bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4598: Androgen receptor acetylation at K618 by ARD1 is essential for dissociation from HSP90 in prostate tumorigenesis
Authors: DePaolo John S. , Wang Zehua , Guo Jianhui , Zhang Guanyi , Zhang Haitao , Zabaleta Jovanny , Liu Wanguo
Published: July 2016

Prostate cancer is an androgen receptor (AR)-driven disease and post-translational modification of AR is critical for its activation in both hormone-sensitive and castration-resistant disease. We previously reported that ARD1 is an oncoprotein in prostate cancer. It acetylates and activates AR to promote prostate tumorigenesis. However, the target residue within AR and the roles and mechanisms of the acetylation event in prostate tumorigenesis remained unknown. Here we show that ARD1 interacts with the AR DNA-binding domain and acetylates AR at lysine 618 (K618) in vivo and in vitro. Inhibition of K618 acetylation by replacing lysine with arginine significantly reduces AR transcriptional activity, prostate cancer cell growth, and xenograft tumor formation due to attenuation of AR nuclear translocation. Constitutive acetylation at K618 by replacing lysine with glutamine reverses these effects. Mechanistically, ARD1 forms a complex with both AR and HSP90. Expression of ARD1 increases levels of AR acetylation and AR-HSP90 dissociation in a dose dependent manner. Moreover, the AR acetylation-defective K618R mutant is unable to dissociate from HSP90 following ligand exposure, and HSP90-dissociated AR is acetylated. This work identifies a new mechanism for ligand-induced AR-HSP90 dissociation and AR activation. Targeting ARD1-mediated AR acetylation may be a potent intervention for AR-dependent prostate cancer therapy. Given that K618 is also present in AR variants, the significance of ARD1-dependent acetylation of AR variants for castration-resistant PCa will be discussed.

Citation Format: John S. DePaolo, Zehua Wang, Jianhui Guo, Guanyi Zhang, Haitao Zhang, Jovanny Zabaleta, Wanguo Liu. Androgen receptor acetylation at K618 by ARD1 is essential for dissociation from HSP90 in prostate tumorigenesis. [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 4598.
Abstract 460: Diagnostic and prognostic value of expression of CCND1 splice variant cyclin D1b in papillary thyroid carcinoma
Authors: Jung Chan Kwon , Jeon Sora , Kim Yourha , Lee Ahwon , Park Gyeong Sin
Published: July 2016

Background: An active cyclin D1 isoform, cyclin D1b, arises as a result of a failure to splice the fourth intron of the CCND1 pre-mRNA transcript. The expression of cyclin D1b has been associated with tumorigenesis and tumor progression in many types of human cancer. However, no studies with cyclin D1b have yet been reported in the thyroid cancer.

Methods: We investigated the CCND1 rs9344 (cDNA 870) polymorphism by TaqMan SNP genotyping assay and expression profiles of mRNA and protein of full-length cyclin D1 (cyclin D1a) and cyclin D1b by quantative real-time PCR and immunohistochemistry in 286 thyroid tumors including 16 nodular hyperplasias, 33 follicular adenomas (FA), 179 papillary thyroid carcinomas (PTC), 32 follicular thyroid carcinomas (FTC), 5 poorly differentiated carcinomas (PDC), 4 anaplastic thyroid carcinomas (ATC), and 17 medullary thyroid carcinomas (MTC). The relationships between cyclin D1 expression levels and clinicopathological features were analyzed.

Results: The cyclinD1b mRNA expression level in all thyroid tumors was significantly higher in the AA and GA genotype groups compared with the GG genotype group (p<0.001 and p = 0.006, respectively). The mRNA expression levels of cyclin D1b was significantly higher in PTCs when compared to benign or other malignant tumors (p<0.001) while no significant difference in cyclin D1a mRNA expression was observed according to the genotypes or tumor types. PTCs showed similar expression levels of cyclin D1b mRNA regardless of histologic subtypes, even when compared with follicular variant. In immunohistochemistry analysis, cyclin D1b overexpression was observed in PTC, PDC, ATC, and MTC, but not in FA and FTC. Cytoplasmic overexpression of cyclin D1b was significantly associated with advanced tumor stage in all PTCs (p = 0.025). Nuclear overexpression of cyclin D1b was associated with an increased incidence of lymph node metastases (p = 0.014) in classic PTCs.

Conclusion: The A allele of the rs9344 polymorphism predisposes for cyclin D1b production in thyroid cancer. Cyclin D1b overexpression has a diagnostic biomarker utility to differentiate PTC from benign follicular nodules and may be functionally involved in the progression of the disease.

