essential regulator in Fas-mediated apoptosis pathway and a metastasis suppressor in solid tumors and that metastatic tumor cells use DNA hypermethylation to repress IRF8 expression to evade apoptotic cell death and to acquire a metastatic phenotype. [Cancer Res 2007;67(7):3301–9]

Background and Purpose: Previous literature and studies from our laboratory indicate that pharmacological inhibition and enhancement of autophagy are both effective in reducing infarct volume after stroke injury. Methods: Mice were treated with rapamycin to enhance, and chloroquine to suppress autophagy in mice immediately after stroke injury and again 24 hours post stroke injury in a murine stroke model. Survival data and neurological score were taken before sacrifice at 48 hours and lesion size was determined by TTC. HT22 neuronal mouse hippocampal cells were treated for one hour with H2O2 to mimic an oxidative stress injury and treated concurrently with rapamycin to enhance autophagy, chloroquine to inhibit autophagy or ZVAD to inhibit apoptosis. The treatment with pharmacological agents continued for 24 hours. Cells were counted with trypan blue on a hemocytometer. Western blot analysis was used to monitor protein changes. shRNA to Atg5 was used to genetically inhibit autophagy. Results: Both pharmacological agents showed decreased lesion size, though enhancement of autophagy with rapamycin exhibited a greater reduction in lesion size as well as increased survival and an improved neurological score. Rapamycin improved survival of neurons after oxidative injury and genetic inhibition of autophagy abrogated rapamycin's positive effect. Conclusions: Rapamycin improves stroke outcome by enhancing autophagy in neuronal cells.

Cisplatin induces renal cell injury and death, resulting in nephrotoxicity that limits its use in cancer therapy. Using cell culture models, recent work has suggested the involvement of p53 in renal cell apoptosis during cisplatin treatment. However, the signals upstream of p53 remain elusive. ATM and ATR are critical regulators of p53 under various conditions of DNA damage. Here, we show that ATM, and not ATR, was proteolytically cleaved into specific fragments of ∼210 and 150 kDa during cisplatin-induced tubular cell apoptosis. ATM cleavage was paralleled by the development of apoptosis. VAD, a broad-spectrum inhibitor of caspases, attenuated the cleavage of ATM, whereas the inhibitors of specific caspases were less effective. In caspase-3-deficient MCF-7 cells, ATM was cleaved, releasing the 210- but not the 150-kDa fragment. Recombinant caspase-3 was much more effective than caspase-7 in cleaving ATM that was immunoprecipitated from cell lysates. During cisplatin incubation, VAD protected ATM and enhanced p53 phosphorylation. In vitro assay of protein kinase activity further showed that ATM immunoprecipitated from cisplatin-treated cells had significantly lower kinase activity toward p53 than that from control cells. Importantly, the protein kinase activity was restored in ATM that was protected by VAD during cisplatin incubation. ATM deficiency sensitized the cells to cisplatin-induced apoptosis, suggesting a cytoprotective role of ATM in this experimental model. Thus proteolysis of ATM by caspases may inactivate this regulatory molecule to facilitate the progression of apoptosis.

Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs., Author Summary Among the most successful of human microbes are intracellular pathogens. By entering the intracellular milieu, these pathogens are protected from harsh environmental factors in the host, including the humoral and cellular immune responses. Porphyromonas gingivalis is an opportunistic pathogen that colonizes the oral mucosa and accesses the bloodstream and distant sites such as the blood vessel walls, brain, placenta and other organs. Still unclear is how P. gingivalis traverses from oral mucosa to these distant sites. Dendritic cells are highly migratory antigen presenting cells that “patrol” the blood, skin, mucosa and all the major organ systems. Capture of microbes by dendritic cells activates a tightly regulated series of events, including directed migration towards the secondary lymphoid organs, where processed antigens are ostensibly presented to T cells. Autophagy is now recognized as an integral component of microbial clearance, antigen processing and presentation by dendritic cells. We report here that P. gingivalis is able to subvert autophagic destruction within dendritic cells. This occurs through its glycoprotein fimbriae, called Mfa-1, which targets the C-type lectin DC-SIGN on dendritic cells. The other major fimbriae on P. gingivalis, FimA, targets TLR2, which promotes autophagic destruction of P. gingivalis. We conclude that DC-SIGN-TLR2 crosstalk determines the intracellular fate of this pathogen within dendritic cells, and may have profound implications for the treatment of many chronic diseases involving low-grade infections.

Purpose: A major clinical problem in the treatment of breast cancer is the inherent and acquired resistance to antiestrogen therapy. In this study, we sought to determine whether antiprogestin treatment, used as a monotherapy or in combination with antiestrogen therapy, induced growth arrest and active cell death in antiestrogen-resistant breast cancer cells. Experimental Design: MCF-7 sublines were established from independent clonal isolations performed in the absence of drug selection and tested for their response to the antiestrogens 4-hydroxytamoxifen (4-OHT) and ICI 182,780 (fulvestrant), and the antiprogestin mifepristone (MIF). The cytostatic (growth arrest) effects of the hormones were assessed with proliferation assays, cell counting, flow cytometry, and a determination of the phosphorylation status of the retinoblastoma protein. The cytotoxic (apoptotic) effects were analyzed by assessing increases in caspase activity and cleavage of poly(ADP-ribose) polymerase. Results: All of the clonally derived MCF-7 sublines expressed estrogen receptor and progesterone receptor but showed a wide range of antiestrogen sensitivity, including resistance to physiological levels of 4-OHT. Importantly, all of the clones were sensitive to the antiprogestin MIF, whether used as a monotherapy or in combination with 4-OHT. MIF induced retinoblastoma activation, G1 arrest, and apoptosis preceded by caspase activation. Conclusions: We demonstrate that: (a) estrogen receptor+progesterone receptor+, 4-OHT-resistant clonal variants can be isolated from an MCF-7 cell line in the absence of antiestrogen selection; and (b) MIF and MIF plus 4-OHT combination therapy induces growth arrest and active cell death of the antiestrogen-resistant breast cancer cells. These preclinical findings show potential for a combined hormonal regimen of an antiestrogen and an antiprogestin to combat the emergence of antiestrogen-resistant breast cancer cells and, ultimately, improve the therapeutic index of antiestrogen therapy.

Antiestrogen resistance is the major impediment to the eradication of estrogen receptor positive (ER+) breast cancer. Approximately 30% of ER+ breast tumors initially responsive to antiestrogen therapy will acquire resistance. One approach to reducing the occurrence of acquired antiestrogen resistance is to identify and target key signaling nodes that specifically attenuate the ability of antiestrogens to kill cancer cells. Toward this goal, we identified MEK/MAPK1/2 as a key molecular target based on the general inability of antiestrogens to effectively block MEK1/MAPK1/2-mediated phosphorylation of BimEL in breast cancer cells. BimEL is a pro-apoptotic member of the BH3 family of proteins that is inactivated (degraded by the proteasome) when phosphorylated by MAPK1/2. The combination of an antiestrogen and MEK1 inhibitor robustly-induced BimEL-dependent breast cancer cell apoptosis (Periyasamy-Thandavan et al., 2012). However, a subpopulation of breast cancer cells survive this combined treatment via an autophagy-dependent mechanism. Based on the key role of JNK in regulating autophagy in cancer cells via BimEL phosphorylation, we hypothesized that JNK was a key effector of autophagy in breast cancer cells surviving antiestrogen treatment when used as a single agent or in combination with a MEK inhibitor. To test this hypothesis, we utilized the selective JNK inhibitor SP600125 as a single agent or in combination with estradiol (E2), 4-hydroxytamoxifen (4-OHT), and/or U0126 (a selective MEK1 inhibitor). Treatments were conducted with the antiestrogen-sensitive cell lines MCF-7 and T-47D and the antiestrogen resistant cell line TR5, previously derived in our laboratory by a step-wise 4-OHT selection. Effects on cell death (determination of apoptosis) and autophagy (determination of autophagy levels and flux) were evaluated for the various hormonal treatments conducted in the presence or absence of JNK inhibition. These studies showed that: (1) targeting JNK in antiestrogen sensitive breast cancer cells induces apoptosis with caspase-dependent cleavage of PARP as a surrogate marker of apoptosis; (2) JNK activity is elevated in antiestrogen resistant TR5 cells and JNK inhibition induces TR5 cell death; and (3) antiestrogen sensitive and resistant breast cancer cells dying as a result of JNK inhibition can show extensive cytosolic vacuolization with the autophagy protein LC3 (ATG8) localized to the aberrant vacuoles. Overall, our results support the conclusion that JNK plays a key autophagy-dependent survival role in breast cancer cells. Ongoing studies aim to identify the specific JNK protein(s) that disrupt autophagy and enhance death of breast cancer cells toward the goal of identifying specific molecular targets to block autophagy in breast cancer cells. Citation Format: Rohit Munagala, Carol Joseph, Annie Liu, Kebin Liu, Muthusamy Thangaraju, Patricia V. Schoenlein. A critical role for c-Jun N-terminal kinase in autophagy and cell survival of breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3318. doi:10.1158/1538-7445.AM2017-3318

Background and purpose The role of autophagy in response to ischemic stroke has been confusing with reports that both enhancement and inhibition of autophagy decrease infarct size and improve post-stroke outcomes. We sought to clarify this by comparing pharmacologic modulation of autophagy in two clinically relevant murine models of stroke. Methods We used rapamycin to induce autophagy, and chloroquine to block completion of autophagy, by treating mice immediately after stroke and at 24 hours post-stroke in two different models; permanent Middle Cerebral Artery Ligation (MCAL), which does not allow for reperfusion of distal trunk of middle cerebral artery, and Embolic Clot Middle Cerebral Artery Occlusion (eMCAO) which allows for a slow reperfusion similar to that seen in most human stroke patients. Outcome measures at 48 hours post-stroke included infarct size analysis, behavioral assessment using Bederson neurological scoring, and survival. Results Chloroquine treatment reduced the lesion size by approximately 30% and was significant only in the eMCAO model, where it also improved the neurological score, but did not increase survival. Rapamycin reduced lesion size by 44% and 50% in the MCAL and eMCAO models, respectively. Rapamycin also improved the neurological score to a greater degree than chloroquine and improved survival. Conclusions While both inhibition and enhancement of autophagy by pharmacological intervention decreased lesion size and improved neurological scores, the enhancement with rapamycin showed a greater degree of improvement in outcomes as well as in survival. The protective action seen with chloroquine may be in part due to off-target effects on apoptosis separate from blocking lysosomal activity in autophagy. We conclude pharmacologic induction of autophagy is more advantageous than its blockade in physiologically-relevant permanent and slow reperfusion stroke models.

Tamoxifen (TAM), an antiestrogen and selective estrogen receptor modifier (SERM) has become a widely utilized therapeutic drug for treating ER+ breast cancer, however many cases develop TAM resistance. In previous in vitro studies, TAM combined with the selective Mitogen/Extracellular signal-regulated Kinase pathway (MEK) inhibitor U0126 (U) led to increased levels of the proapoptotic protein BimEL. This combined treatment was more effective at killing estrogen receptor expressing (ER+) breast cancer cells than either agent alone via a Bim-dependent mechanism [Periyasamy-Thandavan el al., Breast Cancer Res. 14(2), 2012]. In this study, we aimed to recapitulate the superior efficacy of this combined treatment strategy with the clinically relevant MEK inhibitor Selumetinib (SEL) using the TAM sensitive, ER+ MCF-7 breast cancer cell line. In addition, we utilized MCF7-TAM resistant cells (TR5), created in vitro through exposures to incremental increases in TAM concentration(100ng to 5.0μM). MCF-7 cells that were treated with TAM, SEL, U, or combination therapies in vitro demonstrated a similar efficacy between U and SEL with the lowest cell viability resulting from TAM plus U or SEL compared to TAM, U, or SEL as single agents. Western blot analysis of U- or SEL-treated MCF7 cells showed a similar robust upregulation of BimEL in the presence or absence of TAM. Comparison of the TR5 TAM resistant cells to parent MCF-7 cells showed elevated levels of active MAPK1/2 protein (p-MAPK) that mediated the phosphorylation and subsequent proteasomal degradation of BimEL. Importantly, inhibition of MEK/MAPK1/2 by SEL or U in TR5 cells in vitro significantly increased BimEL levels and TAM induced apoptosis as evidenced by increased levels of cleaved PARP and/or lamin A. In a pilot in vivo study, we further demonstrated that TR5 cells were highly tumorigenic with the xenografts being estrogen independent and insensitive to TAM inhibition. In contrast, the TR5 xenografts were sensitive to SEL when delivered in the presence or absence of TAM. Further, tissue analyzed from SEL-treated TR5 xenografts showed significantly higher levels of BimEL than tissue analyzed from TAM- or E2-treated xenografts. These initial studies provide strong data that MEK1/MAPK1/2 inhibition will be required in vivo to raise BimEL levels to negatively affect breast cancer growth. These studies also support the concept of combining SEL with antiestrogen treatment (as a combined adjuvant therapy) to reduce the emergence of antiestrogen resistant cells and to include SEL as part of a regimen to treat antiestrogen resistant breast cancer. Citation Format: Jason Conger, Matthew Manning, Kingsley Anosike, Ahmed Elsherbini, Brandon Ware, Emily Bass, Mathusamy Thangaraju, Darren Browning, Patricia V. Schoenlein. Targeting MEK/MAPK1/2 in vivo to eradicate antiestrogen resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4672.

