Supplementary Table 1, Figures 1-4, Methods from Nrdp1-Mediated Regulation of ErbB3 Expression by the Androgen Receptor in Androgen-Dependent but not Castrate-Resistant Prostate Cancer Cells

Cisplatin-based combination chemotherapy is the foundation for treatment of advanced bladder cancer (BlCa), but many patients develop chemoresistance mediated by increased Akt and ERK phosphorylation. However, the mechanism by which cisplatin induces this increase has not been elucidated. Among six patient-derived xenograft (PDX) models of BlCa, we observed that the cisplatin-resistant BL0269 express high epidermal growth factor receptor, ErbB2/HER2 and ErbB3/HER3. Cisplatin treatment transiently increased phospho-ErbB3 (Y1328), phospho-ERK (T202/Y204) and phospho-Akt (S473), and analysis of radical cystectomy tissues from patients with BlCa showed correlation between ErbB3 and ERK phosphorylation, likely due to the activation of ERK via the ErbB3 pathway. In vitro analysis revealed a role for the ErbB3 ligand heregulin1-β1 (HRG1/NRG1), which is higher in chemoresistant lines compared to cisplatin-sensitive cells. Additionally, cisplatin treatment, both in PDX and cell models, increased HRG1 levels. The monoclonal antibody seribantumab, that obstructs ErbB3 ligand-binding, suppressed HRG1-induced ErbB3, Akt and ERK phosphorylation. Seribantumab also prevented tumor growth in both the chemosensitive BL0440 and chemoresistant BL0269 models. Our data demonstrate that cisplatin-associated increases in Akt and ERK phosphorylation is mediated by an elevation in HRG1, suggesting that inhibition of ErbB3 phosphorylation may be a useful therapeutic strategy in BlCa with high phospho-ErbB3 and HRG1 levels.

Prostate cancer is initially regulated by the androgen receptor (AR), a ligand-activated, transcription factor, and is in a hormone -dependent state (hormone -sensitive prostate cancer (HSPC)), but eventually becomes androgen -refractory (castration -resistant prostate cancer (CRPC)) because of mechanisms that bypass the AR, including by activation of ErbB3, a member of the epidermal growth factor receptor family. ErbB3 is synthesized in the cytoplasm and transported to the plasma membrane for ligand binding and dimerization, where it regulates downstream signaling, but nuclear forms are reported. Here, we demonstrate in prostatectomy samples that ErbB3 nuclear localization is observed in malignant, but not benign prostate, and that cytoplasmic (but not nuclear) ErbB3 correlated positively with AR expression but negatively with AR transcriptional activity. In support of the latter, androgen depletion upregulated cytoplasmic, but not nuclear ErbB3, while in vivo studies showed that castration suppressed ErbB3 nuclear localization in HSPC, but not CRPC tumors. In vitro treatment with the ErbB3 ligand heregulin-1 beta (HRG) induced ErbB3 nuclear localization, which was androgen -regulated in HSPC but not in CRPC. In turn, HRG upregulated AR transcriptional activity in CRPC but not in HSPC cells. Positive correlation between ErbB3 and AR expression was demonstrated in AR -null PC -3 cells where stable transfection of AR restored HRG-induced ErbB3 nuclear transport, while AR knockdown in LNCaP reduced cytoplasmic ErbB3. Mutations of ErbB3's kinase domain did not affect its localization but was responsible for cell viability in CRPC cells. Taken together, we conclude that AR expression regulated ErbB3 expression, its transcriptional activity suppressed ErbB3 nuclear translocation, and HRG binding to ErbB3 promoted it.

OBJECTIVE:Assess the effectiveness of a multifaceted stewardship intervention to reduce frequency and duration of inappropriate antibiotic use for emergency department (ED) patients with skin and soft tissue infections (SSTI). We hypothesized the antibiotic stewardship program would reduce antibiotic duration and improve guideline adherence in discharged SSTI patients. DESIGN:Nonrandomized controlled trial. SETTING:Academic EDs (intervention site and control site). PATIENTS OR PARTICIPANTS:Attending physicians and nurse practitioners at participating EDs. INTERVENTION(S):Education regarding guideline-based treatment of SSTI, tests of antimicrobial treatment of SSTI, implementation of a clinical treatment algorithm and order set in the electronic health record, and ED clinicians' audit and feedback. RESULTS:We examined 583 SSTIs. At the intervention site, clinician adherence to guidelines improved from 41% to 51% (aOR = 2.13 [95% CI: 1.20-3.79]). At the control site, there were no changes in adherence during the "intervention" period (aOR = 1.17 [0.65-2.12]). The between-site comparison of these during vs. pre-intervention odds ratios was not different (aOR = 1.82 [0.79-4.21]). Antibiotic duration decreased by 26% at the intervention site during the intervention compared to pre-intervention (Adjusted Geometric Mean Ratio [95% CI] = 0.74 [0.66-0.84]). Adherence was inversely associated with SSTI severity (severe vs mild; adjusted OR 0.42 [0.20-0.89]) and purulence (0.32 [0.21-0.47]). Mean antibiotic prescription duration was 1.95 days shorter (95% CI: 1.54-2.33) in the time period following the intervention than pre-intervention period. CONCLUSIONS:A multifaceted intervention resulted in modest improvement in adherence to guidelines compared to a control site, driven by treatment duration reductions.