Citation Format: Chan Kwon Jung, Sora Jeon, Yourha Kim, Ahwon Lee, Gyeong Sin Park. Diagnostic and prognostic value of expression of CCND1 splice variant cyclin D1b in papillary thyroid carcinoma. [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 460.
Abstract 4602: A83-01 inhibits TGF-β/Smad3 and Wnt/β-catenin-mediated EMT and tumor metastasis in HER2-overexpressing breast cancer cells
Authors: Wu Yanyuan , Trinh Tran , Sarkissyan Marianna , Dwabe Sami , Kim Juri , Farias-Eisner Robin , Vadgama Jay
Published: July 2016

Interactions between Wnt and TGF-β (Transforming growth factor-β) signaling pathways play a critical role in promoting EMT (Epithelial to Mesenchymal Transition) and tumor metastasis. We previously demonstrated that activation of Wnt3/β-catenin signaling pathway promoting EMT is an important mechanism leading to HER2-cells becoming resistant to trastuzumab. In this study, we used HER2-overexpressing breast cancer cells to investigate the interactions between TGF-β and Wnt/β-catenin pathways in regulation of EMT and promoting breast cancer cells invasion. Our data showed that phosphorylation of Smad3 at ser423/425 by TGF-β led to nuclear localization of Smad3 and upregulation of Twist protein expression in HER2-overexpressing breast cancer cells. Cells treated with TGF-β showed poor response to trastuzumab and exhibited EMT-like phenotype; decreased E-cadherin and increased N-cadherin expression. A small molecule inhibitor, A83-01 which inhibits phosphorylation of Smad3 repressed TGF-β-induced nuclear accumulation of Smad3 and Twist expression. The A83-01 also inhibited TGF-β-induced cell migration and invasion and enhanced anti-tumor activity of trastuzumab. The TGF-β/Smad3 induced EMT in HER2-cells was found to be concordant with activation of Wnt3/β-catenin pathway. Following TGF-β treatment, Chromatin immunoprecipitation identified occupancy of Twist at promoter region of Wnt3. This data suggests that Twist in response to TGF-β activated Wnt3 gene transcription in HER2-overexpressing breast cancer cells. Knock-down of Twist by shRNA down-regulated Wnt3 expression and inhibited TGF-β-induced nuclear accumulation of β-catenin. Subsequently, TGF-β-induced matrix metalloproteinases, MMP1, MMP7, MMP9, VEGF (Vascular endothelial growth factors) and activation of Wnt/β-catenin signaling were repressed by the shRNA treatment.

In summary, our data demonstrated for the first time that the TGF-β/Smad3 mediated Twist results in transcriptional upregulation of Wnt3/β-catenin pathway and promotes EMT and cell invasion. The data suggests that therapeutic targeting of Twist by A83-01 and similar small molecules could inhibit the interaction between TGF-β and Wnt pathways and prevent breast cancer progressing to EMT and metastases.

Citation Format: Yanyuan Wu, Tran Trinh, Marianna Sarkissyan, Sami Dwabe, Juri Kim, Robin Farias-Eisner, Jay Vadgama. A83-01 inhibits TGF-β/Smad3 and Wnt/β-catenin-mediated EMT and tumor metastasis in HER2-overexpressing breast cancer cells. [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 4602.
Abstract 462: The role of Her2 overexpression in Middle Eastern papillary thyroid cancer
Authors: Siraj Abdul K. , Beg Shaham , Bu Rong , AlSobhi Saif , Al-Dayel Fouad , Al-Kuraya Khawla
Published: July 2016

Thyroid cancer is the second most common malignancy among females in Saudi Arabia accounting for 7.4% of all cancers and 10.6% of all female malignant cancer. Among different subtypes, papillary thyroid cancer (PTC) is the most common subtype of thyroid cancer. Although the overall cancer related ten year survival rate for PTC has reached 90%, local, regional and distant metastasis does occur in 10% of cases. Therefore, in search for new therapeutic targets for PTC, we investigated the role of Her2 oncogene in PTC as this gene is known to be involved in other cancers such as breast cancer. The aim of this study was to determine the prevalence and prognostic role of Her2 overexpression in Middle Eastern papillary thyroid carcinoma by immunohistochemistry (IHC), Fluorescent In-Situ Hybridization (FISH) and clinical data. A tissue microarray (TMA) containing >1000 PTC cases with follow-up data was used. TMA sections were analysed at protein and DNA level using IHC and FISH. FISH analyses were performed to look for the gain or amplifications in Her2 gene. IHC analysis showed Her2 overexpression in 19.7% of our cases of PTC. Elevated expression was almost exclusively 2+ (194 tumors) and only rarely 3+ (1 tumor). Interestingly, no amplification of Her2 gene was visualized by FISH. Only 3% of our cases showed mildly elevated HER2 gene copy numbers not reaching the threshold for amplification. HER2 overexpression and copy number gains were unrelated to tumor stage, metastasis, patient survival and other clinical and pathological parameters. Our results demonstrated that Her2 overexpression occurs at relevant frequency in papillary thyroid cancer and in the absence of gene amplification. Additionally, expression of Her2 seems to hold no clinical value as prognostic factor in PTC

Citation Format: Abdul K. Siraj, Shaham Beg, Rong Bu, Saif AlSobhi, Fouad Al-Dayel, Khawla Al-Kuraya. The role of Her2 overexpression in Middle Eastern papillary thyroid 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 462.
Abstract 4621: Rapamycin inhibits the phosphorylation of mSin1 by targeting a new mTOR complex
Authors: Huang Shile , Luo Yan , Liu Lei
Published: July 2016

Current knowledge indicates that mTOR functions as two complexes, mTORC1 and mTORC2, in mammalian cells. mTORC1 comprises mTOR, mLST8 and raptor, and is sensitive to acute rapamycin treatment, whereas mTORC2 consists of mTOR, mLST8, rictor, mSin1 and Protor, and is sensitive to chronic rapamycin treatment in some cases. It is well known that mTORC1 phosphorylates 4E-BP1 and S6K1, while mTORC2 phosphorylates Akt and SGK1. Recently, we and others have observed that rapamycin inhibits the phosphorylation of mSin1. However, little is known about the molecular mechanism by which rapamycin inhibits mSin1 phosphorylation. Here we show that rapamycin inhibits mSin1 phosphorylation in mTOR kinase dependent manner. This is strongly supported by the findings that expression of rapamycin-resistant mTOR (mTOR-T), but not rapamycin-resistant and kinase dead mTOR (mTOR-TE) prevented rapamycin from inhibiting mSin1 phosphorylation; Knockdown of mTOR inhibited mSin1 phosphorylation. However, surprisingly, disruption of either mTORC1 or mTORC2 (by silencing raptor and rictor, respectively) failed to inhibit the phosphorylation of mSin1 as rapamycin did. Similarly, downregulation of S6K1 or Akt did not inhibit mSin1 phosphorylation as rapamycin did either. Of interest, knockdown of mLST8, a component shared by mTORC1/2, inhibited mSin1 phosphorylation. Furthermore, rapamycin treatment reduced the physical interaction of mSin1 with mLST8 and mTOR, but not with rictor. The results suggest that rapamycin inhibits mSin1 phosphorylation, which is not by inhibiting mTORC1 or mTORC2, but via inhibiting a new mTOR complex, which contains at least mTOR and mLST8. (Supported by the FWCC, LSU Health Sciences Center)

Citation Format: Shile Huang, Yan Luo, Lei Liu. Rapamycin inhibits the phosphorylation of mSin1 by targeting a new mTOR complex. [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 4621.
Abstract 4640: Doxorubicin induced cardiomyopathy associated with natural reproductive hormone cycling in female tumor-bearing spontaneously hypertensive rats
Authors: Pokrzywinski Kaytee L. , Rosen Elliot T. , Bonnano Julia , Biel Thomas , Moshkelani Delaram , Aryal Baikuntha , Mog Steven , Rao V. Ashutosh
Published: July 2016

Doxorubicin (dox) is a widely used oncology agent known to induce dose-limiting cardiomyopathy, driven partially by the excess production of reactive oxygen species (ROS) in mitochondria. Cardiomyocytes are particularly sensitive to ROS due to higher densities of mitochondria and lower concentrations of antioxidants. The incidence of dox induced cardiomyopathy is lower in reproductively mature women compared to age matched men, while pre-pubescent and post-menopausal females are more sensitive. Previous studies have associated reproductive hormone levels with differences in cardiosensitivity based on gender and age. This is the second part of a two part study investigating the role that reproductive hormones play in dox induced cardiotoxicity in female, tumor-bearing spontaneously hypertensive rats (SHRs). In the current study, we investigated the impact of natural hormone cycling on dox induced cardiomyopathy. Before beginning the experiment, regular hormone cycling was established by monitoring daily changes in the estrous cycle by way of vaginal cytology. The animals were organized by phase (proestrus, estrus, metestrus, or diestrus) and implanted with SST-2 cells, a syngeneic breast cancer cell line derived from a SHR. Tumor-bearing animals were administered a single dose of saline, dox, the cardioprotectant dexrazoxane, or a combination thereof and monitored for 14 days. Daily vaginal cytologies were obtained throughout the experiment to monitor estrous cycling as a means of tracking hormone fluctuations. Serum was collected at various time points to correlate estrous cycling with circulating hormone levels (E2 and P4) and measure the cardiac health indicator cardiac troponin I (cTnI). In all hormone groups, dox treatment resulted in a net loss in bodyweight and significant anti-tumor activity, as measured by tumor volume, compared to respective saline controls. Interestingly, animals treated with saline in metestrus displayed increased tumor volumes compared to animals in different estrous phases. We are currently evaluating cardiac status by echocardiogram, histopathology, and cTnI. We are also measuring serum E2 and P4 hormone levels in an effort to understand the relationship between cardiomyopathy and circulating hormones.

Citation Format: Kaytee L. Pokrzywinski, Elliot T. Rosen, Julia Bonnano, Thomas Biel, Delaram Moshkelani, Baikuntha Aryal, Steven Mog, V. Ashutosh Rao. Doxorubicin induced cardiomyopathy associated with natural reproductive hormone cycling in female tumor-bearing spontaneously hypertensive rats. [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 4640.
Abstract 465: CD44 as a potential therapeutic target and prognosis marker for glioblastoma
Authors: Wang Fengfei , Shen Xin , Pruszynski Jessica , Huang Jason , Kirmani Batool , Wu Erxi , Fonkem Ekokobe
Published: July 2016

Glioblastoma (GBM) is a common and very aggressive primary brain tumor with no cure. The current diagnosis relying on magnetic resonance imagining, computed tomography, and biopsy is a very expensive and aggressive process. More effective therapeutic strategies such as target therapies and simplified early diagnosis tools are urgently needed for GBM management and prognosis. As recent reports suggest that GBM expresses CD44, a type-I transmembrane glycoprotein, is a marker of cancer stem cell and therapy resistance, with a goal of identifying a therapeutic target and a prognosis maker for GBM, we analyzed several data sets previously detected by microarrays using an online R2 Genomics analysis and visualization platform (http://r2.amc.nl). Through examining of the expression levels of CD44 in the tissues of normal cerebellum (without brain tumor) and in GBM tissues, we find that the expression level of CD44 in GBM is higher than that in non-brain tumor tissues. Comparing the expression levels of CD44 in Primary and recurrent GBMs, GBM cell lines, neuron stem cell lines, and normal cortex tissues, we observe that the expression levels of CD44 in recurrent GBMs are higher than primary GBMs; the CD44 levels in GBM cell lines are similar to neuron stem cell lines which are significantly higher than that in the normal cortex tissues. We further evaluated the correlation of CD44 levels with patients’ overall survival in 4 available complete data sets in the R2 database, including 2 from The Cancer Genome Atlas (TCGA) database. We show that an elevated level of CD44 in GBM is associated with a short overall survival time although variations exist from cohort to cohort. In addition, we studied the effect of p53 status on CD44 expression, we reveal that CD44 level is higher in the GBMs carrying p53 mutation compared to those with wild-type p53. Taken together, these results indicate that CD44 could be a potential therapeutic target of GBM and the expression level of CD44 in the GBM may be an indicator of poor outcome for patients with GBM.

Citation Format: Fengfei Wang, Xin Shen, Jessica Pruszynski, Jason Huang, Batool Kirmani, Erxi Wu, Ekokobe Fonkem. CD44 as a potential therapeutic target and prognosis marker for glioblastoma. [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 465.
Abstract 4650: Effect of prescription CYP3A inducers and inhibitors on response to anti-androgen therapy in prostate cancer
Authors: Mitra Ranjana , Goodman Oscar B.
Published: July 2016

Purpose: Prostate cancer affects elderly men who typically have multiple comorbidities requiring concomitant prescription medications, many of which are CYP inducers or inhibitors. Androgen deprivation therapy (ADT) is the mainstay therapy for treating advanced prostate cancer. Recently we demonstrated that CYP3A5 facilitates the nuclear translocation of androgen receptor (AR), promoting prostate cancer growth. As the mechanisms by which intratumoral CYP3A5 regulates AR in prostate cancer are not well understood, the clinical impact of concomitant drugs on the efficacy of ADT is presently unknown. This work describes a platform for assessing the effects of commonly prescribed drugs known to be CYP3A5 inducers or inhibitors on AR signaling and prostate cancer growth.

Methods: We have created a lentiviral LNCaP based reporter system for monitoring changes in AR signaling utilizing a construct comprising a promotor containing AR inducible transcriptional response elements (TREs), fused with firefly luciferase, whereas the negative control (NC) lacks the TREs. We used both a mixed transfected pool and individually selected clones to monitor changes in AR activity.

Results: We tested our assay system by inhibiting CYP3A5 gene expression in LNCaP cells using siRNA and then treating it with DHT. Our data indicate that CYP3A5 siRNA reduced DHT induced AR activity significantly as compared to non-target (NT) siRNA. We further tested our reporter system with rifampicin, phenytoin (both CYP3A inducers) and ritonavir, fluoxetine, amiodarone (CYP3A inhibitors). Rifampicin (antibiotic) and phenytoin (anticonvulsant) showed significant (P≤0.05) induction of AR activity as shown by increased luminescence units. Out of the three inhibitors which were tested amiodarone (antiarrhythmic) and ritonavir (antiretroviral) reduced the AR activity by 50% and 80% respectively whereas fluoxetine (antidepressant) did not result in any significant changes. Both amiodarone and ritonavir also inhibited R1881 (artificial androgen) induced AR activity as compared to control.

Conclusion: Concomitant medications can alter the AR activity thus modifying the response to ADT therapy in prostate cancer patients. These finding support the notion that an CYP3A inhibitor-rich concomitant medical regimen may provide additional clinical benefit for men undergoing ADT and further support the concept of targeting nuclear translocation as a therapeutic adjunct in ADT.

Citation Format: Ranjana Mitra, Oscar B. Goodman Jr. Effect of prescription CYP3A inducers and inhibitors on response to anti-androgen therapy 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 4650.
Abstract 4658: Activation mechanisms of Integrated Stress Response by inhibition of oncogenic BRAF in melanoma cell lines
Authors: Nagasawa Ikuko , Tsukahara Satomi , Kunimasa Kazuhiro , Tomida Akihiro
Published: July 2016

The eIF2α-ATF4 signaling pathway termed “Integrated stress response (ISR)” plays an important role in protecting cells against various cellular stresses such as deprivation of glucose and amino acids, virus infection, and deficiency of heme. Under such stress conditions, eIF2α kinases, including PERK, GCN2, PKR and HRI, phosphorylate eIF2α, resulting in reduction of general protein synthesis while facilitating selective translation of ATF4, a central transcription factor that upregulates expression of stress-induced genes for cellular adaptation. Recently, it was reported that BRAF inhibitor vemurafenib triggers activation of the eIF2α-ATF4 signaling in BRAF-mutant melanoma cell lines; however, activation mechanisms of vemurafenib-induced ISR signaling not fully understood.

In this study, we first compared vemurafenib-induced ISR activation in BRAF-mutant and -wild type melanoma cell lines. Consistent with previous studies, vemurafenib activated ISR signaling preferentially in BRAF-mutant cell lines, as determined by phosphorylation of eIF2α and ATF4 expression. We also found that the ISR activation by vemurafenib was impaired by knockdown of BRAF in BRAF-mutant cell lines. These results indicated that existence of mutated BRAF is important for vemurafenib to trigger activation of ISR signaling pathway. We next examined which eIF2α kinase is involved in the ISR signaling activation by vemurafenib. We found that knockdown of GCN2, an eIF2α kinase sensing depletion of amino acids, impaired phosphorylation of eIF2α and ATF4 induction by vemurafenib. In addition, we confirmed that vemurafenib induced phosphorylation of GCN2 on Thr899 that is known to occur in response to amino acids starvation. In agreement with these results, gene expression profiling with ontology analysis revealed that vemurafenib upregulated genes that are involved in amino acids biosynthesis via ISR signaling. Collectively, our results suggested that vemurafenib activated ISR signaling pathway possibly through mimicking amino acid deprivation in BRAF-mutant melanoma cell lines.

Citation Format: Ikuko Nagasawa, Satomi Tsukahara, Kazuhiro Kunimasa, Akihiro Tomida. Activation mechanisms of Integrated Stress Response by inhibition of oncogenic BRAF in melanoma cell lines. [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 4658.
Abstract 467: Immunohistochemical multiplex staining strategies with CD8, CD103, PD-1, FOXP3 and pan melanoma cocktail in tumor infiltrating lymphocytes in melanoma
Authors: Tacha David E. , Yuan Wei , Terrell Jillian
Published: July 2016

Introduction

Tumor-associated immune suppression can lead to defective T-cell mediated antitumor immunity. CD8 T-cells play a critical role in the host defense against cancers and infectious diseases. However, the presence of antigen-specific CD8 T-cells does not always imply that cancers and/or pathogens are efficiently eliminated in the body. In tumor infiltrating lymphocytes (TILS), other markers including CD103, PD-1 and FOXP3 are broadly expressed and have shown a wider range of immunoregulatory and important roles in T-cell activation, T- cell regulatory and programmed cell-death checkpoints. Existing methods can either deliver phenotypic information on homogenous samples such as flow cell cytometry or give immunohistochemical information on single biomarker. However, it would be of advantage to be able to visualize the distributions of multiple biomarkers in TILs that would discriminate stromal cells versus solid tumor. Therefore, a multiplex IHC stain utilizing a melanoma marker as a staining mask that separates the malignant tumor cells from stromal cells maybe a good strategy to facilitate better interpretation.

Materials and Methods

Monoclonal mouse antibodies to CD8, FOXP3 and PD-1 and rabbit monoclonal antibodies CD8, CD103 and PD-1 were optimally tittered for single, double and triple stains and visualized with HRP DAB/Black chromogens and/or alkaline phosphatase fast red/blue chromogens. A mouse monoclonal pan melanoma cocktail was visualized with a fast red chromogen and was used as a staining mask for melanoma tumors cells, but not stromal cells.

Results

Single, double and triple stains that included pan melanoma cocktail (mask) and CD8, CD103 FOXP3 and PD-1 were successfully established. All double and triple stains gave comparable results to single stains. The co-expression of TILs including CD8 and CD103 or CD8 or FOXP3 and PD-1 was easily identified with contrasting chromogens. TILs in stroma tissues and in malignant tumors cells were easily discriminated with the pan melanoma (mask) that stained only tumor cells with fast red chromogen, but did not stain TILS or stromal tissues.

Conclusion

The evaluation of TILS in malignant melanoma was enhanced with multiplex stains. TILs in stromal tissues and melanoma tumors cells were easily distinguished with the Pan Melanoma staining mask. These finding may help facilitate important prognostic information and thus may support the rationale for using immunotherapy strategies.

Citation Format: David E. Tacha, Wei Yuan, Jillian Terrell. Immunohistochemical multiplex staining strategies with CD8, CD103, PD-1, FOXP3 and pan melanoma cocktail in tumor infiltrating lymphocytes in melanoma. [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 467.
Abstract 4683: Verapamil and tamoxifen modulate ABCB1 expression in multidrug-resistant cells and resensitize them to conventional chemotherapy
Authors: Limniatis Georgia , Georges Elias
Published: July 2016

Multidrug-resistant (MDR) tumors are an increasingly important obstacle in cancer treatment, wherein many cases are associated with the overexpression of P-glycoprotein 1 (P-gp1, ABCB1). Previous efforts to sensitize ABCB1-expressing tumor cells to anti-cancer drugs were associated with severe side effects, so interest in clinical trials using these drugs has dwindled.

Studies have shown P-glycoprotein-overexpressing cells to be hyper sensitive (or collaterally sensitive) to a diverse group of non-toxic compounds. Given earlier results demonstrating a significant increase in ABCB1 expression post-chemotherapeutic treatment of cancer patients (e.g., breast, ovarian, myeloma, and acute myeloid leukemia), and the role of ABCB1 in drug resistance, it was determined that targeting ABCB1-expressing tumor cells with effective collateral sensitivity drugs should increase the effectiveness of current chemotherapeutic treatments.

In this report, it was of interest to determine the effect of the collateral sensitivity drugs verapamil and tamoxifen on P-glycoprotein 1 expression at the level of cells and individual cell clones. Clones from P-gp1-overexpressing Chinese hamster ovarian cell lines (CHRC5) or triple negative breast cancer cells (MDA-MB-231/400 nM doxo) were treated with varying levels of collateral sensitivity drugs and ABCB1 expression was determined by Western blots and ELISAs. The sensitivity of each cell clone to anti-cancer or collateral sensitivity drugs was determined using cytotoxicity assays.

Our results demonstrate a drop in ABCB1 expression in each clone, together with decreased collateral sensitivity and overall increased sensitivity to anti-cancer drugs in cell lines selected with verapamil and tamoxifen. Work is in progress to study the various mechanisms responsible for this drop in ABCB1 expression and its impact on treatment outcome.

Citation Format: Georgia Limniatis, Elias Georges. Verapamil and tamoxifen modulate ABCB1 expression in multidrug-resistant cells and resensitize them to conventional chemotherapy. [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 4683.
Abstract 4689: Successfully targeting triple negative breast cancer using combination therapy of Didox and Doxorubicin with reduced cardiotoxicity
Authors: Wilson Elizabeth A. , Elford Howard L. , Faridi Jesika S.
Published: July 2016

Triple-negative breast cancer (TNBC) is an aggressive histological subtype that has limited treatment options with chemotherapy being the only systemic therapy for these patients. The most common chemotherapy regimen used includes the anthracycline Doxorubicin (DOXO). Although DOXO is a highly effective agent, its efficacy is limited due to acquired drug resistance and cardiotoxicity. We have previously identified that ribonucleotide reductase M2 (RRM2) is upregulated in TNBC cells and is a key contributor to acquired drug resistance. The ribonucleotide reductase inhibitor Didox has been shown to be a powerful free radical scavenger and iron chelator that provides the capabilities to alleviate the cytotoxic profile of DOXO especially the cardiotoxicity as well as enhance the DOXO efficacy. In this current study, MDA-MB-468 and MDA-MB-231 TNBC cells were treated with DOXO alone or DOXO plus Didox. Synergy assays were performed and IC50 values were determined. Here we report that Didox worked synergistically with DOXO and reduced the DOXO IC50 values by more than one-half in each cell line. In order to determine the in vivo benefit of adding Didox to DOXO therapy, ncr-nude mice implanted with MDA-MB-468 xenografts were treated with (1) vehicle, (2) daily Didox alone (425 mg/kg), (3) DOXO alone (10 mg/kg/week for 2 doses and 2 mg/kg/week for 3 additional doses), or (4) the combination of DOXO and Didox. Tumor volumes over time were determined for each of the treatment groups. At endpoint, we performed necropsy, collected serum, and dried and weighed the hearts. Treatment with DOXO alone prevented tumor growth and provided about a 70% lower tumor volume as compared to vehicle treatment. Treatment with Didox alone yielded about 50% lower tumor volume as compared to vehicle treatment. However, adding Didox in combination with DOXO significantly inhibited the tumor size and resulted in a 90% lower final tumor volume as compared to vehicle treatment. One indicator of DOXO cardiotoxicity is increased heart weight. We found that the heart weight of DOXO alone treated animals was significantly increased and the inclusion of Didox in the treatment prevented this increase. Collectively, these results identify that adding the ribonucleotide reductase inhibitor Didox in combination with DOXO may be used to treat TNBC by enhancing the efficacy on tumor inhibition and by alleviating the cardiotoxic effects of DOXO.

Citation Format: Elizabeth A. Wilson, Howard L. Elford, Jesika S. Faridi. Successfully targeting triple negative breast cancer using combination therapy of Didox and Doxorubicin with reduced cardiotoxicity. [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 4689.
Abstract 4690: Novel Ring1B-Bmi1 small molecule inhibitors target leukemia stem cells
Authors: Shukla Shirish , Gray Felicia , Ying Weijang , Cho Hyoje , Zhao Qingjie , Miao Hongzhi , Purohit Trupta , Grembecka Jolanta , Cierpicki Tomasz
Published: July 2016

Leukemia is a clonal neoplastic disorder which possesses a rare population of cells, termed leukemia stem cells (LSCs), capable of limitless self-renewal and are necessary to initiate, sustain or regenerate the disease. Targeting LSCs self-renewal not only would offer substantial therapeutic benefit but also likely to be essential and sufficient for selective eradication of LSCs. BMI1, polycomb group protein, has been identified and shown to be required for self-renewal of LSCs. Bmi1 alongwith Ring1B constitute enzymatic active unit of Poly-comb Repressive Complex 1(PRC1) and execute gene silencing through histone H2A lysine119 ubiquitination (H2AK119ub). Considering Bmi1 as a key regulator of self-renewal, targeting Bmi1 activity could impair LSCs self-renewal ability and may represent a new therapeutic approach for leukemia. Thus, in this study we aimed to develop and characterize novel small molecule inhibitors for Ring1B - Bmi1. Using NMR fragment-based screening and extensive medicinal chemistry optimization we developed WY332 that binds to Ring1B-Bmi1 with low micromolar affinity. Further characterization of the WY332 demonstrated selective reduction in Ring1B-Bmi1 mediated H2AK119ub levels in in vitro and in leukemic cells. Contrary, structurally similar and less potent compound displayed similar effect at much higher concentrations establishes that higher binding affinity of potent inhibitor is associated with blocking Ring1B-Bmi1 E3 ligase activity. Further evaluation of lead inhibitor, WY332, in colony formation assay using leukemic cells demonstrated significant decrease in the number and size of the colonies indicated that interference with Ring1B- Bmi1 activity alter the frequency of LSCs colony forming potential. Moreover, expression analysis of CD11b and CD34, cell surface markers associated with myeloid differentiation and AML LSCs phenotype, in WY332 treated TEX cells revealed marked increase in CD11b expression and significantly decreased CD34 expression establishes the specific effect of our Ring1B-Bmi1 small molecule inhibitors on induction of differentiation in leukemic cells. Interestingly, gene expression analysis of TEX cells treated with WY332 revealed significant increase in expression of genes suppressed in LSCs. Overall, these compounds represent the novel small molecule inhibitors of Ring1B-Bmi1 which inhibit E3 ubiquitin ligase activity and impair leukemic stem cell self-renewal capacity.

Citation Format: Shirish Shukla, Felicia Gray, Weijang Ying, Hyoje Cho, Qingjie Zhao, Hongzhi Miao, Trupta Purohit, Jolanta Grembecka, Tomasz Cierpicki. Novel Ring1B-Bmi1 small molecule inhibitors target leukemia stem cells. [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 4690.
Abstract 4692: Targeting the BET bromodomain proteins and anti-apoptotic protein BCL2 in small cell lung cancer
Authors: Lam Lloyd T. , Lin Xiaoyu , Faivre Emily , Yang Ziping , Huang Xiaoli , Bellin Ricky , Lu Xin , Shen Yu , Uziel Tamar
Published: July 2016

10% to 15% of all lung cancers are small cell lung cancer (SCLC), named for the size of the cancer cells when seen under a microscope. SCLC often starts in the bronchi near the center of the chest. It usually grows and spreads quickly to distant parts of the body before it is diagnosed. Unfortunately, no new treatment has been identified in the past 30 years for patients with SCLC. The BET (bromodomain and extra-terminal) proteins bind acetylated histones and recruit protein complexes to promote transcription initiation and elongation. In hematologic cancers, BET proteins have been shown to regulate expression of MYC and other oncogenic transcription factors that drive disease pathology. Pharmacologic inhibition of BET binding to acetylated proteins has been shown to inhibit tumor growth in MYC-dependent cancers, such as acute myeloid leukemia, multiple myeloma and neuroblastoma. Here, we demonstrate that ∼40% of SCLC cell lines are exquisitely sensitive to growth inhibition by the BET inhibitor ABBV-075. Whereas most SCLC cell lines undergo cell cycle arrest with ABBV-075 treatment, a few cell lines undergo apoptosis. Sensitivity does not correlate with MYC status (amplification/expression). However, downregulation of anti-apoptotic protein BCLxL and BCL2 by ABBV-075 was observed in sensitive SCLC cells. Synergy was observed when co-treating BET inhibitor and BCL2 inhibitors, venetoclax (ABT-199) or navitoclax (ABT-263), and it positively correlated with BCL2 expression. Thus, high BCL2 protein expression could potentially be a biomarker predictive of these therapeutic combinations. The potential higher synergy with venetoclax may be explained by downregulation of BCLxL with BET inhibition and potent activity of venetoclax against BCL2. Our studies have provided evidence for treating SCLC by BET inhibition and/or combination with BCL2 inhibitors and a rationale for the clinical development of BET inhibitors in this disease with high unmet medical need.

Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication.

Citation Format: Lloyd T. Lam, Xiaoyu Lin, Emily Faivre, Ziping Yang, Xiaoli Huang, Ricky Bellin, Xin Lu, Yu Shen, Tamar Uziel. Targeting the BET bromodomain proteins and anti-apoptotic protein BCL2 in small cell lung 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 4692.
Abstract 470: Application of sequencing, liquid biopsies and patient derived xenografts for personalized medicine in melanoma
Authors: Girotti Maria R. , Gremel Gabriela , Lee Rebecca , Galvani Elena , Rothwell Dominic , Viros Amaya , Mandal Amit Kumar , Lim Kok Haw Jonathan , Saturno Grazia , Furney Simon J , Baenke Franziska , Pedersen Malin , Rogan Jane , Swan Jacqueline , Smith Matthew , Fusi Alberto , Oudit Deemesh , Dhomen Nathalie , Brady Ged , Dive Caroline , Marais Richard
Published: July 2016

BRAF/MEK inhibitors and immunotherapies have revolutionized care for patients with advanced melanoma, improving expected median survival from 9 months to 25-30 months, but the majority of patients still die of their disease. Personalized medicine strives to individualize and improve patient care. To individualize treatment decisions in advanced melanoma we analyzed 364 samples from 214 patients. We performed whole exome sequencing (WES) and circulating tumor DNA (ctDNA) analysis, and we developed patient derived xenografts (PDX). WES and targeted sequencing of ctDNA allowed us to predict responses to therapy and to identify and monitor mechanisms of resistance. WES of tumors revealed new therapeutic strategies in BRAF V600 wild-type and BRAF inhibitor-resistant melanoma and we validated these in patient derived xenografts (PDX). Thus, we describe a powerful combination of techniques for personalized medicine to improve the management of melanoma patients.

