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We previously reported that single cells from a human colorectal cancer (CRC) cell line (HCA-7) formed either hollow single-layered polarized cysts or solid spiky masses when plated in 3D in type-I collagen. To begin in-depth analyses into whether clonal cysts and spiky masses possessed divergent properties, individual colonies of each morphology were isolated and expanded. The lines thus derived faithfully retained their parental cystic and spiky morphologies and were termed CC (cystic) and SC (spiky), respectively. Although both CC and SC expressed EGF receptor (EGFR), the EGFR-neutralizing monoclonal antibody, cetuximab, strongly inhibited growth of CC, whereas SC was resistant to growth inhibition, and this was coupled to increased tyrosine phosphorylation of MET and RON. Addition of the dual MET/RON tyrosine kinase inhibitor, crizotinib, restored cetuximab sensitivity in SC. To further characterize these two lines, we performed comprehensive genomic and transcriptomic analysis of CC and SC in 3D. One of the most up-regulated genes in CC was the tumor suppressor 15-PGDH/HPGD , and the most up-regulated gene in SC was versican ( VCAN ) in 3D and xenografts. Analysis of a CRC tissue microarray showed that epithelial, but not stromal, VCAN staining strongly correlated with reduced survival, and combined epithelial VCAN and absent HPGD staining portended a poorer prognosis. Thus, with this 3D system, we have identified a mode of cetuximab resistance and a potential prognostic marker in CRC. As such, this represents a potentially powerful system to identify additional therapeutic strategies and disease-relevant genes in CRC and possibly other solid tumors.

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 that AR, 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 ligase Nrdp1 is a model AR target modulated by androgens in hormone-naïve PCa but not in CRPC. Using Nrdp1, we investigated how AR switches transcription programs during CRPC progression. The proximal Nrdp1 promoter 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 between Nrdp1 and AR expression, supporting AR regulation of NRDP1 levels in androgen-sensitive tissue. However, despite sustained AR levels, AR binding to the Nrdp1 promoter and Nrdp1 expression 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 to Nrdp1 ARE, increased its transcription, and augmented NRDP1 protein expression and responsiveness to ADT, indicating that nuclear FLNA controls AR-mediated androgen-sensitive Nrdp1 transcription. 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., (© 2015 Society for Endocrinology.)

Lrig1 is the founding member of the Lrig family of transmembrane leucine-rich repeat proteins, which also includes Lrig2 and Lrig3. Lrig1 is a negative regulator of oncogenic receptor tyrosine kinases, including ErbB and Met receptors, and promotes receptor degradation. Lrig1 has recently emerged as both a tumor suppressor and a key regulator of epidermal and epithelial stem cell quiescence. Despite this, little is known of the mechanisms by which Lrig1 is regulated. Lrig3 was recently reported to increase ErbB receptor expression suggesting that it may function in a manner opposite to Lrig1. In this study, we explore the interaction between Lrig1 and Lrig3 and demonstrate that Lrig1 and Lrig3 functionally oppose one another. Lrig3 opposes Lrig1 negative regulatory activity and stabilizes ErbB receptors. Conversely, Lrig1 destabilizes Lrig3, limiting Lrig3's positive effects on receptors and identifying Lrig3 as a new target of Lrig1. These studies provide new insight into the regulation of Lrig1 and uncover a complex cross-talk between Lrig1 and Lrig3.

Lrig1 is the founding member of the Lrig family and has been implicated in the negative regulation of several oncogenic receptor tyrosine kinases including ErbB2. Lrig1 is expressed at low levels in several cancer types but is overexpressed in some prostate and colorectal tumors. Given this heterogeneity, whether Lrig1 functions to suppress or promote tumor growth remains a critical question. Previously, we found that Lrig1 was poorly expressed in ErbB2-positive breast cancer, suggesting that Lrig1 has a growth-inhibitory role in this tumor type. However, breast cancer is a complex disease, with ErbB2-positive tumors accounting for just 25% of all breast cancers. To gain a better understanding of the role of Lrig1 in breast cancer, we examined its expression in estrogen receptor α (ERα)-positive disease which accounts for the majority of breast cancers. We find that Lrig1 is expressed at significantly higher levels in ERα-positive disease than in ERα-negative disease. Our study provides a molecular rationale for Lrig1 enrichment in ERα-positive disease by showing that Lrig1 is a target of ERα. Estrogen stimulates Lrig1 accumulation and disruption of this induction enhances estrogen-dependent tumor cell growth, suggesting that Lrig1 functions as an estrogen-regulated growth suppressor. In addition, we find that Lrig1 expression correlates with prolonged relapse-free survival in ERα-positive breast cancer, identifying Lrig1 as a new prognostic marker in this setting. Finally, we show that ErbB2 activation antagonizes ERα-driven Lrig1 expression, providing a mechanistic explanation for Lrig1 loss in ErbB2-positive breast cancer. This work provides strong evidence for a growth-inhibitory role for Lrig1 in breast cancer.

The ErbB3 receptor tyrosine kinase contributes to a variety of developmental processes, and its overexpression and aberrant activation promote tumor progression and therapeutic resistance. Accumulating evidence suggests that tumor overexpression may be mediated by the loss of posttranscriptional negative regulatory mechanisms, such as protein degradation, that normally keep receptor levels in check. Our previous studies indicate that the RING finger E3 ubiquitin ligase Nrdp1, a protein lost in breast and other tumor types, suppresses ErbB3 levels by mediating ligand-independent receptor ubiquitination and degradation. Here we demonstrate that Nrdp1 preferentially associates with the nascent form of ErbB3 to accelerate its degradation, and we show that the two proteins colocalize at the endoplasmic reticulum (ER). Blocking the exit of ErbB3 from the ER does not affect the ability of Nrdp1 to mediate receptor ubiquitination or degradation, while functional disruption of the conserved ER-associated degradation (ERAD) pathway ATPase VCP/p97 leads to the Nrdp1-dependent accumulation of ubiquitinated ErbB3 but blocks receptor degradation. Further evidence indicates that the ErbB3 targeted by Nrdp1 for degradation is properly folded and fully functional. Collectively, these observations point to a novel mechanism of receptor tyrosine kinase quantity control wherein steady-state levels of signaling-competent receptor are dictated by an ER-localized degradation pathway.

