Activation of the intrinsic apoptotic pathway represents a major mechanism for breast cancer regression resulting from anti-estrogen therapy. The BH3-only protein BIK is inducible by estrogen-starvation and anti-estrogen treatment and plays an important role in anti-estrogen induced apoptosis of breast cancer cells. BIK is predominantly localized to the endoplasmic reticulum where it regulates BAX/BAK-dependent release of Ca2+ from the endoplasmic reticulum stores and cooperates with other BH3-only proteins such as NOXA to cause rapid release of cytochrome c from mitochondria and activate apoptosis. BIK is also known to inactivate BCL-2 through complex formation. Previously, we demonstrated that apoptosis triggered by BIK in estrogen-starved human breast cancer cells is suppressed by GRP78, a major endoplasmic reticulum chaperone. Here we described the isolation of a novel clonal human breast cancer cell line (MCF-7/BUS-10) resistant to long-term estrogen deprivation. These cells exhibit elevated level of GRP78, which protects them from estrogen starvation-induced apoptosis. Our studies revealed that overexpression of GRP78 suppresses apoptosis induced by BIK and NOXA, either alone or in combination. Surprisingly, the interaction of GRP78 with BIK does not require its BH3 domain, which has been implicated in all previous BIK protein interactions. We further showed GRP78 and BCL-2 form independent complex with BIK and that increased expression of GRP78 decreases BIK binding to BCL-2. Our findings provide the first evidence that GRP78 can decrease BCL-2 sequestration by BIK at the endoplasmic reticulum, thus uncovering a potential new mechanism whereby GRP78 confers endocrine resistance in breast cancer. Activation of the intrinsic apoptotic pathway represents a major mechanism for breast cancer regression resulting from anti-estrogen therapy. The BH3-only protein BIK is inducible by estrogen-starvation and anti-estrogen treatment and plays an important role in anti-estrogen induced apoptosis of breast cancer cells. BIK is predominantly localized to the endoplasmic reticulum where it regulates BAX/BAK-dependent release of Ca2+ from the endoplasmic reticulum stores and cooperates with other BH3-only proteins such as NOXA to cause rapid release of cytochrome c from mitochondria and activate apoptosis. BIK is also known to inactivate BCL-2 through complex formation. Previously, we demonstrated that apoptosis triggered by BIK in estrogen-starved human breast cancer cells is suppressed by GRP78, a major endoplasmic reticulum chaperone. Here we described the isolation of a novel clonal human breast cancer cell line (MCF-7/BUS-10) resistant to long-term estrogen deprivation. These cells exhibit elevated level of GRP78, which protects them from estrogen starvation-induced apoptosis. Our studies revealed that overexpression of GRP78 suppresses apoptosis induced by BIK and NOXA, either alone or in combination. Surprisingly, the interaction of GRP78 with BIK does not require its BH3 domain, which has been implicated in all previous BIK protein interactions. We further showed GRP78 and BCL-2 form independent complex with BIK and that increased expression of GRP78 decreases BIK binding to BCL-2. Our findings provide the first evidence that GRP78 can decrease BCL-2 sequestration by BIK at the endoplasmic reticulum, thus uncovering a potential new mechanism whereby GRP78 confers endocrine resistance in breast cancer. IntroductionAnti-estrogen therapy represents a major advance in the treatment of estrogen receptor-positive breast cancer that can produce significant clinical responses and delay of progression. However, their efficacy is limited by intrinsic and acquired therapeutic resistance (1Musgrove E.A. Sutherland R.L. Nat. Rev. Cancer. 2009; 9: 631-643Crossref PubMed Scopus (968) Google Scholar, 2Tripathy D. J. Clin. Oncol. 