Background. Acute upper respiratory tract infections are a common cause of emergency department (ED) visits and often result in unnecessary antibiotic treatment. Methods. We conducted a randomized clinical trial to evaluate the impact of a rapid, multipathogen respiratory panel (RP) test vs usual care (control). Patients were eligible if they were >= 12 months old, had symptoms of upper respiratory infection or influenza-like illness, and were not on antibiotics. The primary outcome was antibiotic prescription; secondary outcomes included antiviral prescription, disposition, and length of stay (ClinicalTrials.gov# NCT02957136). Results. Of 191 patients enrolled, 93 (49%) received RP testing; 98 (51%) received usual care. Fifty-three (57%) RP and 7 (7%) control patients had a virus detected and reported during the ED visit (P = .0001). Twenty (22%) RP patients and 33 (34%) usual care patients received antibiotics during the ED visit (-12%; 95% confidence interval, -25% to 0.4%; P = .06/0.08); 9 RP patients received antibiotics despite having a virus detected. The magnitude of antibiotic reduction was greater in children (-19%) vs adults (-9%, post hoc analysis). There was no difference in antiviral use, length of stay, or disposition. Conclusions. Rapid RP testing was associated with a trend toward decreased antibiotic use, suggesting a potential benefit from more rapid viral tests in the ED. Future studies should determine if specific groups are more likely to benefit from testing and evaluate the relative cost and effectiveness of broad testing, focused testing, and a combined diagnostic and antimicrobial stewardship approach.

BACKGROUND:Despite overexpression of the ErbB (EGFR/HER2/ErbB3/ErbB4) family in castration-resistant prostate cancer (CRPC), some inhibitors of this family, including the dual EGFR/HER2 inhibitor lapatinib, failed in Phase II clinical trials. Hence, we investigated mechanisms of lapatinib resistance to determine whether alternate ErbB inhibitors can succeed.METHODS:The CWR22 human tumour xenograft and its CRPC subline 22Rv1 and sera from lapatinib-treated CRPC patients from a previously reported Phase II trial were used to study lapatinib resistance. Mechanistic studies were conducted in LNCaP, C4-2 and 22Rv1 cell lines.RESULTS:Lapatinib increased intratumoral HER2 protein, which encouraged resistance to this treatment in mouse models. Sera from CRPC patients following lapatinib treatment demonstrated increased HER2 levels. Investigation of the mechanism of lapatinib-induced HER2 increase revealed that lapatinib promotes HER2 protein stability, leading to membrane localisation, EGFR/HER2 heterodimerisation and signalling, elevating cell viability. Knockdown of HER2 and ErbB3, but not EGFR, sensitised CRPC cells to lapatinib. At equimolar concentrations, the recently FDA-approved pan-ErbB inhibitor dacomitinib decreased HER2 protein stability, prevented ErbB membrane localisation (despite continued membrane integrity) and EGFR/HER2 heterodimerisation, thereby decreasing downstream signalling and increasing apoptosis.CONCLUSIONS:Targeting the EGFR axis using the irreversible pan-ErbB inhibitor dacomitinib is a viable therapeutic option for CRPC.

Skin and soft-tissue infections (SSTIs) are common infections seen in the Emergency Department (ED). However, adherence to IDSA guidelines for treatment, in terms of antibiotic selection and duration, is poor. Objective. To evaluate the ability of a multifaceted intervention to improve adherence to the 2014 IDSA guidelines for the management of SSTIs in an academic adult and pediatric ED. A non-randomized study of a multifaceted intervention for SSTI in the ED was employed which included educational presentations, implementation of an electronic order set based on 2014 IDSA guidelines, dissemination of ED-specific antibiograms, monthly departmental peer comparisons, and bimonthly individual feedback. SSTI visits were identified using ICD-10 codes L00, L02.x1, L03, L08.89, and L08.9. The primary endpoint was adherence to IDSA guidelines. Outcomes during the preintervention and intervention periods, adjusting for patient demographics and infection characteristics, were compared with the same time periods at a control site (CS) that did not implement the intervention. There were 583 SSTIs included in the study [intervention site (IS) =283, CS =300] split over three time periods: preintervention (October 2015–March 2016; IS = 130, CS = 150), intervention (October 2016–March 2017; IS = 99, CS = 150), and postintervention (April 2017–July 2017; IS = 54, CS = 0). At the IS, adherence was 41% prior to the intervention and 51% during the intervention. At the CS adherence was 19% and 25% during the two time periods. In the adjusted model, adherence at the IS was higher during the intervention compared with the preintervention period [adjusted odds ratio (aOR) 2.26 (95% CI 1.24–4.10)]. Adherence in the postintervention period was similar to the preintervention period [aOR 0.94 (0.45–1.97)]. No changes were seen during the two time periods at the CS [aOR 1.00 (0.53–1.89)]. Implementation of an antimicrobial stewardship intervention for SSTI significantly improved adherence to IDSA guidelines; however, adherence regressed after the intervention ended. Additionally, adherence was generally poor in all time periods and at both sites. Further research is needed to understand barriers and challenges to implementation of SSTI guidelines in ED settings. L. G. Miller, Merck: Grant Investigator, Research grant

