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Supplementary Table 1 from ErbB2 Trafficking and Degradation Associated with K48 and K63 Polyubiquitination

Supplementary Figure 3 from ErbB2 Trafficking and Degradation Associated with K48 and K63 Polyubiquitination

Supplementary Figure 2 from ErbB2 Trafficking and Degradation Associated with K48 and K63 Polyubiquitination

Supplementary Figure Legends 1-3 from ErbB2 Trafficking and Degradation Associated with K48 and K63 Polyubiquitination

Supplementary Figure 1 from ErbB2 Trafficking and Degradation Associated with K48 and K63 Polyubiquitination

The overexpressed ErbB2/HER2 receptor is a clinically validated cancer target whose surface localization and internalization mechanisms remain poorly understood. Downregulation of the overexpressed 185-kDa ErbB2 receptor is rapidly (2–6 hours) induced by the HSP90 chaperone inhibitor geldanamycin (GA), whereas its downregulation and lysosomal degradation are more slowly (24 hours) induced by the proteasome inhibitor bortezomib/PS341. In PS341-treated SK-BR-3 cells, overexpressed ErbB2 coprecipitates with the E3 ubiquitin ligase c-Cbl and also with the deubiquitinating enzyme USP9x; moreover, siRNA downregulation of USP9x enhances PS341-induced ErbB2 downregulation. Because polyubiquitin linkages via lysine 48 (K48) or 63 (K63) can differentially address proteins for 26S proteasomal degradation or endosome trafficking to the lysosome, multiple reaction monitoring (MRM)/mass spectrometry (MS) and polyubiquitin linkage–specific antibodies were used to quantitatively track K48-linked and K63-linked ErbB2 polyubiquitination following either GA or PS341 treatment of SK-BR-3 cells. MRM/MS revealed that unlike the rapid, modest (4-fold to 8-fold), and synchronous GA induction of K48 and K63 polyubiquitinated ErbB2, PS341 produces a dramatic (20-fold to 40-fold) sequential increase in polyubiquitinated ErbB2 consistent with K48 polyubiquitination followed by K63 editing. Fluorescence microscopic imaging confirmed that PS341, but not GA, induces colocalization of K48-linked and K63-linked polyubiquitin with perinuclear lysosome-sequestered ErbB2. Thus, ErbB2 surface overexpression and recycling seem to depend on its polyubiquitination and deubiquitination; as well, the contrasting effects of PS341 and GA on ErbB2 receptor localization, polyubiquitination, and degradation point to alternate cytoplasmic trafficking likely regulated by different K48 and K63 polyubiquitin editing mechanisms. Cancer Res; 70(9); 3709–17. ©2010 AACR.

A major challenge to broadening oncology applications for inhibitors of the ubiquitin-proteasome system (UPS) is the identification of UPS-dependent cancer pathways predictive of tumors responsive to peptidomimetic inhibitors of its 20S core protease activity. To inform clinical studies evaluating UPS inhibitors as breast cancer therapeutics, seven phenotypically diverse human breast cancer cell line models were characterized for their cellular and molecular responses to the clinically approved 20S inhibitor bortezomib (PS341; Velcade), focusing on those overexpressing estrogen receptor (ER) or ERBB2/HER2, because these oncogenic receptor pathways are constitutively activated in approximately 80% of all breast cancers. All models demonstrated dose-dependent bortezomib reduction in intracellular 20S activity correlating with cell growth inhibition, and bortezomib IC(50) values (concentrations producing 50% growth inhibition) varied directly with pretreatment 20S activities (r = 0.74; *, p0.05), suggesting that basal 20S activity may serve as a clinical predictor of tumor responsiveness to UPS inhibition. Reduction in 20S activity (60%) was associated with early (24 h) intracellular relocalization of ER (nucleus to cytoplasm) and ERBB2 (plasma membrane to perinuclear lysosomes), buildup of ubiquitinated and Hsp70-associated receptor, degradation and loss of ER and ERBB2 function, and induction of cellular apoptosis. These models were also used to screen a pharmacologic panel of pathway-targeted anticancer agents [4-hydroxy-3-methoxy-5-(benzothiazolylthiomethyl)benzylidenecyanoacetamide (AG825), 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzoimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide (AZD6244/ARRY142886), 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride (LY294002), 17-N-allylamino-17-demethoxy geldanamycin (17AAG), and (2E)-N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]amino]methyl]phenyl]-2-propenamide (LAQ824)] for those capable of sensitizing to bortezomib. In keeping with the observation that 20S reduction has little effect on mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling in either ER-positive or ERBB2-positive models, only the MEK-1/2 inhibitor AZD6244 consistently improved the antitumor activity of bortezomib.