2009; 27: 2580-2582Crossref PubMed Scopus (3) Google Scholar). To overcome this limitation, it is important to understand the resistance mechanisms and identify new therapeutic targets. Estrogen is required for the proliferation of estrogen receptor-positive breast cancer cells (3Thiantanawat A. Long B.J. Brodie A.M. Cancer Res. 2003; 63: 8037-8050PubMed Google Scholar). When subjected to estrogen starvation, exposure to anti-estrogens, or treatment with aromatase inhibitors, significant apoptosis of breast cancer cells was observed. A key regulator of apoptosis is the B-cell lymphoma (BCL) 2The abbreviations used are: BCLB-cell lymphomaaaamino acidsGRPglucose-regulated proteinBHBCL-2 homologyBIKBCL-2-interacting killer7-AAD7-amino actinomycin.-2 family of proteins which has been reported to localize to the membranes of various organelles (4Youle R.J. Strasser A. Nat. Rev. Mol. Cell Biol. 2008; 9: 47-59Crossref PubMed Scopus (3465) Google Scholar). Pro-survival members of the BCL-2 family such as BCL-2, share three or four of the conserved domains known as BCL-2 homology (BH) regions. BCL-2 and BCL-XL lower the Ca2+ store in the endoplasmic reticulum and antagonize the pro-apoptotic function of BAX to promote cell survival (5Teles A.V. Ureshino R.P. Dorta D.J. Lopes G.S. Hsu Y.T. Smaili S.S. Neurosci. Lett. 2008; 442: 96-99Crossref PubMed Scopus (8) Google Scholar). Pro-apoptotic members such as BAX and BAK share two or three BH domains. Upon activation, BAX translocates to mitochondria and initiates the release of cytochrome c into the cytosol (6Hsu Y.T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 3668-3672Crossref PubMed Scopus (1023) Google Scholar). A third group of apoptotic regulators, referred to as BH3-only proteins, share only the 9 amino acid BH3 region. In their active conformation, BH3-only proteins can induce BAX/BAK activation and initiate apoptosis. They can also bind directly to BCL-2 or BCL-XL through the BH3 domain and inhibit their pro-survival activities.BCL-2-interacting killer (BIK), the founding member of the BH3-only proteins, is a pro-apoptotic tumor suppressor in several human tissues and has also been used as a therapeutic target for anti-cancer drugs (7Chinnadurai G. Vijayalingam S. Rashmi R. Oncogene. 2008; 27: S20-29Crossref PubMed Scopus (83) Google Scholar). BIK plays a critical role in promotion of anti-estrogen-induced cell death in human breast cancer cells (8Hur J. Chesnes J. Coser K.R. Lee R.S. Geck P. Isselbacher K.J. Shioda T. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2351-2356Crossref PubMed Scopus (74) Google Scholar). Utilizing MCF-7/BUS cells that have been vigorously characterized as highly dependent on estrogen for growth, BIK mRNA and protein were strongly induced by estrogen-starvation or anti-estrogen treatment while the other BCL-2 family proteins, such as BCL-2, BCL-XL, and BAX were not affected (8Hur J. Chesnes J. Coser K.R. Lee R.S. Geck P. Isselbacher K.J. Shioda T. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2351-2356Crossref PubMed Scopus (74) Google Scholar, 9Coser K.R. Chesnes J. Hur J. Ray S. Isselbacher K.J. Shioda T. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 13994-13999Crossref PubMed Scopus (133) Google Scholar, 10Hur J. Bell D.W. Dean K.L. Coser K.R. Hilario P.C. Okimoto R.A. Tobey E.M. Smith S.L. Isselbacher K.J. Shioda T. Cancer Res. 2006; 66: 10153-10161Crossref PubMed Scopus (45) Google Scholar). Conversely, knockdown of BIK by siRNA significantly inhibited apoptosis caused by anti-estrogen treatment. BIK is predominantly localized to the outer membrane of the endoplasmic reticulum and mediates apoptosis through the mitochondrial pathway (11Mathai J.P. Germain M. Marcellus R.C. Shore G.C. Oncogene. 2002; 21: 2534-2544Crossref PubMed Scopus (109) Google Scholar). Although BIK does not interact directly with pro-apoptotic BAX and BAK, it regulates BAX/BAK-dependent release of Ca2+ from the endoplasmic reticulum stores, and cooperates with other BH3-only proteins such as NOXA, to cause rapid release of cytochrome c from mitochondria and activate caspases (12Mathai J.P. Germain M. Shore G.C. J. Biol. Chem. 2005; 280: 23829-23836Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). BIK is also known to form complex with BCL-2 at the endoplasmic reticulum and modulate BCL-2 activity; when in excess, endoplasmic reticulum-localized BCL-2 is able to protect against BIK-induced apoptosis (13Breckenridge D.G. Germain M. Mathai J.P. Nguyen M. Shore G.C. Oncogene. 