ErbB2-positive mammary tumors can escape PI3K-p110α loss through downregulation of the Pten tumor suppressor.

Breast cancer is the most common cancer among women and 30% of patients will be diagnosed with an ErbB2-positive tumor. Forty percent of ErbB2-positive breast tumors have an activating mutation in p110α, a catalytic subunit of phosphoinositide 3-kinase. Clinical and experimental data show that breast tumors treated with a p110α-specific inhibitor often circumvent inhibition and resume growth. To understand this mechanism of resistance, we crossed a p110α conditional (p110αflx/flx) mouse model with mice that overexpress the ErbB2/Neu-IRES-Cre transgene (NIC) specifically in the mammary epithelium. Although mammary-specific deletion of p110α dramatically delays tumor onset, tumors eventually arise and are dependent on p110ß. Through biochemical analyses we find that a proportion of p110α-deficient tumors (23%) display downregulation of the Pten tumor suppressor. We further demonstrate that loss of one allele of PTEN is sufficient to shift isoform dependency from p110α to p110ß in vivo. These results provide insight into the molecular mechanism by which ErbB2-positive breast cancer escapes p110α inhibition.

The ErbB2ΔEx16 splice variant is a major oncogenic driver in breast cancer that promotes a pro-metastatic tumor microenvironment.

Amplification and overexpression of erbB2/neu proto-oncogene is observed in 20-30% human breast cancer and is inversely correlated with the survival of the patient. Despite this, somatic activating mutations within erbB2 in human breast cancers are rare. However, we have previously reported that a splice isoform of erbB2, containing an in-frame deletion of exon 16 (herein referred to as ErbB2ΔEx16), results in oncogenic activation of erbB2 because of constitutive dimerization of the ErbB2 receptor. Here, we demonstrate that the ErbB2ΔEx16 is a major oncogenic driver in breast cancer that constitutively signals from the cell surface. We further show that inducible expression of the ErbB2ΔEx16 variant in mammary gland of transgenic mice results in the rapid development of metastatic multifocal mammary tumors. Genetic and biochemical characterization of the ErbB2ΔEx16-derived mammary tumors exhibit several unique features that distinguish this model from the conventional ErbB2 ones expressing the erbB2 proto-oncogene in mammary epithelium. Unlike the wild-type ErbB2-derived tumors that express luminal keratins, ErbB2ΔEx16-derived tumors exhibit high degree of intratumoral heterogeneity co-expressing both basal and luminal keratins. Consistent with these distinct pathological features, the ErbB2ΔEx16 tumors exhibit distinct signaling and gene expression profiles that correlate with activation of number of key transcription factors implicated in breast cancer metastasis and cancer stem cell renewal.

PIK3CA(H1047R)- and Her2-initiated mammary tumors escape PI3K dependency by compensatory activation of MEK-ERK signaling.

Human breast cancers that have HER2 amplification/overexpression frequently carry PIK3CA mutations, and are often associated with a worse prognosis. However, the role of PIK3CA mutations in the initiation and maintenance of these breast cancers remains elusive. In the present study, we generated a compound mouse model that genetically mimics HER2-positive breast cancer with coexisting PIK3CA(H1047R). Induction of PIK3CA(H1047R) expression in mouse mammary glands with constitutive expression of activated Her2/Neu resulted in accelerated mammary tumorigenesis with enhanced metastatic potential. Interestingly, inducible expression of mutant PIK3CA resulted in a robust activation of phosphatidylinositol-3-kinase (PI3K)/AKT signaling but attenuation of Her2/Her3 signaling, and this can be reversed by deinduction of PIK3CA(H1047R) expression. Strikingly, although these Her2(+) PIK3CA(H1047R)-initiated primary mammary tumors are refractory to HER2-targeted therapy, all tumors responded to inactivation of the oncogenic PIK3CA(H1047R), a situation closely mimicking the use of a highly effective inhibitor specifically targeting the mutant PIK3CA/p110a. Notably, these tumors eventually resumed growth, and a fraction of them escaped PI3K dependence by compensatory ERK activation, which can be blocked by combined inhibition of Her2 and MEK. Together, these results suggest that PIK3CA-specific inhibition as a monotherapy followed by combination therapy targeting MAPK and HER2 in a timely manner may be an effective treatment approach against HER2-positive cancers with coexisting PIK3CA-activating mutations.