Power as the ability to determine action is critical to any organizational player in order to achieve goals and set strategic directions. For marketing departments power perspectives have only been scarcely applied with analyses focusing on structural and contingency determinants, thus lacking a thorough understanding of the drivers of marketing power. Particularly personal power sources have gone unmentioned or been explicitly excluded in marketing power research (Homburg et al. 1999). However, insight on the underlying mechanisms is needed to shed light on the effectiveness of power sources and to design empowering means that counter marketing’s strategic decline and enable marketing to reflect its attributed organizational importance. Acknowledging the shortcomings in previous research, this study proposes a combination of person- and position-based power sources to explore the marketing department’s power. The study applies a framework by Kenny and Wilson (1984) that includes a) expertise and charisma as personal power sources to reveal the role of individual characteristics, b) access to resources, access to information, and relational capital to account for hierarchical or position-related derivation of power, and c) additionally incorporates asset specificity as a context specific moderator. In doing so, the study picks up on research of micro-foundations by investigating the contributions that individuals make to departmental power beyond unit-level or firm-level factors and accounts for the relevance of contextual factors, as they influence the function of power sources. The research propositions are derived from Resource Dependence Theory (RDT) and follow the understanding that a department acquires power by providing resources to the firm that top management depends on in order to achieve its goals. By providing necessary information, business relationships, and expertise, the marketing department’s person-based and position-based factors function as sources of power. The less substitutable and more critical these factors are to overall organizational success, the more power the marketing department gains.

Market orientation, an important organizational concept, is fundamentally driven by the marketing department, as marketing exerts its power to spread a market-oriented mindset throughout the organization. While the determinants and success-related benefits of market orientation have been broadly studied, the conditions under which it is established have been largely neglected. Addressing this gap, this paper investigates the associated role of the marketing department’s power in the creation of market orientation and how it is moderated by means of managerial control. Based on a Resource Dependence Theory (RDT) perspective, it is argued that top management depends on marketing to exert its power in order to induce market-oriented behaviors and values firm-wide, and that to manage this dependence top management iniates control. To observe the moderating effect of managerial control, we differentiate between formal and informal controls as Organizational Control Theory (OCT) suggests that the feasibility and effectiveness of controls is affected by the respective task context. Previous literature noted that establishing market orientation reflects a process of cultural transformation (Gebhardt et al. 2006) and therefore describes a changing and uncertain environment. Following OCT, formal controls are impaired in uncertain context settings, whereas informal controls serve as a “fall-back” option that is effective in such contexts. Therefore, we advance current conceptualizations of management control in market orientation research by applying formalization, centralization and output control as forms of formal control, and being the first to include socialization and selection & training as informal controls. The study aims to show that informal controls are suitable mechanisms to foster marketing departmental power in the creation of market orientation.

The marketing department’s power is an important topic in current marketing debate. Previous scholars have focused largely on exploring its capability-related and contingency-related antecedents, its dispersion, and its performance implications. Customer connection, as a critical organizational capability, has been related to increasing marketing’s power. However, this capability has seen equivocal results as a source of power. For instance, Moorman and Rust (1999) confirm its relevance to the emergence of marketing power, while Verhoef and Leeflang (2009) do not. The mixed results indicate that different context factors must be at work and they underscore the need to explore contextual moderators (e.g., Verhoef et al., 2011). As marketing literature has mostly neglected the close link between Resource Dependence Theory (RDT) and Strategic Contingency Theory (SCT), our study advances previous work by combining RDT and SCT to argue that the relationship between power sources and power is contingent on context factors. Based on RDT, the customer connection capability represents a critical resource to firm success, so that firm’s depend on marketing’s customer-connecting role and marketing thus gains power. According to SCT, however, this relationship is additionally affected by contextual circumstances under which capabilities like customer connection are enhanced or hindered to function as sources of power. The study develops and tests a model based on Yan and Gray’s (2001) framework comprising of six context factors—asset specificity, selection and training, market orientation, environmental dynamism, differentiation strategy, and political skill of the head of marketing—that affect the availability of alternatives and strategic importance of resources like the customer connection capability.