2003; 22: 8608-8618Crossref PubMed Scopus (647) Google Scholar). The discovery that BIK is a key mediator of estrogen-starvation and anti-estrogen induced apoptosis implies that inhibition of BIK expression or activity at the endoplasmic reticulum may represent a novel mechanism for the development of endocrine resistance in human breast cancer.Toward understanding how BIK function is regulated at the endoplasmic reticulum, a search for novel interactive protein partners of BIK revealed that endogenous BIK selectively forms complex with the glucose-regulated protein GRP78 but not other endoplasmic reticulum chaperones (14Fu Y. Li J. Lee A.S. Cancer Res. 2007; 67: 3734-3740Crossref PubMed Scopus (249) Google Scholar). GRP78, also referred to as BiP/HSPA5, is a central regulator of endoplasmic reticulum function due to its role in protein folding and assembly, targeting misfolded proteins for degradation, endoplasmic reticulum Ca2+ binding and controlling the activation of transmembrane endoplasmic reticulum stress inducers (15Lee A.S. Cancer Res. 2007; 67: 3496-3499Crossref PubMed Scopus (687) Google Scholar, 16Ni M. Zhou H. Wey S. Baumeister P. Lee A.S. PLoS One. 2009; 4: e6868Crossref PubMed Scopus (119) Google Scholar, 17Wang M. Ye R. Barron E. Baumeister P. Mao C. Luo S. Fu Y. Luo B. Dubeau L. Hinton D.R. Lee A.S. Cell Death Differ. 2010; 17: 488-498Crossref PubMed Scopus (139) Google Scholar). GRP78 is induced by a wide variety of physiologic and pathologic stress (18Pfaffenbach K.T. Lee A.S. Curr. Opin. Cell Biol. 2011; 23: 150-156Crossref PubMed Scopus (221) Google Scholar) and is up-regulated in many types of cancer including breast cancer (15Lee A.S. Cancer Res. 2007; 67: 3496-3499Crossref PubMed Scopus (687) Google Scholar, 19Gazit G. Lu J. Lee A.S. Breast Cancer Res. Treat. 1999; 54: 135-146Crossref PubMed Scopus (136) Google Scholar, 20Lee E. Nichols P. Spicer D. Groshen S. Yu M.C. Lee A.S. Cancer Res. 2006; 66: 7849-7853Crossref PubMed Scopus (245) Google Scholar). Whereas GRP78 is predominantly an endoplasmic reticulum lumen protein, a subfraction can exist as a transmembrane protein, and either directly or indirectly interact with proteins localized at the outer endoplasmic reticulum membrane (21Rao R.V. Peel A. Logvinova A. del Rio G. Hermel E. Yokota T. Goldsmith P.C. Ellerby L.M. Ellerby H.M. Bredesen D.E. FEBS Lett. 2002; 514: 122-128Crossref PubMed Scopus (499) Google Scholar, 22Reddy R.K. Mao C. Baumeister P. Austin R.C. Kaufman R.J. Lee A.S. J. Biol. Chem. 2003; 278: 20915-20924Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar). GRP78 overexpression blocked BIK-induced apoptosis, suppressed estrogen starvation-induced BAX activation, mitochondrial permeability transition, and consequent apoptosis (14Fu Y. Li J. Lee A.S. Cancer Res. 2007; 67: 3734-3740Crossref PubMed Scopus (249) Google Scholar). Conversely, knockdown of endogenous GRP78 by siRNA sensitized human breast cancer cells to estrogen starvation-induced apoptosis. These studies provide the first evidence that GRP78 confers resistance to estrogen starvation-induced apoptosis in human breast cancer cells via a novel mechanism mediated by BIK.Establishment of anti-estrogen-resistant clonal cell lines offers important cell model systems to study the mechanism for resistance to endocrine therapy (23Coser K.R. Wittner B.S. Rosenthal N.F. Collins S.C. Melas A. Smith S.L. Mahoney C.J. Shioda K. Isselbacher K.J. Ramaswamy S. Shioda T. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 14536-14541Crossref PubMed Scopus (57) Google Scholar). Here we described the isolation of a novel clonal human breast cancer cell line (MCF-7/BUS-10) resistant to long-term estrogen deprivation. These cells exhibit an elevated level of GRP78, which protects them from estrogen starvation-induced apoptosis. To further understand the functional relationship between GRP78 and BIK, we showed that BIK acts cooperatively with NOXA to induce apoptosis. However, overexpression of GRP78 suppresses apoptosis induced by both BH3-only proteins, either alone or in combination. Surprisingly, the interaction of GRP78 with BIK does not require its BH3 domain, which has been implicated in all previous BIK protein interactions. GRP78 and BCL-2 form an independent complex with BIK and the increased expression of GRP78 can decrease BIK binding to BCL-2. Our findings provide the first evidence that GRP78 can regulate the amount of BCL-2 sequestered by BIK at the endoplasmic reticulum, thus uncovering a potential new mechanism whereby GRP78 confers endocrine resistance in breast cancer.DISCUSSIONEvidence is accumulating that induction of apoptosis is an important component of breast cancer regression resulting from anti-estrogen therapy (36Riggins R.B. Bouton A.H. Liu M.C. Clarke R. 2005; PubMed Scopus Google Scholar). on the that endocrine the of resistance and the that to therapy resistance is and clinical a major (1Musgrove E.A. Sutherland R.L. Nat. Rev. Cancer. 2009; 9: 631-643Crossref PubMed Scopus (968) Google Scholar). The BH3-only protein BIK, which is inducible by estrogen and has been to a critical role in the anti-estrogen-induced apoptosis of breast cancer cells (8Hur J. Chesnes J. Coser K.R. Lee R.S. Geck P. Isselbacher K.J. Shioda T. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2351-2356Crossref PubMed Scopus (74) Google Scholar, R.B. Bouton A.H. Liu M.C. Clarke R. 2005; PubMed Scopus Google Scholar). we demonstrated apoptosis triggered by BIK in estrogen-starved human breast cancer cells suppressed by GRP78, which complex with BIK (14Fu Y. Li J. Lee A.S. Cancer Res. 2007; 67: 3734-3740Crossref PubMed Scopus (249) Google Scholar). the mechanism is not In the we several on how GRP78 may cells to from intrinsic apoptosis mediated by we a clonal human breast cancer cell line that is resistant to long-term estrogen These cells were from the MCF-7/BUS which showed to estrogen deprivation. exposure of MCF-7/BUS cells to estrogen starvation, while the level of BIK was the level of GRP78 the first with of apoptosis (14Fu Y. Li J. Lee A.S. Cancer Res. 2007; 67: 3734-3740Crossref PubMed Scopus (249) Google Scholar). However, long-term estrogen starvation, the GRP78 level was and showed as by The of cell GRP78 knockdown in the resistant cells further demonstrated that GRP78 to the survival of cells estrogen GRP78 resistance to apoptosis Our studies have new functional between GRP78, BIK, NOXA, and BCL-2. Here we that BIK is a of apoptosis NOXA, and when a was observed. is in with the previous that overexpression of NOXA not induce cytochrome c release but of NOXA and BIK can induce activation of BAX and initiate mitochondrial cytochrome c release Lee A.S. Res. PubMed Scopus Google Scholar). The mechanism the by BIK and NOXA is that NOXA can complex with BAK from the binding of and BIK can activate BAX L. G. A. E. 2005; PubMed Scopus Google Scholar). is that the of apoptosis induction is due to BIK binding with BCL-2 at the endoplasmic reticulum and NOXA can GRP78 is able to both BIK and apoptosis, either or in combination. where GRP78 is induced and at level such as long-term estrogen it apoptosis resulting from BIK it is by BH3-only protein such as studies that BH3-only proteins such as BIK on their BH3 domain to interact with other members of the BCL-2 protein family Curr. Opin. Cell Biol. 2005; 17: PubMed Scopus Google Scholar). In the interactive domains between GRP78 and BIK, we that the BH3 domain of BIK is for its binding to the between to to the is required for binding to the other GRP78 binding to BIK at its amino is with previous findings that a subfraction of GRP78 may exist in a transmembrane with amino to the cytosol (21Rao R.V. Peel