Introduction In this pre-clinical in vitro study conducted in estrogen receptor positive (ER+) breast cancer cells, we have characterized the effects of insulin-like growth factor I (IGF-1) on the cytostatic and cytotoxic action of antiestrogen treatment when used as a single agent or in combination with the antiprogestin mifepristone (MIF). Our goal was to identify new molecular targets to improve the efficacy of hormonal therapy in breast cancer patients that have a poor response to hormonal therapy, in part, due to high circulating levels of unbound insulinIGF-1. Methods IGF-1-mediated effects on cytostasis and apoptotic cell death were determined with cell counts conducted in the presence and absence of trypan blue; enzyme-linked immunosorbent assays to determine the intracellular levels of cleaved cytokeratin 18, a marker of epithelial cancer cell apoptosis; and immunoblot analysis to determine the levels of cleaved poly-ADP ribose polymerase (PARP) and lamin A that result from caspase-dependent apoptosis. Cytotoxicity was further characterized by determination of the levels of reactive oxygen species (ROS) and the percent of mitochondrial membrane depolarization in cell populations treated with the different hormones in the presence and absence of IGF-1. Small molecule inhibitors of the dual-specificity protein kinase MEK1, MEK1 siRNA, Bim siRNA, and vectors overexpressing MEK1 wild type and mutant, dominant negative cDNA were used to identify key IGF-1 downstream prosurvival effectors. Results IGF-1, at physiologically relevant levels, blocked the cytotoxic action(s) of the antiestrogens 4-hydroxytamoxifen (4-OHT) and tamoxifen (TAM) when used as single agents or in combination with the antiprogestin MIF. The antiapoptotic action of IGF-1 was mediated primarily through the action of MEK1. MEK1 expression reduced the levels of ROS and mitochondrial membrane depolarization induced by the hormonal treatments via a mechanism that involved the phosphorylation and proteasomal turnover of the proapoptotic BH3-only Bcl-2 family member Bim. Importantly, small-molecule inhibitors of MEK1 circumvented the prosurvival action of IGF-1 by restoring Bim to levels that more effectively mediated apoptosis in ER+ breast cancer cells. Conclusion his study provides strong support for the use of MEK1 inhibitors in combination with hormonal therapy to effectively affect cytostasis and activate a Bim-dependent apoptotic pathway in ER+ breast cancer cells. We discuss that MEK1 blockade may be a particularly effective treatment for women with high circulating levels of IGF-1, which have been correlated to a poor prognosis.

Mutations having pleiotropic effects on polar organelle development (pod) in Caulobacter crescentus have been identified and shown to occur in at least 13 genes scattered throughout the genome. Mutations at each locus affect a unique combination of polar traits, suggesting that complex interactions occur among these genes. The DNA sequence of one of these genes, pleC, indicates that it is homologous to members of the family of histidine protein kinase genes. Membes of this family include the senor components of the bacterial two-component regulatory systems. Furthermore, in vitro experiments demonstrated that the PleC protein was capable of autophosphorylation. These results suggest that the PleC protein (and perhaps the proteins encoded by the other pod genes as well) regulates the expression of genes involved in polar organelle development through the phosphorylation of key regulatory proteins. The use of a phosphorelay system cued to internal changes in the cell would provide a mechanism for coordinating major changes in gene expression with the completion of specific cell cycle events.

Many human cancers that are initially responsive to chemotherapy eventually fail to respond to treatment. For some drugs, dose escalation that may be required for a cure cannot be achieved because sensitive tissues such as bone marrow (BM) limit cytotoxic therapy. Approaches to prevent or circumvent BM toxicity are therefore a high priority of research on dose escalation protocols. In this study, we have transplanted BM cells from transgenic mice that constitutively express physiologic amounts of a functional human multidrug resistance (MDR1) cDNA to lethally irradiated C57BL/6 x SJL F1 mice (n = 36). From 6 weeks to 10 months after the transplant, all animals contained MDR1 DNA in spleen and BM specimens as indicated by Southern blot analysis, and expressed MDR1 messenger RNA in BM samples as detected by slot blot analysis. In addition, these animals were resistant to the myelosuppressive effect of doxorubicin, daunomycin, taxol, vinblastine, vincristine, etoposide, and actinomycin D, whereas control animals that were reconstituted with normal BM were drug sensitive. Finally, the chemoprotection afforded by the MDR1 gene could readily be reversed by adding chemosensitizers such as cyclosporin A and R-verapamil to chemotherapy. Hence, it appears that BM cells expressing the human MDR1 gene maintain this function after transplantation to host animals for a minimum of 10 months, and confer multidrug resistance to these BM recipients. This selective advantage conferred by expression of the MDR1 cDNA suggests a strategy for the use of MDR1 gene therapy in cancer chemotherapy and for the introduction of otherwise nonselectable genes into BM.

The Caulobacter crescentus flaFG region encodes trans -acting, regulatory factors that modulates flagellin synthesis during flagellum biogenesis. In this study, sequence analysis and experiments utilizing a promoterless cat gene demonstrated that the flaF and flbT genes have overlapping transcripts with the same orientation. In addition, the 5′ ends of the flgL and flbA genes were located. A sequence resembling an Rho-factor-independent terminator was found in the 3′ region of the flaF gene. This region was uniquely A + T-rich and the encoded mRNA contained an inverted repeat sequence which could form a stable stem-loop strucuture followed by nine U-residues. The codon usage of C. crescentus genes was examined and indicated a preference for specific codons from each of the synonymous codon groups. Furthermore, comparison to the codon usage of other organisms with G + C-rich genomes indicated a strong preference for the same codons prefered by C. crescentus .