Aspergillus thyroiditis: first antemortem case diagnosed by fine-needle aspiration culture in a pediatric stem cell transplant patient.

Aspergillus thyroiditis (AT) has historically been considered a postmortem diagnosis in immunocompromised patients; most have disseminated disease. This report summarizes the clinical challenge of diagnosing AT. It also highlights the value of the early use of thyroid fine-needle aspiration culture and the need for a high index of suspicion to reach the final diagnosis before disease dissemination.

Phosphorylation of eIF4E promotes EMT and metastasis via translational control of SNAIL and MMP-3.

The progression of cancers from primary tumors to invasive and metastatic stages accounts for the overwhelming majority of cancer deaths. Understanding the molecular events which promote metastasis is thus critical in the clinic. Translational control is emerging as an important factor in tumorigenesis. The messenger RNA (mRNA) cap-binding protein eIF4E is an oncoprotein that has an important role in cancer initiation and progression. eIF4E must be phosphorylated to promote tumor development. However, the role of eIF4E phosphorylation in metastasis is not known. Here, we show that mice in which eukaryotic translation initiation factor 4E (eIF4E) cannot be phosphorylated are resistant to lung metastases in a mammary tumor model, and that cells isolated from these mice exhibit impaired invasion. We also demonstrate that transforming growth factor-beta (TGFß) induces eIF4E phosphorylation to promote the translation of Snail and Mmp-3 mRNAs, and the induction of epithelial-to-mesenchymal transition (EMT). Furthermore, we describe a new model wherein EMT induced by TGFß requires translational activation via the non-canonical TGFß signaling branch acting through eIF4E phosphorylation.

Impaired cell death and mammary gland involution in the absence of Dock1 and Rac1 signaling.

Throughout life, the tight equilibrium between cell death and the prompt clearance of dead corpses is required to maintain a proper tissue homeostasis and prevent inflammation. Following lactation, mammary gland involution is triggered and results in the death of excessive epithelial cells that are rapidly cleared by phagocytes to ensure that the gland returns to its prepregnant state. Orthologs of Dock1 (dedicator of cytokinesis 1), Elmo and Rac1 (ras-related C3 botulinum toxin substrate 1) in Caenorhabditis elegans are part of a signaling module in phagocytes that is linking apoptotic cell recognition to cytoskeletal reorganization required for engulfment. In mammals, Elmo1 was shown to interact with the phosphatidylserine receptor Bai1 and relay signals to promote phagocytosis of apoptotic cells. Still, the role of the RacGEF Dock1 in the clearance of dying cells in mammals was never directly addressed. We generated two mouse models with conditional inactivation of Dock1 and Rac1 and revealed that the expression of these genes is not essential in the mammary gland during puberty, pregnancy and lactation. We induced mammary gland involution in these mice to investigate the role of Dock1/Rac1 signaling in the engulfment of cell corpses. Unpredictably, activation of Stat3 (signal transducer and activator of transcription 3), a key regulator of mammary gland involution, was impaired in the absence of Rac1 and Dock1 expression. Likewise, failure to activate properly Stat3 was coinciding with a significant delay in the initiation and progression of mammary gland involution in mutant animals. By using an in vitro phagocytosis assay, we observed that Dock1 and Rac1 are essential to mediate engulfment in epithelial phagocytes. In vivo, cell corpses accumulated at late time points of involution in Dock1 and Rac1 mutant mammary glands. Overall, our study demonstrated an unsuspected role for Dock1/Rac1 signaling in the initiation of mammary gland involution, and also suggested a role for this pathway in the clearance of dead cells by epithelial phagocytes.

eIF4F suppression in breast cancer affects maintenance and progression.