Abstract Introduction: Despite ample evidence for the overexpression/overactivation of the epidermal growth factor receptor (EGFR) family, the dual EGFR/ErbB2 inhibitor lapatinib failed in patients with castration-resistant prostate cancer (CRPC). Our preclinical data show that the pan-ErbB inhibitor dacomitinib succeeds where lapatinib fails by decreasing membrane-localized EGFR family protein expression. Experimental Procedures: Nude mice were implanted with CWR22 tumors (human-patient-derived, androgen-dependent) and its CRPC subline 22Rv1 (relapsed CWR22). Mice were castrated or left intact and gavaged daily with 100 mg/kg lapatinib or vehicle. Tumors were analyzed for EGFR/ErbB2/ErbB3/androgen receptor (AR) proteins by both immunohistochemistry in paraffin-embedded tumor sections and Western blots in tumor lysates. Observations were validated in human-derived androgen-dependent and CRPC cell lines treated with lapatinib or dacomitinib by immunoblots and by immunofluorescence. Results: Intratumoral EGFR and ErbB2 (but not ErbB3) increased in intact, lapatinib-treated 22Rv1 tumor-bearing mice compared to placebo-treated ones. Elevated EGFR/ErbB2 was also observed in vitro in lapatinib-treated LNCaP, C4-2, PC-346C cells compared to vehicle-treated cells. EGFR phosphorylation increased from 1/5 intact, lapatinib-treated Rv1 tumors (20%) to 3/5 castrated, lapatinib-treated 22Rv1 tumors (60%). Lapatinib caused an increase in cytoplasmic EGFR and ErbB2 in LNCaP (androgen-dependent) and 22Rv1 (CRPC) cells. Low, physiological doses of dacomitinib overcame lapatinib resistance by suppressing cell viability and this effect was heightened by EGFR family knockdown or when AR activity was simultaneously inhibited. High-magnification microscopy revealed that dacomitinib possibly exerted its effects by decreasing the presence of membrane-localized EGFR, ErbB2 and ErbB3 proteins in comparison to lapatinib. Conclusions: We hypothesize that increased EGFR/ErbB2 heterodimerization is one cause of lapatinib resistance. Activated EGFR family proteins are typically localized in the plasma membrane and dacomitinib, by disrupting this membrane-localization, decreases cell viability. Future studies will attempt to refine the mechanism by which lapatinib and dacomitinib induce changes in RTK localization and how this may impact therapeutic response and prostate tumor progression. Citation Format: Maitreyee K. Jathak, Thomas M. Steele, Salma Siddiqui, Benjamin A. Mooso, Leandro S. D'Abronzo, Christiana M. Drake, Paramita M. Ghosh. The pan-ErbB inhibitor dacomitinib but not the dual EGFR/ErbB2 inhibitor labatinib disrupts membrane localization of the EGFR family of receptor tyrosine kinases [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 868.