In this study, human MCF-7 breast cancer cells, which express functional estrogen and progesterone receptors, were used to compare the efficacy of combined antiestrogen plus antiprogestin therapy to antiestrogen monotherapy. Cells were treated with the antiestrogen 4-hydroxytamoxifen (4-OHT) and/or the antiprogestin mifepristone (MIF) and effects on cell proliferation (cytostatic action), cell cycle phase, the phosphorylation state of the tumor suppressor retinoblastoma protein (Rb), and induction of active cell death (cytotoxic action) were determined. Combination hormonal therapy showed both increased cytostatic and cytotoxic activity as compared to either monotherapy. The increased cytostatic action was mediated by Rb activation; whereas, the cytotoxic (pro-apoptotic) action of combined hormonal therapy correlated to a significant reduction in Rb protein levels. To test the apparent role of Rb protein loss in the pro-apoptotic action of combined hormonal therapy, Rb was downregulated in MCF-7 cells using siRNA-targeting. The siRNA-mediated knockdown of Rb combined with 4-OHT therapy resulted in a pro-apoptotic action similar to that resulting from 4-OHT and MIF combination treatment, which included increased cell detachment from the monolayer, high-molecular-weight genomic DNA fragmentation, and cleavage of poly ADP-ribose polymerase (PARP) and lamin A. From these studies, we conclude that Rb protein downregulation is required for 4-OHT-treated, estrogen receptor positive (ER+) breast cancer cells to undergo active cell death. We discuss the potential of using an antiprogestin such as MIF plus antiestrogen treatment to more effectively downregulate Rb in ER+ breast cancer cells to increase the overall cytotoxic action of hormonal therapy.

In past studies, we showed that pro-survival autophagy mediates resistance to antiestrogen-induced apoptosis and facilitates the emergence of antiestrogen resistant breast cancer cells (Sammadar et al., Molecular Cancer Ther. 9, 2008). We also identified the pro-apoptotic BimEL protein as a key death effector of antiestrogen treatment and determined that BimEL pro-apoptotic activity is abrogated by a MEK1/MAPK1/2-dependent phosphorylation (Periyasamy-Thandavan et al., Breast Cancer Res. 14, 2012). In this study, we tested the hypothesis that the MEK1/MAPK1/2/BimEL signaling node simultaneously regulates antiestrogen- induced pro-survival autophagy and apoptosis in ER+ breast cancer cells. In our approach, we utilized T47-D and MCF-7 breast cancer cells and modulated the activity of MEK1/MAPK1/2/ BimEL expression with U0126, a small molecule inhibitor of MEK1, BimEL cDNA over-expression, or siRNA targeting under a variety of endocrine treatments. We also conducted studies in the presence or absence of the pan-caspase inhibitor ZVAD-fmk so that autophagy levels/function could be analyzed independent of effects mediated by BimEL-dependent caspase activation. Autophagic flux was measured by comparing the levels of lipidated LC3II in cell populations undergoing the different treatments in the presence or absence of chloroquine (CQ), a lysosomotropic agent that prevents autolysosomal turnover. These studies determined that: 1) dephosphorylated BimEL significantly attenuates autophagic flux, but primarily as a consequence of BimEL-induced caspase-activation; (2) BimEL-dependent apoptotic death induced in antiestrogen-treated ER+ breast cancer cells by MEK1 inhibition is not dependent on autophagy; and (3) agents that selectively target autophagy (i.e. CQ and spautin) enhance apoptosis induced by MEK1 blockade in breast cancer cells undergoing endocrine treatment. Based on these pre-clinical in vitro studies, we postulate that the eradication of antiestrogen resistant breast cancer will require the targeting of MEK1/MAPK1/2 to activate BimEL and pro-survival autophagy to eradicate cells surviving BimEL-dependent apoptosis. Citation Format: Mathew Manning, Suchreet Takhar, Sudharsan Periyasamy-Thandavan, Michael Cheng, Thomas Barrett, William Hill, Darren Browning, Muthusamy Thangaraju, Meghan McGee-Lawrence, Patricia V. Schoenlein. Dual targeting of MEK/MAPK1/2 and pro-survival autophagy to optimize antiestrogen treatment toward the eradication of antiestrogen resistant breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1005. doi:10.1158/1538-7445.AM2015-1005

In a recent study, we identified the dephosphorylated form of BimEL as a key death effector of antiestrogen treatment of ER+ breast cancer cells and further showed that MEK1/MAPK1/2 blockade was required to produce high levels of dephosphorylated BimEL, particularly under conditions of insulin like growth factor 1 (IGF1) stimulation (Periyasamy-Thandavan et al., Breast Cancer Res. 14, 2012). Studies by others have identified MEK1/MAPK1/2 activation as essential to autophagy, a catabolic process induced by multiple stresses including ROS, ceramide accumulation, and nutrient deprivation. Autophagy induction results in autophagosome formation, trafficking of damaged proteins and mitochondria to the autophagosomes, and ultimately fusion with the lysosomes resulting in autolysosome formation. The autolysosome and its contents are degraded by the hydrolytic enzymes of the lysosome. Of particular interest to antiestrogen treatment of breast cancer, we and others have shown that pro-survival autophagy facilitates the emergence of antiestrogen resistant breast cancer cells. Thus, we are keenly interested in how MEK1/MAPK1/2 signaling affects pro-survival autophagy and if MEK blockade would be an effective approach toward blocking pro-survival autophagy in ER+ breast cancer cells undergoing hormonal treatment. In this study, we hypothesized that the requirement of MEK1/MAPK1/2 for pro-survival autophagy is due, in part, to its role in blocking the intracellular accumulation of dephosphorylated BimEL. To test this hypothesis, we modulated the expression of dephosphorylated BimEL with either a BimEL cDNA expression vector, siRNA targeting of BimEL, or MEK1 blockade with the small molecule inhibitor U0126 and determined the levels of the autophagic flux in ER+ breast cancer cells undergoing antiestrogen treatment. The determination of autophagic flux was made by comparing the levels of two proteins involved in autophagy -the LC3 /Atg8 and p62 (SQSTM1) proteins- in cell populations undergoing the different treatments in the presence or absence of chloroquine (CQ). The lipidated form of LC3, designated LC3II, is typically increased in cells undergoing autophagy, facilitates the formation of the mature autophagosomal membranes, and is subsequently degraded in the autolysosome. The p62 protein is required for the delivery of ubiquitinated protein complexes to the autophagosome and is degraded along with the ubiquitinated complex of proteins. CQ is a lysosomotrophic agent routinely used in autophagic flux assays because it blocks the turnover of autolysosomes with accumulation of LC3 II and p62, allowing the total levels of LC3II and p62 to be ascertained under all treatment conditions. These studies showed that siRNA targeting of BimEL increased basal and tamoxifen-induced autophagy in ER+ MCF-7 breast cancer cells. In contrast, the overexpression of dephosphorylated BimEL led to an increase in LC3 II and p62 levels due to a significant attenuation of autophagic flux (approximately 50%) in antiestrogen-treated cell populations. Current studies are focused on the mechanism of BimEL-mediated blockade of pro-survival autophagy, with the long term goal of optimizing this “downstream effector” function of MEK1/MAPK1/2 blockade in ER+ breast cancer cells for improved therapeutic outcome. Citation Format: S. Takhar, M. Manning, A. Eason, M. Dix, S. Periyasamy-Thandavan, R. Padi, E. Bieberich, W. Hill, D. Browning, V. Ganapathy, M. Thangaraju, P. V. Schoenlein. MEK inhibitors mount a two-pronged attack to kill estrogen receptor positive (ER+) breast cancer cells undergoing hormonal therapy: Attenuated autophagy and induction of apoptosis. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B50. doi: 10.1158/1557-3125.RASONC14-B50

Transduction of MDR1 may be of use in chemoprotection of normal bone marrow (BM) cells during treatment of malignancies, or as a selectable marker for the transfer of other genes into the BM, a critical target for the cure of many diseases. To that aim, the human multidrug resistance gene MDR1 was cloned into an SV40 pseudoviral vector containing the SV40 origin of replication (ori) and encapsidation signal (ses), and the plasmid was encapsidated in COS cells as SV40/MDR1 pseudovirions. Expression of the human MDR1 gene was demonstrated in murine MEL cells infected with SV40/MDR1 pseudovirions, using a monoclonal antibody (MPK16) specific for the human 170-kD P-glycoprotein. Functional P-glycoprotein was demonstrated by resistance to colchicine in NIH-3T3 cells infected with SV40/MDR1 pseudovirions. Activity of P-glycoprotein was assayed by rhodamine-123 dye exclusion and fluorescence-activated cell sorter analysis (FACS) in various cell types including hematopoietic cells. Highly efficient gene transfer and expression was demonstrated in all murine and human cell types tested, including primary human BM cells. Using multiplicities of infection (moi) of 1-2, over 95% of cells were found to become MDR1+. The percent of MDR1+ cells was proportional to the moi. We conclude that the SV40 pseudoviral vector is efficient for gene transmission into human hematopoietic cells.

Paclitaxel functions by preventing microtubule degradation, leading to mitotic arrest and apoptotic death. Of particular interest, paclitaxel-induced death in breast cancer cells is dependent, in part, on the levels of BimEL, a pro-apoptotic member of the Bcl-2 family of proteins. In addition, our recent studies demonstrated that BimEL is required for 4-hydroxytamoxifen-induced apoptosis of estrogen receptor positive (ER+) MCF-7 breast cancer cells [Breast Cancer Res. 2012 Mar 19;14(2):R52]. In contrast, we demonstrated low-level BimEL expression in ER+ T47D breast cancer cells that do not undergo antiestrogen-induced apoptosis. Thus, low-level BimEL expression in ER+ breast cancer may predict a poor apoptotic threshold which ultimately would facilitate the development of acqured resistance to paclitaxel, as well as antiestrogen therapy. Based on the ability of HDAC inhibitors to increase the transcription of pro-apoptotic genes, we hypothesized that the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) would increase BimEL expression in T47D breast cancer cells and induce a robust apoptotic response to paclitaxel chemotherapy and antiestrogen treatment. In this study, we now demonstrate that SAHA does significantly up-regulate BimEL expression in T47D cells, as well as in MCF-7 cells. Concomitant with BimEL upregulation, SAHA sensitizes T47D and MCF-7 cells to paclitaxel-induced apoptosis. Similarly, SAHA sensitizes T-47D cells to antiestrogen-induced apoptosis, while augmenting the level of antiestrogen-induced apoptosis in MCF-7 cells. These studies indicate that the pro-apoptotic protein BimEL is required for SAHA-induced sensitization of breast cancer cells to paclitaxel and/or antiestrogen-induced apoptosis. Currently, siRNA studies are being conducted to determine if BimEL is a key death effector in response to SAHA treatment and if the increased death from SAHA and paclitaxel or SAHA and antiestrogens is synergistic or additive. Our results provide strong support for the use of HDAC inhibitors when designing novel combination therapies to reduce the emergence of acquired resistance in breast cancer cells undergoing chemo- or antihormonal therapy. Acknowledgement: this work was supported by teh MCG foundation and NIHRO1 CA121438 to P.V.S. Citation Format: Aric Berning, Alexander Eason, Nathan Gilley, Suchreet Takhar, Sally ElShafey, Muthusamy Thangaraju, Patricia V. Schoenlein. HDAC inhibition induces Bim expression and apoptosis in breast cancer cells undergoing paclitaxel or antiestrogen treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1725. doi:10.1158/1538-7445.AM2013-1725

In an effort to understand the underlying mechanisms of inherent antiestrogen resistance in estrogen receptor positive (ER+) breast cancer cells, we have been studying the pro-survival role of insulin-like growth factor 1 (IGF-1) in protecting breast cancer cells from death induced by hormonal therapy. In past studies, we demonstrated that IGF-1, at physiologically relevant doses binds the insulin like growth factor receptor (IGF-IR), blocks antiestrogen- and antiprogestin- induced apoptosis in ER+ MCF-7 breast cancer cells. We further showed that the mechanism of protection was dependent on the action of the dual specificity protein kinase MEK1 (Abstract #4846, Proc Am Assoc Cancer Res, 51, 2010, p1178-79); however we did not elucidate the specific death effector pathway being blocked by MEK1/MAPK signaling. For this study, we sought to identify the specific death pathway blocked by the IGF-IR/MEK1/MAPK prosurvival signaling. We now present strong data identifying the pro-apoptotic BH3 family member Bim as a critical death effector activated by MEK1 blockade in ER+ breast cancer cells. We utilized Bim siRNA, which effectively down-regulated Bim intracellular protein expression by five-to ten-fold. Bim downregulation significantly attenuated the pro-apoptotic action(s) of U0126, as well as that of the antiestrogen 4-hydroxytamoxifen (4-OHT) and/or the antiprogestin mifepristone (MIF). Bim knockdown reduced the generation of ROS that results from treatment with 4-OHT, MIF, and U0126 (whether used as monotherapies or in combination) and is required for MCF-7 apoptotic cell death under these treatment conditions. We are currently elucidating how Bim action induces cell death in breast cancer cells with compromised MEK1 action. These studies support the targeting of MEK1 to activate a Bim-dependent apoptotic pathway during hormonal therapy of early-stage breast cancer, particularly in patients with high circulating levels of IGF-1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2. doi:10.1158/1538-7445.AM2011-2