Citation Format: Maria R. Girotti, Gabriela Gremel, Rebecca Lee, Elena Galvani, Dominic Rothwell, Amaya Viros, Amit Kumar Mandal, Kok Haw Jonathan Lim, Grazia Saturno, Simon J Furney, Franziska Baenke, Malin Pedersen, Jane Rogan, Jacqueline Swan, Matthew Smith, Alberto Fusi, Deemesh Oudit, Nathalie Dhomen, Ged Brady, Caroline Dive, Richard Marais. Application of sequencing, liquid biopsies and patient derived xenografts for personalized medicine in melanoma. [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 470.
Abstract 4707: Investigating the role of EZH2 as a therapeutic target in clear cell renal cell carcinoma (ccRCC)
Authors: Wei Darmood , Ricketts Christopher J. , Schmidt Laura S. , Yang Youfeng , Vocke Cathy D. , Linehan William M.
Published: July 2016

Enhancer of zeste homolog 2 (EZH2) is a key component of the polycomb repressive complex 2 (PRC2). EZH2 is frequently overexpressed in a wide variety of human malignancies including non-Hodgkin lymphoma, gastric cancer, pancreatic cancer, and lung cancer. Thus it has potential to become a therapeutic target. Characterization of EZH2 as a therapeutic target in clear cell renal cell carcinoma (ccRCC) has not been fully explored. ccRCC have been defined by mutation of the von Hippel-Lindau (VHL) tumor suppressor gene in combination with chromosome 3p loss. Recent sequencing efforts have revealed that several chromatin remodeling genes encoded on chromosome 3p are often mutated, of which PBRM1 is the most frequent (41%). The PBRM1 gene codes for the BAF180 protein, a SWI/SNF chromatin remodeling complex subunit. Loss of BAF180 in ccRCC may disrupt the PBAF variant of the SWI/SNF complex. The SWI/SNF complex remodels the chromatin landscape by either sliding or evicting the nucleosomes from the chromatin. This chromatin remodeling modulates the accessibility to promoter regions by transcriptional machinery. It is through this mechanism that the SWI/SNF complex can regulate a range of cellular processes. It has been demonstrated that the SWI/SNF complex can act antagonistically to the PRC2 complex by evicting PRC2 complex from the promoters of tumor suppressors such as CDKN2A/p16. Disruption of the SWI/SNF complex would impede the eviction of the PRC2 complex, similarly observed in SNF5-deficient malignant rhabdoid tumors. Therefore, we hypothesize that PBRM1 inactivation disrupts specific SWI/SNF complexes allowing EZH2 to bind and repress target tumor suppressor genes. Thus inhibition of EZH2 in ccRCC may present as a targeted therapeutic option in tumors with PBRM1 mutations. We have investigated EZH2 in ccRCC cell lines with PBRM1 mutations and observed that these cells lines have overexpression of EZH2 in comparison to RPTEC (renal cortex proximal tubule epithelium cell line). We examined the effects on two EZH2 inhibitors (GSK126 and EPZ6438) on ccRCC cell lines both in vitro and in vivo. Our preliminary data suggests EZH2 inhibition results in reduced growth of ccRCC cell lines with PBRM1 mutations.

Citation Format: Darmood Wei, Christopher J. Ricketts, Laura S. Schmidt, Youfeng Yang, Cathy D. Vocke, William M. Linehan. Investigating the role of EZH2 as a therapeutic target in clear cell renal cell carcinoma (ccRCC). [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 4707.
Abstract 472: A digital sorting-based detection and isolation assay for single disseminated cancer cells from lymph nodes of melanoma patients
Authors: Scheitler Sebastian , Weidele Kathrin , Alberter Barbara , Werno Christian , Klein Christoph A. , Polzer Bernhard
Published: July 2016

We recently showed that sentinel lymph node (SLN) disaggregation followed by immunocytology enables precise quantification of disseminated cancer cells (DCC). We demonstrated that this approach has a 20-fold higher sensitivity to detect melanoma DCCs than routine histopathology and that the DCC density (DCCD, defined as the number of DCCs per million lymph node cells) together with ulceration state and tumor thickness enables individual risk prediction superior to current AJCC guidelines (Ulmer et al., PLoS Medicine, 2014). Here, we present the adaptation of this method to a semi-automated workflow, including automated single cell detection and isolation by DEPArrayTM technology for subsequent molecular analysis of DCCs from SLN of melanoma patients.

The developed workflow includes a mechanical disaggregation of lymph node tissue and collection of the mononuclear cells after Percoll density centrifugation. Several tumor cell enrichment methods were tested (CellSearch® and size-based enrichment technologies), however, failed to process cell suspensions generated from lymph nodes due to clumping of cells during the enrichment procedure. Tumor cell enrichment using MACS depletion of CD45+ cells delivered approx. 50% of spiked-in cell line cells with a mean depletion rate of lymphocytes and erythrocytes > 98%. To prevent limitations in DCC detection caused by phenotypic heterogeneity of tumor cells, we established a double staining against two melanoma-associated markers gp100 and MCSP. Individual melanoma cells are then detected and isolated by DEPArrayTM technology enabling single cell whole genome amplification (Ampli1TM) for subsequent molecular analysis as assessment of copy number alterations (e.g. by arrayCGH) or sequence analysis for melanoma specific point mutations (e.g. BRAF or NRAS). We successfully applied the workflow to first SLN samples from melanoma patients.

The established workflow enables a standardized detection of single DCCs from lymph nodes of melanoma patients applicable in clinical diagnostic labs. Automated single cell isolation and subsequent molecular analysis of DCCs is feasible within short time after SLN biopsy. In the future, this approach could help to select individualized therapies for melanoma patients.

Citation Format: Sebastian Scheitler, Kathrin Weidele, Barbara Alberter, Christian Werno, Christoph A. Klein, Bernhard Polzer. A digital sorting-based detection and isolation assay for single disseminated cancer cells from lymph nodes of melanoma patients. [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 472.