Abstract Background: Prostate cancers (PCa) rely on androgenic ligands and the androgen receptor (AR) for their growth and survival, making AR inhibition a predominant therapeutic strategy for these tumors. Some prostate tumors however fail this therapy due to ‘bypass’ mechanisms that emerge as a result of prolonged AR targeting. This in vivo study attempted to assess the expression and activation of the epidermal growth factor receptor (EGFR) family (whose role is well-documented in PCa) in response to androgen deprivation therapy (ADT). Methods: Nude mice were implanted (s.c.) with CWR22 tumors (human-patient-derived, androgen-dependent ‘AD’) and its castration-resistant (‘CR’) subline CWR22-Rv1 (relapsed CWR22). Androgen deprivation (i.e. AR inhibition) was achieved by surgical or ‘sham’ castration of mice. Tumors were analyzed (immunohistochemistry/immunoblot) for EGFR/ErbB2/ErbB3/AR proteins and proliferative/apoptotic markers. Results: Castration caused significant tumor regression in AD but not CR tumors. in vitro viability assays demonstrated that castration (mimicked by using charcoal-stripped serum, ‘css’) did not slow down CR cells to the same degree as it did AD cells. At baseline, intratumoral EGFR protein was unchanged in R22 tumors, ErbB2 levels decreased and ErbB3 protein increased in Rv1 tumors. Castration increased ErbB3 but not EGFR or ErbB2 proteins in CWR22 tumors. Phosphorylated forms of these receptors were generally difficult to detect but there was more phosphorylated ErbB3 protein in Rv1 tumors. Downstream of the EGFR family, there was less phosphorylated Erk but not Akt protein in CWR22-Rv1 tumors. Castration decreased Erk protein in AD tumors but increased it in CR tumors. Immunohistochemical quantification revealed that cytoplasmic EGFR and ErbB3 proteins were elevated in CR tumors but reduced in AD tumors. Castration greatly decreased Ki-67 staining in AD but not in CR tumors while the number of TUNEL-positive nuclei and intensity of PARP staining decreased in castrated CR but not in AD tumors. ErbB3 and AR proteins were significantly correlated with DNA damage and proliferation in CWR22 tumors but only nuclear AR levels and proliferation were significantly correlated in CR tumors. Conclusions: We conclude that androgen deprivation therapy may alter EGFR and ErbB3 protein levels and localization in androgen-dependent and castration-resistant tumors. The EGFR family is typically activated at the cell surface hence their presence and activity there, in response to castration, may initiate signalling pathways encouraging tumor cell proliferation and survival. Citation Format: Maitreyee K. Jathal, Thomas M. Steele, Salma Siddiqui, Benjamin A. Mooso, Leandro S. D’Abronzo, Christiana M. Drake, Paramita M. Ghosh. In vivo analysis of EGFR family signalling as a bypass mechanism in prostate cancer. [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 1303.

Abstract The p14ARF tumor suppressor is often deleted or silenced in malignancies. Prostate tumors are an exception, where p14 expression is elevated. To understand this phenomenon, we assessed the expression of p53 pathway members, which are most effected by p14ARF. The expression of androgen receptor (AR), a pivotal prostate cancer regulator, which is also affected by p14arf and MDM2 was analyzed as well. The studies used archival prostate tumor tissues obtained from prostatectomies performed at the Veterans Affairs-Northern California Health Care System in Mather California between 1996 and 2002 to better define the relationship between these interrelated networks. A prostate tumor tissue array consisting of 78 tumors of differing stages and grades was constructed to evaluate correlations between multiple parameters. Immunohistochemical studies assessed expression of the proliferation marker Ki67, p53, MDM2, MDM4, p14ARF, and the AR in the nuclear and cytoplasmic compartments of tumor and adjacent cells. p53, MDM4, p14ARF and AR were detected in nuclear and cytoplasmic compartments of tumor and non-tumor cells, but were predominantly nuclear. MDM2 expression was primarily cytoplasmic in tumor cells. Multivariate analysis of the immunohistochemical markers identified a strong correlation between expression of p14ARF and AR. Studies utilizing the prostate CWR22 xenograft and LNCaP cell line models revealed that castration or androgen deprivation resulted in reduced p14arf levels and that this effect correlated with a precipitous decline in E2F1-3a levels. In a reciprocal analysis, RB ablation enhanced p14ARF transcription, arguing that the E2F/RB pathway mediates AR-dependent p14ARF expression. However, we also identified an AR binding site located ∼40 KB upstream of the p14ARF gene. Chromatin immunoprecipitation (ChIP) studies showed that in prostate cells this site was bound by AR. ChIP studies also revealed E2F1 and E2F3 were present at the p14ARF promoter. Together, the studies argue p14ARF is a direct transcriptional target of AR and that AR and E2F collaborate to promote p14ARF expression. . Citation Format: Maria Mudryj, Salma Siddiqui, Stephen J. Libertini, Alan P. Lombard, Benjamin Mooso, Leandro D'Abronzo, Frank Melgoza, Alexander Borowsky, Christiana Drake, LiHong Qi, Paramita M. Ghosh. Androgen receptor-mediated regulation of p14ARF transcription in prostate tumor cells. [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 5051. doi:10.1158/1538-7445.AM2015-5051

Prostate cancer (PCa) progression is regulated by the androgen receptor (AR); however, patients undergoing androgen-deprivation therapy (ADT) for disseminated PCa eventually develop castration-resistant PCa (CRPC). Results of previous studies indicated thatAR, a transcription factor, occupies distinct genomic loci in CRPC compared with hormone-naïve PCa; however, the cause of this distinction was unknown. The E3 ubiquitin ligaseNrdp1is a model AR target modulated by androgens in hormone-naïve PCa but not in CRPC. UsingNrdp1, we investigated how AR switches transcription programs during CRPC progression. The proximalNrdp1promoter contains an androgen response element (ARE); we demonstrated AR binding to this ARE in androgen-sensitive PCa. Analysis of hormone-naive human prostatectomy specimens revealed correlation betweenNrdp1and AR expression, supporting AR regulation of NRDP1 levels in androgen-sensitive tissue. However, despite sustained AR levels, AR binding to theNrdp1promoter andNrdp1expression were suppressed in CRPC. Elucidation of the suppression mechanism demonstrated correlation of NRDP1 levels with nuclear localization of the scaffolding protein filamin A (FLNA) which, as we previously showed, is itself repressed following ADT in many CRPC tumors. Restoration of nuclear FLNA in CRPC stimulated AR binding toNrdp1ARE, increased its transcription, and augmented NRDP1 protein expression and responsiveness to ADT, indicating that nuclear FLNA controls AR-mediated androgen-sensitiveNrdp1transcription. Expression of other AR-regulated genes lost in CRPC was also re-established by nuclear FLNA. Thus, our results indicate that nuclear FLNA promotes androgen-dependent AR-regulated transcription in PCa, while loss of nuclear FLNA in CRPC alters the AR-regulated transcription program.