In our past studies, we have implicated autophagy as a mechanism of cell survival that contributes to the development of antiestrogen resistance in breast cancer cells (Autophagy. 2009 Apr; 5(3): 400-3). The breast cancer model system used to establish this link between autophagy and antiestrogen resistance was the estrogen-receptor positive MCF-7 cell line. We subjected this cell line to a step-wise selection in increasing concentrations of 4-hydroxytamoxifen (active metabolite of tamoxifen) in the absence of clonal selection and established the antiestrogen resistant TR5 cell line. Analysis of antiestrogen resistant TR5 cells showed induction of ceramide levels during the drug selection that correlated with autophagy induction. Thus, we have begun to characterize the role of key enzymes of the ceramide pathway in pro-survival autophagy relative to the development of antiestrogen resistance. In this study, we show that the inhibition of sphingosine kinase activity effectively kills antiestrogen-sensitive MCF-7 cells and antiestrogen-resistant TR5 cells. Specifically, blockade of spingosine kinase action in MCF-7 and TR5 cells reduced cell number, increased the percentage of cells in the population that showed uptake of trypan blue, induced caspase-9 activation and increased the cleavage of poly-ADP-ribose polymerase and lamin A. Thus, blockade of sphingosine kinase resulted in a robust induction of apoptotic cell death. SIP, the product of sphingosine kinase 1, did not appear to block the death-promoting action of sphingosine kinase blockade. Thus, the pro-survival action(s) of SIP did not compensate for loss of sphingosine kinase action in hormonally-treated breast cancer cells. Importantly, blockade of sphingosine kinase in both MCF-7 and TR5 cells blocked autophagic flux with a marked accumulation of the autophagy proteins LC3 and p62 which are normally turned-over in active autolysosomes. Blockade of autophagic flux was determined using the long-lived protein turnover assay. Although autophagic flux was impaired, autophagosomes were readily seen in the cytoplasm of cells and the lipidated LC3 protein was localized within the membrane of these autophagosomes. Further, studies utilizing electron microscopy to examine cells treated with sphingosine kinase inhibitors did not readily show impaired autophagosome-lysosome fusion. These studies provide strong evidence that sphingosine kinase activity is required for an autolysosomal function downstream of autophagosome-to-lysosome fusion. These studies also support the approach of targeting sphingosine kinase (1 and/or 2) as an effective means to block pro-survival autophagy in breast cancer cells undergoing antiestrogen therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3783. doi:10.1158/1538-7445.AM2011-3783

Introduction: SLC6A14 is an amino acid transporter that transports all essential amino acids as well as glutamine and arginine, and is energized by Na+ gradient, Cl- gradient, and membrane potential. The expression of SLC6A14 is upregulated in ER-positive breast cancer cells but not in ER-negative breast cancer cells. Since cancer cells have an increased need for essential amino acids (protein synthesis), Gln (nucleotide synthesis/glutaminolysis), and Arg (essential amino acid in cancer), we hypothesize that cancer cells satisfy their need for these amino acids by upregulating SLC6A14, and that blockade of SLC6A14 offers a rational strategy for treatment of ER-positive breast cancer. Methods: The transport of Leu, Gln, and Arg via SLC6A14 was studied in X. laevis oocyte expression system. SLC6A14 expression was monitored by RT-PCR and immunohistochemistry in normal and tumors (ER-positive and ER-negative) from primary breast cancer specimens. The influence of estrogen on SLC6A14 expression was investigated in ER-positive BT474 breast cancer cells. α-MT was used as the blocker of SLC6A14. The differential effects of α-MT on MCF7 (ER-positive), MB231 (ER-negative) and MCF10A (non-malignant) cells were investigated in terms of mTOR activity, autophagy, and apoptosis. The ability of α-MT to inhibit the growth of breast cancer cells was evaluated by xenograft in nude mice. α-MT was administered to mice in drinking water (2 mg/ml). Plasma levels of α-MT were measured by HPLC. Results: The transport of Leu, Gln, and Arg via SLC6A14 was dependent on Na+ and Cl- with a Na+:Cl-:amino acid stoichiometry of 2:1:1, and was blocked by α-MT. SLC6A14 expression was upregulated in ER-positive breast cancer. SLC6A14 was not expressed in ER-negative breast cancer. Estrogen induced SLC6A14 expression in ER-positive breast cancer cells. Exposure of ER-positive MCF7 breast cancer cells to α-MT induced the expression of asparagine synthetase and CHOP, inhibited mTOR activity, and induced autophagy and apoptosis. When combined with an autophagy inhibitor, α-MT induced apoptosis in MCF7 cells more potently. These effects were specific for ER-positive breast cancer cells, and were not seen in ER-negative cells or in non-malignant cells. In mouse xenografts, α-MT reduced the growth of ER-positive ZR-75-1 breast cancer cells, but did not have any effect on the growth of ER-negative MB231 cells. MCF7 cells did not grow in nude mice unless estrogen was administered, and under these conditions, α-MT did not block the growth of the cells. Plasma concentration of α-MT in treated mice was 8.5 ± 1.5 μM. α-MT was not detectable in the plasma of untreated mice. Conclusions: SLC6A14 is an ideal and effective drug target for treatment of ER-positive breast cancer. α-MT has potential for use as an anticancer drug, and may also serve as a lead compound in the design/development of newer blockers of SLC6A14 for use in humans. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4459. doi:10.1158/1538-7445.AM2011-4459

In recent studies, we have demonstrated that autophagy is essential for the survival of estrogen receptor positive (ER+) breast cancer cells during anti-estrogen therapy and facilitates the development of antiestrogen resistance. For these studies, we subjected ER+ MCF-7 cells to a step-wise drug selection protocol, with 4 hydroxytamoxifen (4-OHT) as the selecting drug and established an antiestrogen resistant subline, designated TR5 (4-OHT resistant to 5μM). TR5 cells, unlike the parent MCF-7 cells, do not die in response to 4-OHT treatment and tolerate high levels of antiestrogen-induced autophagosome formation. Microarray analysis of 4-OHT-treated TR5 cells shows an approximate 5-10 -fold increase in TONDU (TDU) mRNA compared to that of the parent MCF-7 cells. TDU, also known as vestigial like gene-1, was initially identified as a transcription factor required for wing development in Drosophilia, but specific roles for TDU in mammalian cells have not been identified. Based on the upregulation of TDU in antiestrogen-resistant TR5 cells, we hypothesized that TDU played a role in pro-survival autophagy. To test this hypothesis, we analyzed the levels of TDU in cultured MCF-7 and TR5 cells under conditions that induce autophagy and further determined how modulation of TDU levels (TDU cDNA overexpression and TDU attenuation by RNAi targeting) affected autophagy and death. 4-OHT and rapamycin, an mTOR inhibitor that induces macroautophagy, increased TDU expression within 24 hours of treatment and increased TDU expression persisted for 96 hours. Further, quantitative Real-Time (RT) PCR analysis of cells overexpressing TDU revealed that TDU upregulated the transcription of autophagy genes (i.e. upregulation of LC3, Atg5 and Atg6). Importantly, the TDU-mediated increased expression of autophagy genes was blocked by TDU RNAi. Consistent with this apparent TDU-induced autophagy, TDU overexpression significantly increased autophagic proteolysis (long-lived protein turnover). In addition, down regulation of TDU by RNAi induced cell death in E2- and 4-OHT-treated MCF-7 and TR5 cells, with increased mitochondrial membrane permeability, cleavage of PARP, and cleavage of lamin-A as read-outs of cell death. To our knowledge, these studies uniquely identify TDU as a transcription factor involved in the regulation of autophagy in ER+ breast cancer cells and as a potential molecular target to block pro-survival autophagy and enhance the cytotoxicity of antiestrogen therapy. Our current studies are aimed at determining whether TDU expression levels can serve as a prognostic indicator of breast cancer response to hormonal therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4845.

SLC6A14 (also known as ATB0,+) is a Na+/Cl− -coupled amino acid transporter that transports 18 of the 20 proteinogenic amino acids, including all essential amino acids. Normal tissues express this transporter at low levels, whereas tumor tissues (colon, breast, and cervix) upregulate this transporter several-fold. The unique functional features of SLC6A14 (broad substrate specificity and multiple driving forces) make it an ideal transporter for tumor cells to maintain amino acid nutrition necessary for enhanced cell proliferation. We speculate that SLC6A14 could not only be an ideal tumor-specific delivery system for amino acid-based anti-cancer drugs but also be a direct drug target for cancer chemotherapy. Recently we identified alpha-methyltryptophan (ALT) as a blocker of SLC6A14. Here we examine the potential of this blocker as an anti-cancer agent in SLC6A14-positive breast and colon cancer cells. Treatment of MCF7 (a breast cancer cell line), HCT116 and LS174T (colon cancer cell lines) with ALT causes amino acid starvation as evidenced by the upregulation of asparagine synthetase and CHOP. These three cell lines are SLC6A14-positive. In contrast, ALT treatment of breast cancer cell line MDA-MB231, which is SLC6A14-negative, does not show any evidence of amino acid starvation. Similarly, ALT treatment has no effect on normal breast and colon epithelial cell lines (MCF10A and CCD841, respectively). These cell lines also do not express SLC6A14. These data show that ALT-induced amino acid starvation is mediated specifically through the blockade of SLC6A14. Since nutrient signaling is also associated with changes in mTOR pathway, we examined the influence of ALT treatment on phosphorylation of S6 and S6kinase. Blockade of SLC6A14 with ALT in SLC6A14-positive cancer cell lines leads to decreased mTOR signaling as evidenced by decreased phosphorylation of S6 and S6kinase. Again, such changes are not seen in SLC6A14-negative cancer or normal cell lines. The initial response of the SLC6A14-positive cancer cell lines to ALT treatment is induction of autophagy. ALT-induced autophagy is documented in these cells by light and electron microscopic examination as well as by biochemical means. Inhibition of autophagy with 3-methyladenine under these conditions leads to apoptosis. We conclude that SLC6A14 is a potential drug target for cancer chemotherapy and that ALT could serve as a lead compound for the future design of high-affinity blockers of SLC6A14. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4838.

In recent studies, we and others showed that autophagy is one of the prosurvival mechanisms during anti-estrogen therapy that plays a significant role in the development of resistance. In this study, we sought to determine if autophagy plays a role in the protective effects of insulin like growth factor 1 (IGF-1)/MEK1 signaling on breast cancer cells treated with hormonal therapy. In initial studies, we established that IGF1 protects estrogen receptor-positive (ER+) breast cancer cells from cell death induced by the anti-estrogen 4-hydroxytamoxifen (4-OHT) and the anti-progestin mifepristone (MIF) and that this protection was mediated by MEK1 signaling. To determine if the mechanism of MEK1-mediated protection involved modulation of autophagy, ER+ breast cancer cells alone or transfected with MEK1-GFP or MEK1 dominant negative (MEK1-DN) expression vectors were treated with 4-OHT and/or MIF, in the presence or absence of IGF1. Effects on autophagy were monitored using immunoblotting with antibodies to key autophagy proteins, electron microscopy, GFP-LC3 reporter-based expression analysis, quantitative PCR, and autophagic proteolysis (turnover of long-lived proteins). Apoptosis was monitored with the JC-1 assay (mitochondrial membrane permeability determination), trypan blue exclusion, cytokeratin 18 cleavage assay, and determination of the levels of cleaved PARP and lamin A. The results showed that IGF1 significantly increased the levels of several autophagy genes, including Atg8, Atg6, and Atg5. However, even with the increased levels of these autophagy proteins, IGF-1 was shown to clearly suppress hormonally-induced long-lived protein turnover or macroautophagy. Thus, the levels of key autophagy proteins were increased, while the catabolic function of the mature autolysosomes was reduced. MEK1 overexpression also significantly protected breast cancer cells from death, while MEK1DN expression significantly enhanced 4-OHT and/or MIF induced apoptotic cell death. Our interpretation of the concomitant attenuation of macroautophagy and cell death by IGF1/MEK signaling is not that autophagy is a prerequisite to cell death, but that autophagic catabolism is a readout of the significant stress experienced by MEK1 inhibition in breast cancer cells. Based on our results, we are now addressing three key questions: (1) will blockade of autophagy enhance the cytotoxicity of MEK1 inhibitors; (2) is the elevated expression of autophagy genes in IGF-1-treated breast cancer cells mediated by MEK1 signaling; and (3) is mitophagy enhanced in breast cancer cells overexpressing MEK1. This knowledge will be used to support the proposed use of MEK1 blockade to enhance the cytotoxic action of hormonal therapy and to circumvent the occurrence of anti-estrogen resistance of breast cancer, particularly in patients with high circulating levels of IGF-1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4846.