Levels of eukaryotic initiation factor 4E (eIF4E) are frequently elevated in human cancers and in some instances have been associated with poor prognosis and outcome. Here we utilize transgenic and allograft breast cancer models to demonstrate that increased mammalian target of rapamycin (mTOR) signalling can be a significant contributor to breast cancer progression in vivo. Suppressing mTOR activity, as well as levels and activity of the downstream translation regulators, eIF4E and eIF4A, delayed breast cancer progression, onset of associated pulmonary metastasis in vivo and breast cancer cell invasion and migration in vitro. Translation of vascular endothelial growth factor (VEGF), matrix metallopeptidase 9 (MMP9) and cyclin D1 mRNAs, which encode products associated with the metastatic phenotype, is inhibited upon eIF4E suppression. Our results indicate that the mTOR/eIF4F axis is an important contributor to tumor maintenance and progression programs in breast cancer. Targeting this pathway may be of therapeutic benefit.

Acetylcholinesterase activity in the freshwater shrimp Caridina nilotica as a biomarker of Roundup(®) herbicide pollution of freshwater systems in South Africa.

The use of Caridina nilotica whole-body acetylcholinesterase (AChE) activity as a potential biomarker of Roundup(®) pollution of aquatic ecosystems was investigated. Forty days post hatch (dph) shrimps were exposed to different concentrations of 0.0, 4.3, 6.7, 10.5, 16.4, 25.6 and 40.0 mg/L in a 96 h acute toxicity test; and 0.0, 2.2, 2.8, 3.4, 4.3 and 5.4 mg/L in a 21 d chronic toxicity test. Whole-body AChE activities were determined at the end of the exposure periods by spectrophotometric assay of sample extract; activities were then normalized against protein contents in the samples and expressed in nanomoles of substrate hydrolyzed. Results of both tests showed that AChE activity was concentration-dependent. Mean AChE activities and standard deviations (±SD) for 96 h acute toxicity were 3.6239 (± 0.4185), 3.4157 (± 1.1842), 2.537 (± 1.3989), 2.4253 (± 1.4202), 2.4127 (± 1.9097), 2.0017 (± 1.1080) and 2.316 (± 0.4001) nmol/min/mg protein; while activity levels for 21 d test were 3.6907(± 0.3401), 2.8473 (± 0.713), 2.9134 (± 0.9879), 2.6738 (± 0.7117), 2.3019 (± 0.4464) and 2.1478 (± 0.864) nmol/min/mg protein. Reference basal AChE activity for 40 dph C. nilotica based on the two control groups was estimated as 3.6907 (± 0.3401) nmol/min/mg proteins. The present work provides ecotoxicological basis for the possible use of AChE activity in C. nilotica as a biomarker for monitoring Roundup(®) pollution in freshwater systems.

Lipid peroxidation in the freshwater shrimp Caridina nilotica as a biomarker of Roundup(®) herbicide pollution of freshwater systems in South Africa.

Glyphosate-based herbicides used to control weeds and invading alien plant species in South Africa ultimately end up in freshwater ecosystems, but no South African environmental water quality guideline exists to regulate these bio-active chemicals. Ecotoxicological tests to assess the possibility of using lipid peroxidation (LPx) in Caridina nilotica as a potential biomarker of Roundup(®), a glyphosate-based herbicide, pollution were conducted. In two separate tests, 40 days post hatch shrimps were exposed to different concentrations of 4.3, 6.7, 10.5, 16.4, 25.6 and 40.0 mg/L in a 96 h acute toxicity test; and 2.2, 2.8, 3.4, 4.3 and 5.4 mg/L in a 21 d chronic toxicity test, using static-non renewal and static-renewal methods, respectively. Shrimp whole body LPx was estimated by thiobarbituric acid reactive species (TBARS) assay, performed by a malondialdehyde (MDA) reaction with 2-thiobarbituric acid (TBA) measured spectrophotometrically. Final MDA concentrations were expressed as nmol MDA produced/mg protein. Results showed that LPx was significantly lower in control animals than in animals exposed to different Roundup(®) concentrations, (p < 0.05). The present work provides an ecotoxicological basis for the possible use of LPx in Caridina nilotica as a biomarker for monitoring Roundup(®) pollution in freshwater ecosystems.

The ShcA SH2 domain engages a 14-3-3/PI3'K signaling complex and promotes breast cancer cell survival.

The ShcA adapter protein transmits activating signals downstream of receptor and cytoplasmic tyrosine kinases through the establishment of phosphotyrosine-dependent complexes. In this regard, ShcA possesses both a phosphotyrosine-binding domain (PTB) and Src homology 2 domain (SH2), which bind phosphotyrosine residues in a sequence-specific manner. Although the majority of receptor tyrosine kinases expressed in breast cancer cells bind the PTB domain, very little is known regarding the biological importance of SH2-driven ShcA signaling during mammary tumorigenesis. To address this, we employed transgenic mice expressing a mutant ShcA allele harboring a non-functional SH2 domain (ShcR397K) under the transcriptional control of the endogenous ShcA promoter. Using transplantation approaches, we demonstrate that SH2-dependent ShcA signaling within the mammary epithelial compartment is essential for breast tumor outgrowth, survival and the development of lung metastases. We further show that the ShcA SH2 domain activates the AKT pathway, potentially through a novel SH2-mediated complex between ShcA, 14-3-3ζ and the p85 regulatory subunit of phosphatidylinositol 3 (PI3') kinase. This study is the first to demonstrate that the SH2 domain of ShcA is critical for tumor survival during mammary tumorigenesis.

beta1-integrin is dispensable for the induction of ErbB2 mammary tumors but plays a critical role in the metastatic phase of tumor progression.

Cross-talk between integrin receptors and activated growth factor receptors has been hypothesized to play a critical role in the initiation and progression of cancer. Despite in vitro evidence documenting the important role of integrin receptors in the regulation of cancer cell proliferation, the relative contribution of the integrin receptors to the initiation and progression of tumors remains unclear. Previous studies with a polyomavirus middle T mammary tumor model have indicated that targeted disruption of beta1-integrin in the mammary glands of these mice completely blocks tumor induction. To further explore the general significance of these observations, we have crossed these conditional beta1-integrin strains to a strain of mice carrying mouse mammary tumor virus/activated erbB2 (herein referred to as the NIC strain). In contrast to the tumor induction block in the polyomavirus middle T model, tumor onset in the beta1-integrin-deficient NIC mice was delayed by only 30 d and was 100% penetrant. This modest effect on tumor induction was not a result of inefficient excision, as all tumors were confirmed as beta1-integrin-null. Animals bearing beta1-integrin-deficient ErbB2 tumors exhibited significantly reduced tumor volume, which was associated with increased tumor cell apoptosis and a reduction in tumor angiogenesis. In addition, beta1-integrin-deficient tumors were compromised in their capacity to metastasize to the lung, a deficiency associated with abrogation of adhesion signaling. Taken together, these observations suggest that, although beta1-integrin is dispensable for the initiation of ErbB2 tumor induction, it plays a critical role in metastatic phase of tumor progression.

Integrin-linked kinase has a critical role in ErbB2 mammary tumor progression: implications for human breast cancer.

Elevated expression of the integrin-linked kinase (ILK) has been observed in a variety of cancers and has been further correlated with poor clinical outcome. Here, we show that mammary epithelial disruption of ILK results in a profound block in mammary tumor induction. Consistent with these observations, inhibition of ILK function in ErbB2-expressing cells with small molecule inhibitor or RNA interference resulted in profound block in their in vitro invasive properties due to the induction of apoptotic cell death. The rare ILK-deficient tumors that eventually arose overcame this block in tumor induction by an upregulation of ErB3 phosphorylation. These observations provide direct evidence that ILK has a critical role in the initiation phase of ErbB2 tumor induction.

Transcription factor regulatory networks in mammary epithelial development and tumorigenesis.

The mouse mammary gland is composed of three epithelial cell types, which include ductal, alveolar and myoepithelial cells. A hierarchy in which the mammary stem cell compartment gives rise to progressively restricted progenitors that ultimately form the luminal (ductal and alveolar) and myoepithelial lineages is now emerging. Although very little is known about the mechanisms controlling the differentiation of the myoepithelial cell lineage, a growing body of work reveals that the luminal cell fate is specified by a network of transcription factors. The precise roles of specific transcription factors in promoting differentiation of luminal progenitors into ductal or alveolar cells are now being elucidated. This review will discuss the importance of these recent observations and place them within the context of other transcription factor networks involved in mammary gland development and tumorigenesis.

Shc is required for ErbB2-induced inhibition of apoptosis but is dispensable for cell proliferation and disruption of cell polarity.

Amplification and overexpression of ErbB2 strongly correlates with aggressive breast cancers. A deeper understanding of pathways downstream of ErbB2 signaling that are required for the transformation of human mammary epithelial cells will identify novel strategies for therapeutic intervention in breast cancer. Using an inducible activation of ErbB2 autophosphorylation qsite mutants and the MCF-10A three-dimensional (3D) culture system, we investigated pathways used by ErbB2 to transform the epithelia. We report that ErbB2 induces cell proliferation and loss of 3D organization by redundant mechanisms, whereas it disrupts apical basal polarity and inhibits apoptosis using Tyr 1201 and Tyr 1226/7, respectively. Signals downstream of Tyr 1226/7 were also sufficient to confer paclitaxel resistance. The Tyr 1226/7 binds Shc, and the knockdown of Shc blocks the ability of ErbB2 to inhibit apoptosis and mediate paclitaxel resistance. Tyr 1226/7 is known to activate the Ras/Erk pathway; however, paclitaxel resistance did not correlate with the activation of Erk or Akt, suggesting the presence of a novel mechanism. Thus, our results show that targeting pathways used by ErbB2 to inhibit cell death is a better option than targeting cell proliferation pathways. Furthermore, we identify a novel function for Shc as a regulator of apoptosis and drug resistance in human mammary epithelial cells transformed by ErbB2.

The Ste20-like kinase SLK is required for ErbB2-driven breast cancer cell motility.

The Ste20-like kinase, SLK, is involved in the control of cell motility through its effects on actin reorganization and focal adhesion turnover. Here we investigated the role of SLK in chemotaxis downstream of the tyrosine kinase receptor, HER2/ErbB2/Neu, which is frequently overexpressed in human breast cancers. Our results show that SLK is required for the efficient cell migration of human and mouse mammary epithelial cell lines in the presence of the Neu activator, heregulin, as a chemoattractant. SLK activity is stimulated by heregulin treatment or by overexpression of activated Neu. Phosphorylation of tyrosine 1201 or tyrosines 1226/7 on Neu is a key event for SLK activation and cell migration, and cancer cell invasion mediated by these tyrosines is inhibited by kinase-inactive SLK. Signaling pathway inhibitors show that Neu-mediated SLK activation is dependent on MEK, PI3K, PLCgamma and Shc signaling. Furthermore, heregulin-stimulated SLK activity requires signals from the focal adhesion proteins, FAK and src. Finally, phospho-FAK analysis shows that SLK is required for Neu-dependent focal adhesion turnover. Together, these studies define an interaction between Neu and SLK signaling in the regulation of cancer cell motility.

Effect of laundry activities on in-stream concentrations of linear alkylbenzene sulfonate in a small rural South African river.