Purpose: Conventional platinum based chemotherapy for advanced urothelial carcinoma is plagued by common resistance to this regimen. Several studies implicate the EGFR family of RTKs in urothelial carcinoma progression and chemoresistance. Many groups have investigated the effects of inhibitors of this family in patients with urothelial carcinoma. This review focuses on the underlying molecular pathways that lead to urothelial carcinoma resistance to EGFR family inhibitors.Materials and Methods: We performed a PubMed (R) search for peer reviewed literature on bladder cancer development, EGFR family expression, clinical trials of EGFR family inhibitors and molecular bypass pathways. Research articles deemed to be relevant were examined and a summary of original data was created. Meta-analysis of expression profiles was also performed for each EGFR family member based on data sets accessible via Oncomine (R).Results: Many clinical trials using inhibitors of EGFR family RTKs have been done or are under way. Those that have concluded with results published to date do not show an added benefit over standard of care chemotherapy in an adjuvant or second line setting. However, a neoadjuvant study using erlotinib before radical cystectomy demonstrated promising results.Conclusions: Clinical and preclinical studies show that for reasons not currently clear prior treatment with chemotherapeutic agents rendered patients with urothelial carcinoma with muscle invasive bladder cancer resistant to EGFR family inhibitors as well. However, EGFR family inhibitors may be of use in patients with no prior chemotherapy in whom EGFR or ERBB2 is over expressed.

Urothelial cell carcinoma of the bladder (UCCB) is the most common form of bladder cancer and it is estimated that ~15,000 people in the United States succumbed to this disease in 2013. Bladder cancer treatment options are limited and research to understand the molecular mechanisms of this disease is needed to design novel therapeutic strategies. Recent studies have shown that microRNAs play pivotal roles in the progression of cancer. miR-148a has been shown to serve as a tumor suppressor in cancers of the prostate, colon, and liver, but its role in bladder cancer has never been elucidated. Here we show that miR-148a is down-regulated in UCCB cell lines. We demonstrate that overexpression of miR-148a leads to reduced cell viability through an increase in apoptosis rather than an inhibition of proliferation. We additionally show that miR-148a exerts this effect partially by attenuating expression of DNA methyltransferase 1 (DNMT1). Finally, our studies demonstrate that treating cells with both miR-148a and either cisplatin or doxorubicin is either additive or synergistic in causing apoptosis. These data taken together suggest that miR-148a is a tumor suppressor in UCCB and could potentially serve as a novel therapeutic for this malignancy.