At a specific time in the Caulobacter crescentus cell cycle, a single flagellar filament and multiple receptor sites for the swarmer-specific phage phi Cbk are assembled at one pole of the predivisional cell. One cluster of genes required for this morphogenesis, the flaYG region, includes the flgJKL genes, which encode structural proteins of the flagellar filament. These flagellin genes are flanked by genes required for filament assembly, the flaYE genes at one end and the flaF-flbT-flbA-flaG genes at the other. In this study, we characterized mutants carrying large chromosomal deletions within this region. Several of these strains are phi CbK resistant and produce a novel 22-kDa flagellin that is not assembled into flagella. Merodiploid strains containing either the entire flaFG region or individual fla transcription units from this region were constructed. These strains were used to correlate the presence or absence of specific gene products to changes in flagellin synthesis, filament assembly, or phage sensitivity. As a result of these studies, we were able to conclude that (i) the production of the 22-kDa flagellin results from the absence of the flbA and flaG gene products, which appear to be components of a flagellin-processing pathway common to the 25-, 27-, and 29-kDa flagellins; (ii) flbT negatively modulates the synthesis of the 27- and 25-kDa flagellins from two genetically unlinked gene clusters; (iii) flgL is the only flagellin gene able to encode the 27-kDa flagellin, and this flagellin appears to be required for the efficient assembly of the 25-kDa flagellins; (iv) flaF is required for filament assembly; and (v) phi CbK resistance results from the deletion of at least two genes in the flaFG region.

This study characterizes amplified structures carrying the human multidrug resistance (MDR) genes in colchicine-selected multidrug resistant KB cell lines and strongly supports a model of gene amplification in which small circular extrachromosomal DNA elements generated from contiguous chromosomal DNA regions multimerize to form cytologically detectable double minute chromosomes (DMs). The human MDR1 gene encodes the 170-kDa P-glycoprotein, which is a plasma membrane pump for many structurally unrelated chemotherapeutic drugs. MDR1 and its homolog, MDR2, undergo amplification when KB cells are subjected to stepwise selection in increasing concentrations of colchicine. The structure of the amplification unit at each step of drug selection was characterized using both high-voltage gel electrophoresis and pulsed-field gel electrophoresis (PFGE) techniques. An 890-kb submicroscopic extrachromosomal circular DNA element carrying the MDR1 and MDR2 genes was detected in cell line KB-ChR-8-5-11, the earliest step in drug selection in which conventional Southern/hybridization analyses detected MDR gene amplification. When KB-ChR-8-5-11 was subjected to stepwise increases in colchicine, this circular DNA element dimerized as detected by PFGE with and without digestion with Not 1, which linearizes the 890-kb amplicon. This dimerization process, which also occurred at the next step of colchicine selection, resulted in the formation of cytologically detectable DMs revealed by analysis of Giemsa-stained metaphase spreads.

Cloning vectors for studies of Caulobacter crescentus genes should be transferrable between Escherichia coli and C. crescentus since a transformation system has not been developed for C. crescentus. We have tested a large number of vectors containing IncP or IncQ replicons and found that many of the vectors containing IncQ replicons, and all but one of the vectors containing IncP replicons, are readily transferred by conjugation into C. crescentus. All of the plasmids tested were maintained in C. crescentus at 1 to 5 copies per cell, but plasmids containing IncP replicons were more stable than plasmids containing IncQ replicons. Further studies with a derivative of the IncQ plasmid R300B showed that when a promoterless kanamycin (Km)-resistance gene (npt2) was inserted into the intercistronic region of the sul-aphC (SuR-SmR) operon, Km resistance was expressed only when the npt2 gene was inserted such that it would be transcribed from the sul promoter. These data indicate that R300B does not contain sequences which would provide promoter function in C. crescentus in the orientation opposite to that of the sul operon and that any genes cloned in this orientation would require native promoters for expression. To provide greater versatility for cloning into R300B, additional vectors were constructed by the addition of multiple cloning sites in the intercistronic region of the sul-aphC operon. In addition, chromosomal DNA libraries were constructed in R300B and in the cosmid vector pLAFR1-7. Specific clones from these libraries containing genes of interest were identified by complementation of the appropriate C. crescentus mutants.

During the Caulobacter crescentus cell cycle, flagellin synthesis and filament assembly are temporally controlled events which require the products encoded by the contiguous flaF, flbT, and flbA-flaG transcription units (P.V. Schoenlein, L.S. Gallman, and B. Ely, J. Bacteriol. 171:000-000, 1989). To better define the functions of these genes, immunoprecipitation studies, Western blot (immunoblot) analyses, and electron microscopic analyses characterized flagellin synthesis and assembly in mutant and merodiploid strains. Mutations in the flaF or flbA-flaG transcription unit resulted in reduced synthesis of the 25- and 27-kilodalton (kDa) flagellins. In contrast, mutations in flbT resulted in overproduction of these flagellins. The FlbT phenotype is unique, since all other identified C. crescentus fla mutations cause a reduction in the levels of the 25- and 27-kDa flagellins. Furthermore, the flbT mutant showed a chemotaxis deficiency even though it was motile. Thus, the flbT gene product appears to be involved in the regulation of both flagellin synthesis and chemotactic function. Mutations in the flbT and flbA-flaG transcription units also resulted in the production of a 22-kDa flagellin species that is not normally detected in wild-type cells. This flagellin species was not detected in the flbT filaments. Furthermore, the 22-kDa flagellin was no longer detected in flbA pseudorevertants that assembled functional filaments. Thus, the 22-kDa flagellin does not appear to be assembled into filaments. Since many of the flbT filaments are shorter than wild-type filaments, we discuss the possibility that the 22-kDa flagellin species may adversely affect flagellin assembly in this mutant.