A. Logvinova A. del Rio G. Hermel E. Yokota T. Goldsmith P.C. Ellerby L.M. Ellerby H.M. Bredesen D.E. FEBS Lett. 2002; 514: 122-128Crossref PubMed Scopus (499) Google Scholar, 22Reddy R.K. Mao C. Baumeister P. Austin R.C. Kaufman R.J. Lee A.S. J. Biol. Chem. 2003; 278: 20915-20924Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar). we that GRP78 protein can bind to BIK, that the protein interaction is mediated by other proteins a we demonstrated that BIK not only forms complex with BCL-2, it also forms complex with GRP78 and BCL-2 form complex with the for binding to Our showed that of GRP78 in cells to of BCL-2 binding to BIK, and of BCL-2 decreases GRP78 binding to was in cell including human breast cancer cells. GRP78 and BCL-2 bind BIK at the in binding due to In to of BIK, it is that the binding between GRP78 and BIK may the of BIK it to the binding of does this apoptosis resulting from estrogen in we that when the breast cancer cells were to estrogen-starvation or anti-estrogen treatment the expression of BIK was the of at the outer of the endoplasmic of the of proteins BCL-2 with the endoplasmic reticulum, to Ca2+ release and of the of apoptosis including BAX to mitochondria and release of cytochrome c to the However, when GRP78 was in level long-term estrogen GRP78 to and BIK through complex formation. binding to BIK, BCL-2 is able to endoplasmic reticulum Ca2+ apoptosis. is with a strongly BCL-2 in the survival pathway by GRP78 overexpression C. M. J. A. C. Cell Death Differ. 2011; Google Scholar). further studies required to the role of GRP78, BIK, NOXA, and BCL-2 in anti-estrogen the complex in clinical which to into the of treatment and A. G. E. S. G. G. R. J. Cancer. PubMed Scopus Google Scholar, C. A. J. Cancer. Full Text Full Text PDF PubMed Scopus Google Scholar). In the cell studies provide the first evidence that GRP78 can the apoptotic of BIK and NOXA and the amount of BCL-2 binding to BIK as a mechanism for GRP78, also provide that of GRP78 may a novel therapeutic to resistance to long-term estrogen in breast cancer. that the apoptotic function of BIK has also been implicated in to and the the mechanisms may also in other systems M. B. J. Oncogene. 2005; PubMed Scopus Google Scholar, M. T. H. L. J. A.S. Mol. Cancer 2005; 4: PubMed Google Scholar). IntroductionAnti-estrogen therapy represents a major advance in the treatment of estrogen receptor-positive breast cancer that can produce significant clinical responses and delay of progression. However, their efficacy is limited by intrinsic and acquired therapeutic resistance (1Musgrove E.A. Sutherland R.L. Nat. Rev. Cancer. 2009; 9: 631-643Crossref PubMed Scopus (968) Google Scholar, 2Tripathy D. J. Clin. Oncol. 2009; 27: 2580-2582Crossref PubMed Scopus (3) Google Scholar). To overcome this limitation, it is important to understand the resistance mechanisms and identify new therapeutic targets. Estrogen is required for the proliferation of estrogen receptor-positive breast cancer cells (3Thiantanawat A. Long B.J. Brodie A.M. Cancer Res. 2003; 63: 8037-8050PubMed Google Scholar). When subjected to estrogen starvation, exposure to anti-estrogens, or treatment with aromatase inhibitors, significant apoptosis of breast cancer cells was observed. A key regulator of apoptosis is the B-cell lymphoma (BCL) 2The abbreviations used are: BCLB-cell lymphomaaaamino acidsGRPglucose-regulated proteinBHBCL-2 homologyBIKBCL-2-interacting killer7-AAD7-amino actinomycin.-2 family of proteins which has been reported to localize to the membranes of various organelles (4Youle R.J. Strasser A. Nat. Rev. Mol. Cell Biol. 2008; 9: 47-59Crossref PubMed Scopus (3465) Google Scholar). Pro-survival members of the BCL-2 family such as BCL-2, share three or four of the conserved domains known as BCL-2 homology (BH) regions. BCL-2 and BCL-XL lower the Ca2+ store in the endoplasmic reticulum and antagonize the pro-apoptotic function of BAX to promote cell survival (5Teles A.V. Ureshino R.P. Dorta D.J. Lopes G.S. Hsu Y.T. Smaili S.S. Neurosci. Lett. 2008; 442: 96-99Crossref PubMed Scopus (8) Google Scholar). Pro-apoptotic members such as BAX and BAK share two or three BH domains. Upon activation, BAX translocates to mitochondria and initiates the release of cytochrome c into the cytosol (6Hsu Y.T. Wolter K.G. Youle R.J. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 3668-3672Crossref PubMed Scopus (1023) Google Scholar). A third group of apoptotic regulators, referred to as BH3-only proteins, share only the 9 amino acid BH3 region. In their active conformation, BH3-only proteins can induce BAX/BAK activation and initiate apoptosis. They can also bind directly to BCL-2 or BCL-XL through the BH3 domain and inhibit their pro-survival activities.BCL-2-interacting killer (BIK), the founding member of the BH3-only proteins, is a pro-apoptotic tumor suppressor in several human tissues and has also been used as a therapeutic target for anti-cancer drugs (7Chinnadurai G. Vijayalingam S. Rashmi R. Oncogene. 2008; 27: S20-29Crossref PubMed Scopus (83) Google Scholar). BIK plays a critical role in promotion of anti-estrogen-induced cell death in human breast cancer cells (8Hur J. Chesnes J. Coser K.R. Lee R.S. Geck P. Isselbacher K.J. Shioda T. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2351-2356Crossref PubMed Scopus (74) Google Scholar). Utilizing MCF-7/BUS cells that have been vigorously characterized as highly dependent on estrogen for growth, BIK mRNA and protein were strongly induced by estrogen-starvation or anti-estrogen treatment while the other BCL-2 family proteins, such as BCL-2, BCL-XL, and BAX were not affected (8Hur J. Chesnes J. Coser K.R. Lee R.S. Geck P. Isselbacher K.J. Shioda T. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 2351-2356Crossref PubMed Scopus (74) Google Scholar, 9Coser K.R. Chesnes J. Hur J. Ray S. Isselbacher K.J. Shioda T. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 13994-13999Crossref PubMed Scopus (133) Google Scholar, 10Hur J. Bell D.W. Dean K.L. Coser K.R. Hilario P.C. Okimoto R.A. Tobey E.M. Smith S.L. Isselbacher K.J. Shioda T. Cancer Res. 2006; 66: 10153-10161Crossref PubMed Scopus (45) Google Scholar). Conversely, knockdown of BIK by siRNA significantly inhibited apoptosis caused by anti-estrogen treatment. BIK is predominantly localized to the outer membrane of the endoplasmic reticulum and mediates apoptosis through the mitochondrial pathway (11Mathai J.P. Germain M. Marcellus R.C. Shore G.C. Oncogene. 2002; 21: 2534-2544Crossref PubMed Scopus (109) Google Scholar). Although BIK does not interact directly with pro-apoptotic BAX and BAK, it regulates BAX/BAK-dependent release of Ca2+ from the endoplasmic reticulum stores, and cooperates with other BH3-only proteins such as NOXA, to cause rapid release of cytochrome c from mitochondria and activate caspases (12Mathai J.P. Germain M. Shore G.C. J. Biol. Chem. 2005; 280: 23829-23836Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). BIK is also known to form complex with BCL-2 at the endoplasmic reticulum and modulate BCL-2 activity; when in excess, endoplasmic reticulum-localized BCL-2 is able to protect against BIK-induced apoptosis (13Breckenridge D.G. Germain M. Mathai J.P. Nguyen M. Shore G.C. Oncogene. 2003; 22: 8608-8618Crossref PubMed Scopus (647) Google Scholar). The discovery that BIK is a key mediator of estrogen-starvation and anti-estrogen induced apoptosis implies that inhibition of BIK expression or activity at the endoplasmic reticulum may represent a novel mechanism for the development of endocrine resistance in human breast cancer.Toward understanding how BIK function is regulated at the endoplasmic reticulum, a search for novel interactive protein partners of BIK revealed that endogenous BIK selectively forms complex with the glucose-regulated protein GRP78 but not other endoplasmic reticulum chaperones (14Fu Y. Li J. Lee A.S. Cancer Res. 2007; 67: 3734-3740Crossref PubMed Scopus (249) Google Scholar). GRP78, also referred to as BiP/HSPA5, is a central regulator of endoplasmic reticulum function due to its role in protein folding and assembly, targeting misfolded proteins for degradation, endoplasmic reticulum Ca2+ binding and controlling the activation of transmembrane endoplasmic reticulum stress inducers (15Lee A.S. Cancer Res. 2007; 67: 3496-3499Crossref PubMed Scopus (687) Google Scholar, 16Ni M. Zhou H. Wey S. Baumeister P. Lee A.S. PLoS One. 2009; 4: e6868Crossref PubMed Scopus (119) Google Scholar, 17Wang M. Ye R. Barron E. Baumeister P. Mao C. Luo S. Fu Y. Luo B. Dubeau L. Hinton D.R. Lee A.S. Cell Death Differ. 