Abstract Virtually all patients undergoing standard-of-care androgen deprivation therapy (ADT) for recurrent prostate cancer (PCa) will develop castration-resistant prostate cancer (CRPC). Despite overexpression of the epidermal growth factor receptor (EGFR) family - especially EGFR, ErbB2 and ErbB3 - in all stages of PCa progression, the dual-kinase EGFR/ErbB2 inhibitor lapatinib (GW572016) failed in Phase 2 trials in hormone-naïve and CRPC patients. Here, we investigated mechanisms of lapatinib resistance using the CWR22 (‘R22’, hormone-naïve) and CWR22-Rv1 (‘Rv1’, CRPC) models of PCa progression. R22 or Rv1 tumors were implanted (s.c.) in male athymic nu/nu mice, castrated or left intact and treated daily with vehicle or 100mg/kg lapatinib (oral gavage). Tumors were collected at the study’s end and analyzed (Western Blot/WB, immunohistochemistry/IHC). With Ki67 staining, compared to R22 tumors, Rv1 tumors were more proliferative and unaffected by castration or lapatinib. R22 tumors regressed after castration but not lapatinib. As in human patients, lapatinib resistance was observed in hormone-naïve R22 and CRPC Rv1 tumors thus validating our experimental model. First we asked why Rv1 tumors proliferated rapidly. Rv1 tumors expressed 2-fold more ErbB3 than R22 tumors (WB). We previously showed that ErbB3 overexpression increased proliferation (Chen et al., Canc. Res, 70(14):5994-6003, 2010) - hence we attribute the rapid growth of Rv1 tumors to high ErbB3. Castration reduced EGFR, phosphorylated Akt and Erk in R22 but not in Rv1 tumors - decreased activation of these kinases may mediate the effects of castration on tumor growth. Rv1 tumors expressed alternately-spliced variants of androgen receptor (AR) that cause castration-resistance and less PSA than R22 tumors. Next, we asked why EGFR/ErbB2 inhibition failed in Rv1 tumors, despite EGFR's prominence in this tumor type. Lapatinib reduced intratumoral PSA levels in intact R22 mice but increased it in Rv1 mice. Lapatinib also increased EGFR and phosphorylated Akt in intact Rv1 but not R22 tumors. We hypothesize that, in Rv1 tumors, increased EGFR induces Akt phosphorylation which, we have shown earlier, regulates AR transcriptional activity and increases PSA expression. In contrast, in castrated Rv1 mice, lapatinib also increased ErbB3 levels. In Rv1 tumors, castration increased nuclear ErbB3 whereas in R22 tumors it increased cytoplasmic ErbB3 (IHC). Our results indicate that lapatinib may have failed in castrated Rv1 mice due to increased overall ErbB3 but in castrated R22 tumors due to increased cytoplasmic ErbB3. Regardless, the result is heightened ErbB3-related activity, which may cause increased proliferation and eventual drug failure. A pan-ErbB drug which also inhibits ErbB3, e.g. dacomitinib, can successfully overcome EGFR/ErbB2 resistance, as demonstrated in vitro. Citation Format: Maitreyee K. Jathal, Thomas M. Steele, Benjamin A. Mooso, Leandro S. D'Abronzo, Salma Siddiqui, Christiana Drake, Paramita M. Ghosh. Increased ErbB3 and EGFR activity mediate lapatinib resistance in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3335. doi:10.1158/1538-7445.AM2014-3335

Abstract Nearly 30% of superficial bladder cancer (BC) cases develop into muscle invasive (MI) cases. With an initial discovery rate of 30% of MI cases, it is clear that treatment of MI disease should be a priority. Currently radical cystectomy is used in MI cases with neoadjuvant platinum based chemotherapy (PBC) highly recommended in the literature. With 1% of MI BC patients receiving this neoadjuvant therapy, metastases are common and PBC is the current treatment for these patients. However, cisplatin resistance is common in patients with MI BC leading to an average survival of only 15 months with a 5 year survival of 20%. Increased expression of epidermal growth factor receptor (EGFR) and related receptors ErbB2 and ErbB3 in BC is well documented. These receptors control cell growth and survival and several EGFR ligands are present in normal human urine. Thus we hypothesized that these receptors may play a role in BC resistance to PBC. EGFR family inhibition (EGFRi) has been investigated in conjunction with PBC in clinical trials. However, these trials did not show and increased benefit due to many of these patients having previously failed PBC therapy, EGFR/ErbB status not being part of the inclusion criteria, and frequent resistance to EGFRi in this patient population. We wanted to determine a mechanism for cisplatin and EGFRi resistance with the hypothesis that this could lead to novel therapeutics for MI BC. The BC cell lines T24, TCCSUP, J82 and RT4 were assayed for cisplatin sensitivity by flow cytometry. The TCCSUP and J82 cells were deemed cisplatin sensitive due to increased apoptosis when treated with cisplatin. The T24 and RT4 cells showed no apoptosis increase and were deemed to be cisplatin resistant. MTT and clonogenic assay determined that cisplatin resistant cells were also resistant to EGFRi. Similarly, cisplatin sensitive cells were sensitive to EGFRi. Additionally, it was determined that treating all cell lines with cisplatin and dacomitinib had a synergistic effect on reducing cell proliferation. Activation of EGFR, ErbB2 and ErbB3 was assayed by western blotting. Cisplatin sensitive cells underwent EGFR driven ErbB activation while cisplatin resistant cells underwent ErbB3 driven ErbB activation. Additionally, activation of EGFR Y845, Y1045, and Y1068 was increased by cisplatin in resistant cells. These results indicate that EGFRi and cisplatin, while working in parallel pathways, can influence one another. EGFR binding may be a prognostic marker for response to EGFRi. If EGFR binds primarily with ErbB2, the cells would likely respond to EGFRi; but, if EGFR binds primarily with ErbB3, the cells would likely be EGFRi resistant. Activation of EGFR Y845 (a Src binding site) may determine the preferential ErbB binding. EGFR Y845 activation may be blocked by EGFRi thus abrogating the effect of cisplatin and returning the cells to a cisplatin sensitive state. Citation Format: Benjamin Asher Mooso, Duanna Challenger, Paramita Ghosh. ErbB receptors and cisplatin resistance: the case for bladder cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 605. doi:10.1158/1538-7445.AM2014-605