In many parts of the world clothes are washed near to or in rivers and streams. Little information is available on resulting concentrations of detergent ingredients or on any potential effects caused. In this study, the fate of a commonly used anionic surfactant, linear alkylbenzene sulphonate (LAS) was investigated in a reach of the Balfour River (Eastern Cape Province, South Africa) which was regularly used as a site for laundry activity. Samples of river water were collected upstream of the main washing site and at a number of locations downstream on several occasions in winter and summer. Sediment samples were also collected and analysed. In addition, a household survey was conducted to ascertain the amount of detergent used and the distribution of washing practices. The results of the survey suggested that the use of riverside locations for laundry activities was seasonal. Most washing tended to be done at home during the winter with riverside sites used more frequently during the summer months. The monitoring data showed that LAS concentrations in water were very variable. They were occasionally high in the immediate vicinity of the laundry site (up to 342 microg L(-1)) but were generally very low (<11 microg L(-1)) at downstream monitoring stations, suggesting that LAS was rapidly dissipated by a combination of degradation, hydrodynamic dispersion and dilution. Concentrations in the immediate vicinity of the washing site were lower than expected on the basis of the household survey because most waste water was disposed of on the river bank rather than directly in the river. No ecological effects are expected from LAS emissions at this site.

National sampling and gross composition of Australian retail pork cuts 2005/2006.

After consideration of five potential sampling designs, 13 retail pork cuts were purchased from randomly selected supermarkets and butchers' stores in urban areas across the socioeconomic scale in three States of Australia in late 2005 and early 2006. They were analysed, raw and cooked, for gross composition (fat, lean, bone and gristle). Gross composition varied considerably within cut associated with large divergences in interpretation of standard pork cuts by butchers. There were no notable differences in gross composition across States, across the socioeconomic range of suburbs of purchase or between outlet types (butcher vs supermarket). Cuts tended to be larger and leaner than those in similar studies in the 80s and 90s. Due to increasing uniformity in breeding and feeding of pigs in Australia, sampling designs in future surveys could be simplified.

Sam68 haploinsufficiency delays onset of mammary tumorigenesis and metastasis.

The Src-associated substrate in mitosis Sam68 is a KH type RNA-binding protein known to be a substrate of numerous tyrosine kinases, and often referred to as a STAR (signal transduction activator of RNA) protein. Herein, we observed that Sam68-null mice display mammary gland and the uterine development defects. Moreover, we report that Sam68 haploinsufficiency impedes mammary tumor onset in vivo driven by the potent mammary-targeted polyoma middle T-antigen (MMTV-PyMT) oncogene. The effect was cell autonomous as the Sam68 knockdown in PyMT-transformed cell lines also delayed tumorigenesis and metastasis formation in nude mice. Interestingly, tumor extracts isolated from PyMT/Sam68(+/-) mice compared with PyMT/Sam68(+/+) mice contained activated Src and FAK kinases. These findings suggest that Sam68 may be a modulator of tyrosine kinase activity in vivo and a signaling requirement for mammary tumorigenesis and metastasis.

Cycloheximide resistance in the Lipomycetaceae revisited.

Cycloheximide (CYH) is a heterocyclic, glutarimide antibiotic that is a potent inhibitor of protein biosynthesis in most eukaryotes. This study demonstrated that yeasts from all species of the Lipomycetaceae, with the exception of Dipodascopsis spp., can grow in the presence of up to 5 g.L(-1) CYH -- a concentration that is five times higher than the accepted "highest" concentration of 1 g.L(-1) used in physiological tests for yeast identification. Lipomycetaceous yeasts are known to utilize heterocyclic nitrogen-containing compounds such as thymine as sole nitrogen source. CYH contains a glutarimide ring, which is chemically similar to thymine. We investigated the possibility that CYH resistance in the Lipomycetaceae might be due to an ability to degrade CYH and use it as the sole nitrogen source. However, we were unable to demonstrate significant growth on CYH as sole nitrogen source. When thymine was used as positive control, we could demonstrate its utilization as sole nitrogen source.