The ErbB2 and ErbB3 receptor tyrosine kinases act synergistically to promote cellular properties associated with tumor development. Previous studies indicate that endogenous ErbB3 protein is markedly elevated in mouse mammary tumors induced by transgenic ErbB2 overexpression. However, this occurs in the absence of elevated ErbB3 transcript, indicating that post-transcriptional regulatory mechanisms play crucial roles in suppressing ErbB3 protein in normal tissue. Our previous studies also demonstrate that protein levels of Nrdp1, an E3 ubiquitin ligase that targets ErbB3 for degradation, are markedly suppressed in tumors from ErbB2 transgenic animals relative to normal tissue. Here we demonstrate that transgenic expression of Nrdp1 cDNA in the mouse mammary gland is not sufficient to suppress elevated ErbB3 levels or tumor initiation and growth in ErbB2 transgenic mice. Unexpectedly, Nrdp1 protein is absent in tumors from Nrdp1/ErbB2 bigenic mice, and real time PCR analysis indicates that Nrdp1 protein levels are suppressed post-transcriptionally. Nrdp1 protein is more resistant to proteasome-dependent degradation when exogenously expressed in cultured MCF10A nontransformed human breast epithelial cells than in breast tumor cells. These observations indicate that mammary tumors use potent post-transcriptional mechanisms to suppress Nrdp1 protein levels and that protein destabilization may play a central role in Nrdp1 loss in tumors.

Aggressive behavior among females is observed in many species, but the mechanisms of this behavior have historically been understudied. In many species of rodents, winter-like short day photoperiods induce increased aggression levels compared to summer-like long day photoperiods. Recent reports in hamsters show that short days also increase aggression in females. We examined the effects of photoperiod on aggression in female California mice, and for the first time compare brain activity of aggression-tested female rodents under different photoperiods. We observed that female California mice were more aggressive when housed in short days versus long days. Intriguingly, we also observed that under long days female attack latency decreases with repeated testing in resident-intruder tests. These data suggest that winner effects that have been described in males may also occur in females. We also used the expression of phosphorylated extracellular signal-regulated kinases (pERK) in the brain to estimate brain activity during aggression tests. pERK can alter neuronal activity in the short term and in the long term can act as a transcription factor. Using immunoblot analyses we observed that aggression-induced pERK expression in the female bed nucleus of the stria terminalis and medial amygdala occurs under both long and short days. Thus, the mechanisms controlling increased aggression under short days are still unclear and additional study is needed., (Copyright 2010 Elsevier B.V. All rights reserved.)

Using a variety of experimental methods, a network of brain areas regulating aggressive behaviors has been identified in several groups of vertebrates. However, aggressive behavior expressed in different contexts is associated with different patterns of activity across hypothalamic and limbic brain regions. Previous studies in rodents demonstrated that short day photoperiods reliably increase both male and female aggression versus long day photoperiods. Here we used immunohistochemistry and western blots to examine the effect of photoperiod on phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK) in male California mice (Peromyscus californicus) during resident-intruder tests. Phosphorylated ERK (pERK) can alter neuronal activity in the short term and in the long term acts as a transcription factor. In the posterior bed nucleus of the stria terminalis (BNST) males tested in aggression tests had more pERK positive cells when housed in short days but not long days. This result was replicated in western blot analyses from microdissected BNST samples. In the medial amygdala (MEA), immunostaining and western analyses showed that pERK expression also was generally increased in short days. Immunostaining was also used to examine phosphorylation of cyclic AMP response element binding protein (CREB). CREB can be phosphorylated by pERK as well as other kinases and functions primarily as a transcription factor. Intriguingly, aggressive interactions reduced the number of cells stained positive for phosphorylated CREB in the infralimbic cortex, ventral lateral septum and MEA. This effect was observed in mice housed in long days but not short days. Overall, these data suggest that different (but overlapping) networks of aggressive behavior operate under different environmental conditions.

The ErbB family of receptor tyrosine kinases engages a wide variety of signaling pathways that collectively direct transcriptional programs controlling organogenesis during development and tissue maintenance in the adult. These receptors are also frequently found overexpressed or aberrantly activated in various cancers, suggesting that ErbB receptor signaling activity must be very tightly regulated. Sufficient levels of ErbB signaling are necessary to mediate tissue homeostasis, for example, but over-signaling can trigger cellular processes that contribute to cancer initiation or progression. Efforts over the last quarter century have led to a thorough understanding of the signaling pathways that are activated by these receptors and the mechanisms by which ErbB receptors engage these pathways. However, the compensatory negative regulatory mechanisms responsible for attenuating receptor activation have only more recently begun to be explored. Here we review the different known mechanisms of ErbB negative regulation, with particular emphasis on those proteins that exhibit some specificity for the ErbB family. We also describe how loss or suppression of ErbB negative regulators may contribute to tumor development, and discuss how restoration or augmentation of these pathways may represent a novel avenue for the development of ErbB-targeted therapies.

When good visualization of the left coronary artery and proximal branches is difficult, the longitudinally angulated projection has proved useful. An accessory for achieving this angulation with a conventional angiographic table and without sacrificing the advantages of video monitoring and the protection of lead vinyl shielding is described.