Yutaka Amemiya

Effects of partner proteins on BCA2 RING ligase activity

BackgroundBCA2 is an E3 ligase linked with hormone responsive breast cancers. We have demonstrated previously that the RING E3 ligase BCA2 has autoubiquitination activity and is a very unstable protein. Previously, only Rab7, tetherin, ubiquitin and UBC9 were known to directly interact with BCA2.MethodsHere, additional BCA2 binding proteins were found using yeast two-hybrid and bacterial-II-hybrid screening techniques with Human breast and HeLa cDNA libraries. Co-expression of these proteins was analyzed through IHC of TMAs. Investigation of the molecular interactions and effects were examined through a series of in vivo and in vitro assays.ResultsTen unique BCA2 interacting proteins were identified, two of which were hHR23a and 14-3-3sigma. Both hHR23a and 14-3-3sigma are co-expressed with BCA2 in breast cancer cell lines and patient breast tumors (n = 105). hHR23a and BCA2 expression was significantly correlated (P = < 0.0001 and P = 0.0113) in both nucleus and cytoplasm. BCA2 expression showed a statistically significant correlation with tumor grade. High cytoplasmic hHR23a trended towards negative nodal status. Binding to BCA2 by hHR23a and 14-3-3sigma was confirmed in vitro using tagged partner proteins and BCA2. hHR23a and 14-3-3sigma effect the autoubiquitination and auto-degradation activity of BCA2. Ubiquitination of hHR23a-bound BCA2 was found to be dramatically lower than that of free BCA2, suggesting that hHR23a promotes the stabilization of BCA2 by inactivating its autoubiquitination activity, without degradation of hHR23a. On the other hand, phosphorylated BCA2 protein is stabilized by interaction with 14-3-3sigma both with and without proteasome inhibitor MG-132 suggesting that BCA2 is regulated by multiple degradation pathways.ConclusionsThe interaction between BCA2 and hHR23a in breast cancer cells stabilizes BCA2. High expression of BCA2 is correlated with grade in breast cancer, suggesting regulation of this E3 ligase is important to cancer progression.

MiR‐301a regulates E‐cadherin expression and is predictive of prostate cancer recurrence

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate gene expression post‐transcriptionally. Dysregulation of miRNA has been implicated in the development and progression of prostate cancer. Through next generation miRNA sequencing, we recently identified a panel of five miRNAs associated with prostate cancer recurrence and metastasis. Of the five miRNAs, miR‐301a had the strongest association with prostate cancer recurrence. Overexpression of miR‐301a in prostate cancer cells, PC3, and LNCaP resulted in increased growth both in vitro and in xenografted tumors. We therefore sought to examine its role in prostate carcinogenesis in greater detail.

Molecular cloning and functional characterisation of chicken Atonal homologue 1: A comparison with human Atoh1

The vertebrate basic helix‐loop‐helix transcription factor Atoh1 is essential for maturation and survival of mechanosensory hair cells of the inner ear, neurogenesis, differentiation of the intestine, homeostasis of the colon and is implicated in cancer progression. Given that mutations in Atoh1 are detected in malignant tumours, study of functionally different Atoh1 alleles and homologues might yield useful avenues for investigation. The predicted sequence of chicken Atoh1 (cAtoh1) has large regions of dissimilarity to that of mammalian Atoh1 homologues. We hypothesise that cAtoh1 might have intrinsic functional differences to mammalian Atoh1.

Loss of Igfbp7 Causes Precocious Involution in Lactating Mouse Mammary Gland

Background Insulin like growth factors (IGFs) and their binding proteins (IGFBPs) are secreted peptides that play major roles in regulating the normal development and maturation of mammary gland. While Igfbp7 has been shown to decrease breast tumor growth, its role in regulating the normal mammary gland development has not been studied. To this end, we generated Igfbp7-null mice and examined the development and maturation of mammary glands in the virgin, pregnant and lactating animals. Results We report here that loss of Igfbp7 significantly retards mammary gland development in the virgin animals. More significantly, the pregnant Igfpb7-null glands contained fewer alveolar structures and that during lactation these glands exhibit the morphological changes that are associated with involution. The transcriptome profile of the Igfbp7-null glands on the lactation day 3 revealed a distinct involution-related gene signature compared to the lactating WT glands. Interestingly, we found that the lactating Igfbp7-null glands exhibit increased expression of Stat3 and enhanced activation of (phosphorylated) Stat3, combined with decreased expression of Stat5 suggesting that the absence of Igfbp7 accelerates the onset of involution. We also found that in absence of Igfpb7, the lactating glands contain increased Igfbp5 protein along with decreased expression of IGF-1 Receptor and Akt activation. Finally, we show that during the normal course of involution, Igfbp7 expression is significantly decreased in the mammary gland. Conclusion Our data suggest that loss of Igfbp7 induces precocious involution possibly through diminished cell survival signals. Our findings identify Igfbp7 as major regulator of involution in the mammary gland.

Direct reprogramming of spiral ganglion non-neuronal cells into neurons: Toward ameliorating sensorineural hearing loss by gene therapy

Primary auditory neurons (PANs) play a critical role in hearing by transmitting sound information from the inner ear to the brain. Their progressive degeneration is associated with excessive noise, disease and aging. The loss of PANs leads to permanent hearing impairment since they are incapable of regenerating. Spiral ganglion non-neuronal cells (SGNNCs), comprised mainly of glia, are resident within the modiolus and continue to survive after PAN loss. These attributes make SGNNCs an excellent target for replacing damaged PANs through cellular reprogramming. We used the neurogenic pioneer transcription factor Ascl1 and the auditory neuron differentiation factor NeuroD1 to reprogram SGNNCs into induced neurons (iNs). The overexpression of both Ascl1 and NeuroD1 in vitro generated iNs at high efficiency. Transcriptome analyses revealed that iNs displayed a transcriptome profile resembling that of endogenous PANs, including expression of several key markers of neuronal identity: Tubb3, Map2, Prph, Snap25, and Prox1. Pathway analyses indicated that essential pathways in neuronal growth and maturation were activated in cells upon neuronal induction. Furthermore, iNs extended projections toward cochlear hair cells and cochlear nucleus neurons when cultured with each respective tissue. Taken together, our study demonstrates that PAN-like neurons can be generated from endogenous SGNNCs. This work suggests that gene therapy can be a viable strategy to treat sensorineural hearing loss caused by degeneration of PANs.

Abstract P5-05-01: IGFBP-7 Reduces Growth of Xenografted Breast Tumors in Mice and Inhibits Breast Cancer Cell Proliferation and Migration through the MEK-ERK Pathway

To identify genes correlated with breast tumorigenesis and eventual metastasis, gene expression profiles were generated using 28K whole genome microarrays. Gene expression profiles of primary breast tumors that grew and metastasized in the NOD/SCID mice were compared with other primary breast tumors that had different growth and metastatic potentials. Five hundred and eighty two genes were significantly differentially expressed by our statistical comparison criteria using the GeneSpring GX 7.3.1 software suite. In the metastatic set eight genes were selectively overexpressed (YB1, MMP7, MMP9, RAB5A, RABGDIB, EPHRB3, WNT2B, CSF1R) and 12 were selectively underexpressed (IGFBP7, GATA3, CST5, CDK6, SERBP1, MGP, TGF1L4, ESE3, ELF3, EDNRB, HECTD1, TINP1). In the present study we focused on the IGFBP-7 gene product which was found to be inversely correlated with disease progression in breast cancer. To further investigate the role of IGFBP-7 in breast tumor suppression, it was overexpressed in the triple negative MDA-MB-468 human breast cancer line. Ectopic overexpression of IGFBP-7 clearly reduced the growth of the MDA-MB-468/IGFBP-7 cells compared to the parental MDA-MB-468 cells. Ectopic overexpression or addition of rIGFBP-7 to breast cancer cell lines in vitro clearly reduced the growth of several breast cancer cell lines and resulted in phenotypic changes characterized by cell aggregation. Investigation of downstream signalling pathways affected by IGFBP-7 revealed that addition of IGFBP-7 to a triple negative breast cancer cell line not only inhibited phosphorylation of ERK-1/2 but also increased expression of the cyclin-dependent kinase inhibitor 1, p21. Similar IGFBP-7 treatment of an ER+ breast cancer line resulted not only in inhibition of ERK-1/2 phosphorylation, but also AKT phosphorylation, suggesting that IGFBP-7 may affect multiple pathways in hormone responsive breast cancer cell lines. IGFBP-7 protein is processed into a shorter form by matriptase. Breast cancer cell lines which were unable to process IGFBP-7 into the shorter form were unaffected by IGFBP-7 mediated growth inhibition, suggesting that cleaved IGFBP-7 may be required for growth inhibitory effects. When injected subcutaneously into NOD/SCID mice, the increased expression of IGFBP-7 in the MDA-MB-468/IGFBP-7 cells reduced the rate of tumor growth in comparison to the parental MDA-MB-468 cells. Furthermore, injection of rIGFBP-7 protein at the tumor site into breast cancer xenografted NOD/SCID mice also resulted in significant tumor growth reduction. These results suggest that the growth of breast cancer could be prevented by the forced expression of IGFBP-7 protein. Work in progress will further uncover the mechanism of IGFBP-7-mediated inhibition on signalling pathways, as well as differentiate the impact of both full length and cleaved IGFBP-7 protein on breast tumor inhibition. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P5-05-01.

Abstract A079: MicroRNA-139 regulates prostate cancer aggressiveness by targeting IGF1R

Background: Dysregulated microRNA (miRNA) expression has been implicated in prostate cancer progression. We previously identified a panel of five miRNAs associated with biochemical recurrence and metastasis following prostatectomy based on NGS-based whole miRNome discovery and qPCR-based validation analysis. In this analysis, we examine the effect of miR-139-5p, one of the downregulated miRNAs identified in the panel, in greater detail. Methods: Using a cohort of 585 patients treated with radical prostatectomy, we examined the prognostic significance of miR-139 (dichotomized around the median) using the Kaplan Meier method and Cox proportional hazard models. We validated these results using The Cancer Genome Atlas (TCGA) data. We created cell lines that overexpressed miR-139 for functional assays. Finally, we examined pathways through which miR-139 may function using prediction algorithms and confirmed targets by Western blotting and reporter assays. Results: MiR-139 downregulation was significantly associated with a variety of accepted prognostic factors in prostate cancer, including Gleason score, pathologic stage, margin positivity, and lymph node status. MiR-139 was associated with prognosis: the cumulative incidence of biochemical recurrence and metastasis was significantly lower among patients with high miR-139 expression (p=0.0004 and 0.038, respectively). After adjusting for known prognostic factors, patients with high miR-139 expression had significantly lower risk of recurrence (HR 0.77, 95% 0.58-1.04). Validation in the TCGA dataset showed a significant association between dichotomized miR-139 expression and biochemical recurrence (OR 0.52, 95% CI 0.33-0.82). Overexpression of miR-139 in PC3 and DU145 prostate cancer cells led to a significant reduction in cell proliferation and migration compared to control cells. IGF1R was identified as a potential target of miR-139 based on previous work in colorectal and non-small cell lung cancers. Reduced luciferase reporter activity was observed upon co-transfection of the 3′ UTR of IGF1Rβ with miR-139 mimic compared to co-transfection with control mimic. Furthermore, Western blotting of PC3 cells overexpressing miR-139 revealed reduced IGF1Rβ protein expression, as well as reduced expression of its downstream pathway proteins pAKT and pERK. Cell cycle analysis indicated a significantly increased number of cells arrested in G2/M phase in PC3 cells overexpressing miR-139. This was accompanied by an increase in β-galactosidase stained senescent cells and p21 protein expression. Conclusions: miR-139 is associated with improved prognosis in patients with localized prostate cancer. This appears to be mediated through an IGF1R pathway leading to increased p21 expression, resulting in prostate cancer cell senescence from G2 arrest. Citation Format: Yutaka Amemiya, Christopher J. Wallis, Tania Benatar, Elizabeth Kobylecky, Linda Sugar, Christopher Sherman, Robert Nam, Arun K. Seth. MicroRNA-139 regulates prostate cancer aggressiveness by targeting IGF1R [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A079.

Identification of a Novel MicroRNA Panel Associated with Metastasis Following Radical Prostatectomy for Prostate Cancer

Background/Aim: This is a case control study designed to identify one or more novel microRNA sequences associated with metastasis following radical prostatectomy for clinically localized prostate cancer. Materials and Methods: Samples were obtained from patients with clinical evidence of metastatic disease following surgery (cases) and patients who showed no evidence of metastasis or biochemical recurrence at least 5 years following surgery (controls) as identified from a single-center, institutional database. Cases and controls were matched for tumor grade and duration of follow-up. Results: Whole miRNome analysis identified 2,792 expressed miRNAs in 19 patient pairs. The 497 miRNA sequences with reads per million over 10, were used for analysis, bootstrapping with backward selection identified a panel of 5-miRNA (miR-17-3p, miR-27a-3p, miR-200a-3p, miR-375, and miR-376b-3p) with a risk score strongly associated with metastasis (AUC=89.5%, 95%CI=79.5-99.5%). Methodologically, most studies use the magnitude of differential expression with or without clinical judgement for selection of predictors for inclusion in panels. In order to strengthen the predictive model, a selection strategy was employed, bootstrapping with automated backwards selection, which relied on the strength of association for inclusion. Conclusion: A genome-wide analysis of microRNA expression identified a panel of 5 miRNAs strongly associated with prostate cancer metastasis following radical prostatectomy.

Novel Ubiquitin E3 Ligases as Targets for Cancer Therapy: Focus on Breast Cancer-Associated Gene 2 (BCA2)

The struggle to find new cancer targets continues unabated. With this in mind, E3 ligases (also called E3 ubiquitin ligases) constitute a large and diverse family of genes that play a role in the ubiquitination of proteins as well as in a myriad of other important activities in cells including, but not limited to, DNA repair and proliferation. Breast cancer-associated protein 2 (BCA2) is an E3 ligase that is expressed in a large number of invasive breast cancers and is involved in several important cellular functions. In this chapter we describe the mechanisms that control the expression and half-life of BCA2 and the association between high expression of BCA2 and breast cancer tumor grade. Furthermore, we explore the role that this E3 ligase may play in cancer progression. Finally, we examine the potential effects of E3 ligases, including BCA2, a novel class of wide-ranging therapeutic cancer targets.

Abstract 5013: Loss of Igfbp7 leads to the expansion of luminal progenitors by altering the stromal fibroblasts’ ability to support luminal cell differentiation.

Insulin-like growth factor binding protein 7, IGFBP7, is a secreted glycoprotein that unlike other IGFBPs, binds insulin with higher affinity than IGFs. Interestingly, high-grade invasive breast cancers lack IGFBP7 expression, and its low expression levels is associated with reduced patient survival. Interestingly, in some cancers expression of IGFBP7 is lost due to promoter methylation or loss of hetrozygosity. Also, in vivo xenograft assays have shown that IGFBP7 can suppress breast cancer cell proliferation. These data together imply that IGFBP7 may act as a potential tumor suppressor. Recent evidence suggests that tumor suppressors and oncogenes play essential roles in regulating the normal development of mammary gland and that alterations to their expression or functions may transform the undifferentiated breast stem cells and progenitors into cancer initiating cells. To examine if IGFBP7 plays essential roles in regulating the normal development of the mammary gland, we developed Igfbp7-null mice and examine the development of the mammary glands. Notably, the pre- and post-pubertal Igfbp7-null glands featured decreased overall size and diminished terminal end bud and alveolar densities. However, the Igfbp7-null glands showed the most startling defects during pregnancy and lactation where lobular sacs were severely deformed and decreased in numbers. To ascertain the molecular mechanism underlying this defective lobular development, we compared the transcriptome profiles of the Igfbp7-null glands and the Wild-Type (WT) glands using RNA-Seq technology. Our transcriptome analysis revealed the decreased expression of a number of key signaling molecules involved in the Notch and IGF/Insulin and other signaling pathways. Interestingly our analysis also revealed the decreased expression of luminal cell differentiation-associated genes such as Gata3 and Pml. Through quantifying, for the first time, the number of luminal progenitors during the different phases of pregnancy and lactation, we determined that loss of Igfbp7 lead to the increased frequency (up to 5±0.3 folds) and thusly, yield (up to 3±0.25 folds) of the luminal progenitors and yet these Igfbp7-null progenitors are unable to differentiate in vivo. We further show that the Igfbp7-null stromal fibroblasts are unable to support the differentiation of the luminal progenitors. For the first time we demonstrate that loss of a tumor suppressor gene, Igfbp7, can suppress the ability of luminal progenitors to differentiate by altering the properties of stromal fibroblasts. It is interesting that the loss of a tumor suppressor gene would result in the expansion of the luminal progenitors since these progenitors and other undifferentiated cells have been envisaged to be prime cellular targets to accumulate transforming mutations that can cause them to act as cancer initiating cells. Citation Format: Sumanta Chatterjee, Stephanie Bacopulos, WenYi Yang, Yutaka Amemiya, Demetri Spyropoulos, Arun Seth, Afshin Raouf. Loss of Igfbp7 leads to the expansion of luminal progenitors by altering the stromal fibroblasts’ ability to support luminal cell differentiation. [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 5013. doi:10.1158/1538-7445.AM2013-5013