2010; 17: 488-498Crossref PubMed Scopus (139) Google Scholar). GRP78 is induced by a wide variety of physiologic and pathologic stress (18Pfaffenbach K.T. Lee A.S. Curr. Opin. Cell Biol. 2011; 23: 150-156Crossref PubMed Scopus (221) Google Scholar) and is up-regulated in many types of cancer including breast cancer (15Lee A.S. Cancer Res. 2007; 67: 3496-3499Crossref PubMed Scopus (687) Google Scholar, 19Gazit G. Lu J. Lee A.S. Breast Cancer Res. Treat. 1999; 54: 135-146Crossref PubMed Scopus (136) Google Scholar, 20Lee E. Nichols P. Spicer D. Groshen S. Yu M.C. Lee A.S. Cancer Res. 2006; 66: 7849-7853Crossref PubMed Scopus (245) Google Scholar). Whereas GRP78 is predominantly an endoplasmic reticulum lumen protein, a subfraction can exist as a transmembrane protein, and either directly or indirectly interact with proteins localized at the outer endoplasmic reticulum membrane (21Rao R.V. Peel A. Logvinova A. del Rio G. Hermel E. Yokota T. Goldsmith P.C. Ellerby L.M. Ellerby H.M. Bredesen D.E. FEBS Lett. 2002; 514: 122-128Crossref PubMed Scopus (499) Google Scholar, 22Reddy R.K. Mao C. Baumeister P. Austin R.C. Kaufman R.J. Lee A.S. J. Biol. Chem. 2003; 278: 20915-20924Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar). GRP78 overexpression blocked BIK-induced apoptosis, suppressed estrogen starvation-induced BAX activation, mitochondrial permeability transition, and consequent apoptosis (14Fu Y. Li J. Lee A.S. Cancer Res. 2007; 67: 3734-3740Crossref PubMed Scopus (249) Google Scholar). Conversely, knockdown of endogenous GRP78 by siRNA sensitized human breast cancer cells to estrogen starvation-induced apoptosis. These studies provide the first evidence that GRP78 confers resistance to estrogen starvation-induced apoptosis in human breast cancer cells via a novel mechanism mediated by BIK.Establishment of anti-estrogen-resistant clonal cell lines offers important cell model systems to study the mechanism for resistance to endocrine therapy (23Coser K.R. Wittner B.S. Rosenthal N.F. Collins S.C. Melas A. Smith S.L. Mahoney C.J. Shioda K. Isselbacher K.J. Ramaswamy S. Shioda T. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 14536-14541Crossref PubMed Scopus (57) Google Scholar). Here we described the isolation of a novel clonal human breast cancer cell line (MCF-7/BUS-10) resistant to long-term estrogen deprivation. These cells exhibit an elevated level of GRP78, which protects them from estrogen starvation-induced apoptosis. To further understand the functional relationship between GRP78 and BIK, we showed that BIK acts cooperatively with NOXA to induce apoptosis. However, overexpression of GRP78 suppresses apoptosis induced by both BH3-only proteins, either alone or in combination. Surprisingly, the interaction of GRP78 with BIK does not require its BH3 domain, which has been implicated in all previous BIK protein interactions. GRP78 and BCL-2 form an independent complex with BIK and the increased expression of GRP78 can decrease BIK binding to BCL-2. Our findings provide the first evidence that GRP78 can regulate the amount of BCL-2 sequestered by BIK at the endoplasmic reticulum, thus uncovering a potential new mechanism whereby GRP78 confers endocrine resistance in breast cancer.
No takes yet. Share an insight, caveat, or question.
Zhou et al. (2011) studied this question.
Synapse has enriched 3 closely related papers on similar clinical questions. Consider them for comparative context: