Loss of MXRA8 Delays Mammary Tumor Development and Impairs Metastasis.

Matrix-remodeling-associated protein 8 or MXRA8 is a transmembrane protein that can bind arthritogenic alpha viruses like the Chikungunya virus and provide viral entry into cells. MXRA8 can also interact with integrin ß3 and thus possibly regulate cell-cell interactions and binding to the extracellular matrix. While MXRA8 has been associated with reduced survival in patients with colorectal and renal clear cell cancers, the role of MXRA8 in breast cancer remains largely unexplored. Therefore, the aim of this research was to determine the role of MXRA8 in breast cancer by knocking out MXRA8 in the human triple-negative breast cancer cell line MDA-MB-231. The loss of MXRA8 reduced cell proliferation in vitro but had no effect on apoptosis or migration in cultured cells. However, the loss of MXRA8 significantly delayed tumor development and reduced metastatic dissemination to the lungs in a xenograft model. RNA sequencing identified three genes, ADMATS1, TIE1, and BMP2, whose expression were significantly reduced in MXRA8-knockout tumors compared to control tumors. MXRA8 staining of a human breast cancer tissue array revealed higher levels of MXRA8 in primary tumors and metastases of aggressive tumor subtypes (TNBC and HER2+) compared to less aggressive, ER+ breast cancers. Our findings demonstrate for the first time that MXRA8 regulates the progression of human TNBC possibly through influencing the interaction of tumor cells with their microenvironment.

Sex Differences in Glomerular Protein Expression and Effects of Soy-Based Diet on Podocyte Signaling.

Background: Kidney disease is a major public health issue arising from loss of glomerular podocyte function, and there are considerable sex differences in its prognosis. Evidence suggests a renoprotective effect of estrogen and soy diet-derived phytoestrogens, although the molecular basis for this is poorly understood. Objective: Here, we aim to assess sex differences in expression of key proteins associated with podocyte survival and determine the effects of dietary soy on glomerular and podocyte signaling. Methods: Male and female FVB mice were fed control, low (1%), and high (20%) doses of isolated soy protein (ISP) in utero and until 100 days of age. Spot urine was collected to measure proteinuria and isolated glomeruli were used to quantify activated and total levels of nephrin, Akt, and ERK1/2. To investigate protective effects of specific soy phytoestrogens, cultured podocytes were treated with or without daidzein and subject to control or high glucose as a model of podocyte injury. Results: Nephrin and Akt were elevated at baseline in glomeruli from females compared to males. Both sexes that were fed 1% and 20% ISP displayed robust increases in total glomerular Akt compared to controls, and these effects were more prominent in females. A similar trend at both doses in both sexes was observed with activated Akt and total nephrin. Notably, males exclusively showed increased phosphorylation of nephrin and extracellular signal-regulated kinase (ERK) at the 1% ISP dose; however, no overt changes in urinary albumin excretion or podocin levels were observed, suggesting that the soy diets did not impair podocyte function. Finally, in cultured male and female podocytes, daidzein treatment suppressed high glucose-induced ERK activation. Conclusions: Together, our findings reveal a putative mechanism to explain the protective influence of sex on kidney disease progression, and they provide further evidence to support a beneficial role for dietary soy in preserving glomerular function.