Activation of nuclear factor-kappaB (NFkappaB) has been linked to the development of hormone-independent, estrogen receptor (ER)-negative human breast cancers. To explore the possibility that activated NFkappaB marks a subset of clinically more aggressive ER-positive breast cancers, NFkappaB DNA-binding was measured in ER-positive breast cancer cell lines and primary breast cancer extracts by electrophoretic mobility shift assay and ELISA-based quantification of specific NFkappaB p50 and p65 DNA-binding subunits. Oxidant (menadione 100 microMx30 min) activation of NFkappaB was prevented by pretreatment with various NFkappaB inhibitors, including the specific IkappaB kinase (IKK) inhibitor, parthenolide (PA), which was found to sensitize MCF-7/HER2 and BT474 but not MCF-7 cells to the antiestrogen tamoxifen. Early stage primary breast cancers selected a priori for lower ER content (21-87 fmol/mg; n=59) and known clinical outcome showed two- to four-fold increased p50 and p65 NFkappaB DNA-binding over a second set of primary breast cancers with higher ER content (100 fmol/mg; n=22). Breast cancers destined to relapse (13/59) showed significantly higher NFkappaB p50 (but not p65) DNA-binding over those not destined to relapse (46/59; p=0.04). NFkappaB p50 DNA-binding correlated positively with several prognostic biomarkers; however, only NFkappaB p50 DNA-binding (p=0.04), Activator Protein-1 DNA-binding (AP-1; por=0.01) and urokinase-type plasminogen activator expression (uPA; p=0.0014) showed significant associations with metastatic relapse and disease-free patient survival. These clinical findings indicate that high-risk ER-positive breast cancers may be prognostically identified by increased NFkappaB p50 DNA-binding, and support preclinical models suggesting that therapeutic inhibition of NFkappaB activation may improve the endocrine responsiveness of high-risk ER-positive breast cancers.

The endosperm and embryo of barley ( Hordeum vulgare L.) grain were investigated to relate thioredoxin h and disulfide changes to germination and seedling development. The disulfide proteins of both tissues were found to undergo reduction following imbibition. Reduction reached a peak 1 day earlier in the embryo than in the endosperm, day 1 vs. day 2. The profile in both cases resembled those observed with wheat and rice, i.e., the reduction of the storage proteins increased initially and then declined during the period of seedling growth. The extent of the increase in reduction observed with barley endosperm was, however, less pronounced than with the other cereals. Also, unlike wheat and rice, the storage proteins of the endosperm were highly reduced in the dry seed and the sulfhydryl content of glutelins showed no appreciable change during this period. The relative abundance of thioredoxin h during germination and early seedling growth differed in the embryo and endosperm: a progressive decrease in the endosperm (as seen with wheat) vs. an increase in the embryo. Thioredoxin h was found in the major seed tissues in characteristic forms. Three forms were found in the scutellum and aleurone, whereas two, which may represent isoforms, were identified in the root and the shoot. Using a recently developed strategy based on two-dimensional gel electrophoresis, several proteins were identified as specific targets for thioredoxin in the embryo following oxidation with H(2)O(2), among them barley embryo globulin 1, peroxiredoxin and acidic ribosomal protein P(3). The results confirm earlier findings with the endosperm of other cereals and extend the importance of thioredoxin-linked redox change to the germinating embryo for functions that potentially include dormancy, protection against reactive oxygen species, translation and the mobilization of storage proteins.

In addition to repressing ERBB2 promoter function, histone deacetylase (HDAC) inhibitors induce the accelerated decay of mature ERBB2 transcripts; the mechanism mediating this transcript destabilization is unknown but depends on the 3′ untranslated region (UTR) of ERBB2 mRNA. Using ERBB2-overexpressing human breast cancer cells (SKBR3), the mRNA stability factor HuR was shown to support ERBB2 transcript integrity, bind and endogenously associate with a conserved U-rich element within the ERBB2 transcript 3′ UTR, coimmunoprecipitate with RNA-associated HDAC activity, and colocalize with HDAC6. HDAC6 also coimmunoprecipitates with HuR in an RNA-dependent manner and within 6 hours of exposure to a pan-HDAC inhibitor dose, that does not significantly alter cytosolic HuR levels or HuR binding to ERBB2 mRNA. Cellular ERBB2 transcript levels decline while remaining physically associated with HDAC6. Knockdown of HDAC6 protein by small interfering RNA partially suppressed the ERBB2 transcript decay induced by either pan-HDAC or HDAC6-selective enzymatic inhibitors. Three novel hydroxamates, ST71, ST17, and ST80 were synthesized and shown to inhibit HDAC6 with 14-fold to 31-fold greater selectivity over their binding and inhibition of HDAC1. Unlike more potent pan-HDAC inhibitors, these HDAC6-selective inhibitors produced dose-dependent growth arrest of ERBB2-overexpressing breast cancer cells by accelerating the decay of mature ERBB2 mRNA without repressing ERBB2 promoter function. In sum, these findings point to the therapeutic potential of HuR and HDAC6-selective inhibitors, contrasting ERBB2 stability effects induced by HDAC6 enzymatic inhibition and HDAC6 protein knockdown, and show that ERBB2 transcript stability mechanisms include exploitable targets for the development of novel anticancer therapies. (Mol Cancer Res 2008;6(7):1250–8)