Abstract Despite availability of improved androgen receptor (AR) inhibitors for the treatment of castration-resistant prostate cancer (CRPC), resistance to treatment develops, and has been traced to activation of multiple signaling pathways suppressed by the AR. We previously showed that the AR maintained castration sensitivity in prostate cancer (PCa) by transcriptional regulation of the E3 ubiquitin ligase Nrdp1, which degrades the receptor tyrosine kinase (RTK) ErbB3; whereas AR inhibition suppressed Nrdp1 levels, thereby activating ErbB3. This resulted in CRPC growth and AR stimulation, but in CRPC, the AR was unable to regulate Nrdp1 or suppress ErbB3, causing uncontrolled progression. Here, we investigate the mechanism by which the AR regulates Nrdp1 transcription and why this regulation is lost in CRPC. Immunohistochemical studies in human PCa tissue and in PCa mouse models demonstrated Nrdp1 localization in both nucleus and cytoplasm. In vitro studies determined cytoplasmic Nrdp1 as 36kDa while nuclear Nrdp1 is 28kDa, the 36kDa form, but not the 28kDa form, negatively correlated with ErbB3 levels, but both forms positively correlated with AR. We demonstrate that Nrdp1 is a direct transcriptional target of the AR in androgen-dependent cells expressing a truncated form of the structural protein Filamin A (FlnA) in the nucleus, but in CRPC cells which have lost nuclear FlnA expression, the AR is no longer able to bind to the Nrdp1 promoter. Restoration of nuclear FlnA restored the ability of AR to regulate Nrdp1 transcription. Thus dual targeting of ErbB3 and AR will be effective in patients whose tumors express nuclear FlnA. Citation Format: Rosalinda M. Savoy, Liqun Chen, Salma Siddiqui, Frank U. Melgoza, Blythe Durbin-Johnson, Mohana Roy, Maitreyee K. Jathal, Swagata Bose, Yu Wang, Benjamin Mooso, Leandro D'Abronzo, William H. Fry, Kermit L. Carraway, Paramita M. Ghosh. Androgen receptor transcriptionally targets the ErbB3 regulator Nrdp1 in the presence of nuclear Filamin A. [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 3578. doi:10.1158/1538-7445.AM2013-3578

Abstract ABSTRACT Lapatinib has been used in multiple clinical trials of either hormone-naïve men with prostate cancer (PCa) or in patients who have progressed to castration resistant prostate cancer (CRPC) following androgen deprivation therapy (ADT). Although lapatinib as a single-agent showed no overall positive effect in hormone-naive PCa, a small subset of unselected CRPC patients experienced PSA decline ∼50% with lapatinib treatment, with one patient remaining on this drug >45 months (Whang, Y.E. et al, 2011. Urol Oncol). The current investigation had been undertaken to determine whether lapatinib may benefit any select group of patients with PCa. Studies in vitro and in animal models supported the observation that lapatinib was ineffective in the presence of androgens; however, in low-androgen conditions, only androgen-dependent LNCaP PCa cells, but not CRPC cells, responded to lapatinib. Since lapatinib is a dual inhibitor of the receptor tyrosine kinase (RTK) epidermal growth factor receptor (EGFR) and a closely related member, ErbB2, hence we investigated the effect of this drug on the EGFR family. Comparison of the various cell lines that did or did not respond to lapatinib demonstrated that those that did not respond to lapatinib had very high inherent EGFR and ErbB3 activity, whereas LNCaP cells did not. More importantly, unlike its CRPC sublines C4-2 and LNCaP-AI, which were less responsive to lapatinib compared to LNCaP, the parental line experienced an increase in ErbB3 during androgen deprivation. Overexpression of ErbB3 induced resistance to lapatinib even in LNCaP cells, indicating an increase in the levels of RTKs as a potential cause for lapatinib resistance. Indeed, lapatinib treatment resulted in an overall increase in the levels of EGFR and/or ErbB2 in all the cell lines tested, even in LNCaP cells that do respond to lapatinib. Thus, our data demonstrate that (i) lapatinib is ineffective in PCa expressing high ErbB3 and (ii) among those with low intrinsic ErbB3, lapatinib is effective only in tumors where ErbB3 levels increase upon androgen withdrawal. Our results show that the critical window of opportunity for employing lapatinib and other dual EGFR/ErbB2 inhibitors is when ErbB3 levels are rising, but not once they have already increased. Since ErbB3 can be detected in the serum of patients with PCa, the increase of this RTK during ADT should be tested in future studies as a predictive marker of lapatinib response. Citation Format: Maitreyee K. Jathal, Benjamin A. Mooso, Leandro S. D'Abronzo, Salma Siddiqui, Liqun Chen, Elyse van Spyck, Anisha Madhav, Paramita M. Ghosh. Identification of conditions of lapatinib effectiveness in prostate cancer. [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 1323. doi:10.1158/1538-7445.AM2013-1323

Abstract Background: Various studies demonstrated increased expression of EGF-receptor (EGFR/HER1/ErbB1) and related receptors ErbB2/HER2/Neu, ErbB3/HER3 and ErbB4/HER4 in bladder cancer. Despite this, clinical trials using drugs inhibiting EGFR (erlotinib), ErbB2 (trastuzumab) or dual EGFR/ErbB2 inhibitors (lapatinib) were all deemed ineffective. Since ErbB receptors require dimerization with other members of this family, each of which regulate cell growth and survival, we hypothesized that all four receptors must be simultaneously suppressed to achieve lasting effects. Here we compare the effects of erlotinib, trastuzumab and lapatinib to a novel pan-ErbB inhibitor dacomitinib in four bladder cancer cell lines expressing high and low levels of these receptors. Methods: The bladder cancer cell lines T24, TCCSUP, J82 and HTB-2FT4 were used to evaluate the efficacy of erlotinib (Tarceva), trastuzumab (Herceptin), lapatinib (Tykerb, GW572016) and dacomitinib (PF00299804). Cell growth was estimated by MTT or crystal-violet staining. Protein expression was determined by western blotting and apoptosis was determined by flow cytometry after seventy-two hour incubation with the indicated treatment. Results: Comparison of the T24, TCCSUP, J82 and HTB-2FT4 bladder cancer cells with C4-2 (prostate) and MCF-7 (breast) cells showed that all but the HTB-2FT4 bladder cancer cells expressed high levels of EGFR and ErbB3 but not ErbB2, while HTB-2FT4 cells expressed only EGFR and TCCSUP cells had higher ErbB levels than T24. Interestingly, ErbB2 and ErbB3 are readily activated by EGF in T24 cells compared to TCCSUP. MTT demonstrated the efficacy of dacomitinib and erlotinib, primarily EGFR inhibitors, compared to lapatinib, primarily an ErbB2 inhibitor with anti-EGFR activity at higher concentrations, while the ErbB2 inhibitor, trastuzumab, had little effect. The trastuzumab and erlotinib combination performed worse than erlotinib alone in T24 cells; hence trastuzumab studies were not pursued further. Crystal-violet staining after treatment with erlotinib, lapatinib and dacomitinib indicated higher efficacy of dacomitinib after seven days in the T24 and TCCSUP cell lines. MTT and flow cytometry showed that lapatinib was ineffective alone but, in combination with dacomitinib, significantly decreased cell viability in TCCSUP cells. In contrast, combining erlotinib, lapatinib and dacomitinib proved most effective in T24 cells. Conclusions: We demonstrate that all ErbB receptors must be simultaneously inhibited to prevent proliferation of both high and low ErbB expressing bladder lines. Inhibition of ErbB2 alone had a deleterious effect, likely due to ErbB2 inhibition stimulating the activation of the other ErbB receptors. While high levels of ErbB kinases sensitized cells to the inhibitors, the drugs were more effective in cells that had lower activation of the receptors. Citation Format: Benjamin A. Mooso, Ralph W. deVere White, Paramita M. Ghosh. ErbB inhibitors in bladder cancer cell lines: Effectiveness in cells that express high levels but low activity of the ErbB receptors. [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 4270. doi:10.1158/1538-7445.AM2013-4270

Abstract Recurrent prostate cancer (PCa) is treated with androgen deprivation therapy (ADT, chemical castration), but patients on this treatment frequently relapse, indicative of the advent of castration resistant prostate cancer (CRPC). Due to the multifocal nature of the disease, any one treatment may not affect all pathways altered in CRPC; therefore, we set out to delay CRPC by increasing the apoptotic rate during ADT. We previously published that Filamin A (FlnA) is localized to the nucleus in androgen-dependent cells, whereas its expression is lost from the nucleus in CRPC. Restoration of FlnA nuclear expression induced apoptosis in the absence of androgens. We now show that genistein combined polysaccharide (GCP), a combination of isoflavones, can induce FlnA translocation to the nucleus. GCP and its main component genistein previously failed to prevent progression in pre-prostatectomy patients with low-grade PCa. However, our new data, both in vitro and in an animal model, show that GCP impedes relapse following castration, and induces apoptosis in PCa cells undergoing ADT, by promoting FlnA cleavage and nuclear translocation. Investigation of the mechanism of GCP's effects on FlnA showed that ADT phosphorylates FlnA and prevents its cleavage, whereas GCP inhibits FlnA phosphorylation, thereby promoting its cleavage and nuclear translocation. Additional data show that nuclear localization of FlnA induces apoptosis during ADT by preventing androgen receptor (AR) stabilization and by suppression of the ErbB3/PI3K/Akt axis. Taken together, our results identify GCP as a putative therapeutic module to prolong the effectiveness of ADT in patients with metastatic prostate cancer. Citation Format: Benjamin A. Mooso, Maria Mudryj, Ralph W. deVere White, Paramita M. Ghosh, Rosalinda M. Savoy, Jean P. Cheung, Ruth L. Vinall, Clifford G. Tepper, Yu Wang, Salma Siddiqui, Roble G. Bedolla, Margarita Mikhailova. Using GCP to improve initial response to androgen deprivation in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr C11.