Statins are cholesterol-lowering drugs with pleiotropic activities including inhibition of isoprenylation reactions and reduction of signals driving cell proliferation and survival responses. The objectives of this study were to examine the effects of statins on breast cancer cells, both in vitro and in vivo, and to begin to determine their mechanism of action. We evaluated the effects of statins on breast cancer cell growth, phosphoprotein signaling intermediates, survival/apoptosis regulators, cell cycle regulators, and activated transcription factors. We also examined the in vivo effect of statin administration in a mouse ErbB2+ breast cancer model. Only lipophilic statins had direct anticancer activity in vitro. Breast cancer cells with activated Ras or ErbB2 pathways seemed to be more sensitive than those overexpressing estrogen receptor, and this correlated with endogenous levels of activated nuclear factor κB (NF-κB). Key intermediates regulating cell survival by NF-κB activation, as well as cell proliferation by the mitogen activated protein kinase cascade, were among the earliest phosphoproteins influenced by statin treatment. These early effects were followed by declines in activator protein-1 and NF-κB activation and concordant changes in other mediators of proliferation and apoptosis. In vivo results showed that oral dosing of statins significantly inhibited the growth of a mouse mammary carcinoma. Lipophilic statins can exert direct anticancer activity in vitro by reducing proliferation and survival signals in susceptible breast cancer phenotypes. Tumor growth inhibition in vivo using a clinically relevant statin dose also seems to be associated with reduced tumor cell proliferation and survival. These findings provide supporting rationale for future statin trials in breast cancer patients. (Cancer Res 2006; 66(17): 8707-13)

A whole cell high-throughput screening assay was developed and tested against2,000 structurally and functionally diverse drug-like small molecules to identify lead compounds capable of cell permeability and selective silencing of ErbB2 transcription. Screening employed reporter sublines clonally selected from ErbB2-negative MCF7 breast cancer cells after stable genomic integration of the ErbB2 proximal promoter driving a luciferase reporter; anti-ErbB2 activities (50% inhibitory concentration values) were compared to inhibition of control MCF7 sublines bearing integrated reporters driven by either a mutated ErbB2 promoter or the cyclin D1 promoter. Of the seven resulting lead compounds, four emerged from the National Cancer Institute (NCI)/ Developmental Therapeutics Program (DTP) Structural Diversity Set (NSC-131547, NSC-176328, NSC-259968, and NSC-321237); three others emerged from a panel of anticancer compounds with known mechanistic actions and included a minor groove DNA-binding antibiotic (NSC-58514, chromomycin A3), a hydroxamic acid inhibitor of histone deacetylases (NSC-709238, trichostatin A), and a tripeptide aldehyde proteasome inhibitor (MG-132). For optimization, 58 scaffold analogs of the four NCI/DTP structural leads and nine functional analogs of the mechanistic leads were secondarily screened to identify seven compounds with comparable or superior activity relative to the leads, including an approved anticancer drug, PS-341 (bortezomib). PS-341 activity was validated against cultured ErbB2-positive breast cancer cell lines (SKBr3 and BT474) and a trastuzumab-resistant ErbB2-positive breast cancer xenograft model (B585), in which PS-341 antitumor activity correlated with selective down-regulation of ErbB2 mRNA and protein levels, confirming the ErbB2- silencing potential of proteasome inhibitors.

Biochemically active wheat thioredoxin h has been overexpressed in the endosperm of transgenic barley grain. Two DNA constructs containing the wheat thioredoxin h gene ( wtrxh ) were used for transformation; each contained wtrxh fused to an endosperm-specific B 1 -hordein promoter either with or without a signal peptide sequence for targeting to the protein body. Twenty-two stable, independently transformed regenerable lines were obtained by selecting with the herbicide bialaphos to test for the presence of the bar herbicide resistance gene on a cotransformed plasmid; all were positive for this gene. The presence of wtrxh was confirmed in 20 lines by PCR analysis, and the identity and level of expression of wheat thioredoxin h was assessed by immunoblots. Although levels varied among the different transgenic events, wheat thioredoxin h was consistently highly expressed (up to 30-fold) in the transgenic grain. Transgenic lines transformed with the B 1 -hordein promoter with a signal peptide sequence produced a higher level of wheat thioredoxin h on average than those without a signal sequence. The overexpression of thioredoxin h in the endosperm of germinated grain effected up to a 4-fold increase in the activity of the starch debranching enzyme, pullulanase (limit dextrinase), the enzyme that specifically cleaves α-1,6 linkages in starch. These results raise the question of how thioredoxin h enhances the activity of pullulanase because it was found that the inhibitor had become inactive before the enzyme showed appreciable activity.