Contexte: L'insuffisance rénale est un problème majeur de santé publique résultant d'une perte de fonction des podocytes glomérulaires, et son pronostic diffère selon le sexe. Bien que le fondement moléculaire en soit mal compris, des données suggèrent que les œstrogènes et des phytoestrogènes dérivés du soja alimentaire auraient un effet néphroprotecteur. Objectifs: Évaluer les différences selon le sexe dans l'expression des protéines clés associées à la survie des podocytes, et déterminer les effets du soja alimentaire sur la signalisation glomérulaire et les podocytaire. Méthodologie: Des souris FVB mâles et femelles ont reçu un régime alimentaire témoin ou un regime à faible dose (1 %) ou à dose élevée (20 %) de protéines de soja isolées (PSI) in utero et jusqu'à l'âge de 100 jours. Des échantillons aléatoires d'urine ont été recueillis pour mesurer la protéinurie et des glomérules isolés ont été utilisés pour quantifier les niveaux activés et totaux de néphrine, d'Akt et d'ERK1/2. Pour évaluer l'effet protecteur de certains phytoestrogènes du soja, des podocytes cultivés ont été traités avec ou sans daidzéine et soumis à une dose témoin ou à une dose élevée de glucose comme modèle de lésion podocytaire. Résultats: Les taux initiaux de néphrine et d'Akt étaient plus élevés dans les glomérules des souris femelles. Les souris mâles et femelles nourries avec des doses de 1 % et de 20 % de PSI ont montré des augmentations significatives de l'Akt glomérulaire totale par rapport aux témoins, et ces effets étaient plus importants chez les femelles. Une tendance semblable a été observée chez les deux sexes et pour les deux doses en ce qui concerne l'Akt activée et la néphrine totale. Seuls les mâles ont montré une augmentation de la phosphorylation de la néphrine et de l'ERK à 1 % de PSI; aucun changement manifeste n'a cependant été observé dans l'excrétion urinaire d'albumine ou dans le taux de podocine, ce qui suggère que le soja alimentaire n'a pas altéré la fonction des podocytes. Dans les podocytes cultivés, tant mâles que femelles, le traitement à la daidzéine a inhibé l'activation de l'ERK induite par une forte dose de glucose. Conclusion: Ensemble, nos résultats révèlent un mécanisme putatif pouvant expliquer l'effet protecteur du sexe du patient sur la progression de l'insuffisance rénale. Ces résultats fournissent des preuves supplémentaires soutenant l'hypothèse d'un rôle bénéfique du soja alimentaire dans la préservation de la fonction glomérulaire.

Elevated Expression of miR-200c/141 in MDA-MB-231 Cells Suppresses MXRA8 Levels and Impairs Breast Cancer Growth and Metastasis In Vivo.

Breast cancer cells with mesenchymal characteristics, particularly the claudin-low subtype, express extremely low levels of miR-200s. Therefore, this study examined the functional impact of restoring miR-200 expression in a human claudin-low breast cancer cell line MDA-MB-231. MDA-MB-231 cells were stably transfected with a control vector (MDA-231EV) or the miR-200c/141 cluster (MDA-231c141). Injection of MDA-231c141 cells into the 4th mammary gland of NCG mice produced tumors that developed significantly slower than tumors produced by MDA-231EV cells. Spontaneous metastasis to the lungs was also significantly reduced in MDA-231c141 cells compared to MDA-231EV cells. RNA sequencing of MDA-231EV and MDA-231c141 tumors identified genes including MXRA8 as being downregulated in the MDA-231c141 tumors. MXRA8 was further investigated as elevated levels of MXRA8 were associated with reduced distant metastasis free survival in breast cancer patients. Quantitative RT-PCR and Western blotting confirmed that MXRA8 expression was significantly higher in mammary tumors induced by MDA-231EV cells compared to those induced by MDA-231c141 cells. In addition, MXRA8 protein was present at high levels in metastatic tumor cells found in the lungs. This is the first study to implicate MXRA8 in human breast cancer, and our data suggests that miR-200s inhibit growth and metastasis of claudin-low mammary tumor cells in vivo through downregulating MXRA8 expression.

Transgenic overexpression of the miR-200b/200a/429 cluster inhibits mammary tumor initiation.

The miR-200 family consists of five members expressed as two clusters: miR-200c/141 cluster and miR-200b/200a/429 cluster. In the mammary gland, miR-200s maintain epithelial identity by decreasing the expression of mesenchymal markers leading to high expression of epithelial markers. While the loss of miR-200s is associated with breast cancer growth and metastasis the impact of miR-200 expression on mammary tumor initiation has not been investigated. Using mammary specific expression of the miR-200b/200a/429 cluster in transgenic mice, we found that elevated expression miR-200s could almost completely prevent mammary tumor development. Only 1 of 16 MTB-IGFIRba429 transgenic mice (expressing both the IGF-IR and miR-200b/200a/429 transgenes) developed a mammary tumor while 100% of MTB-IGFIR transgenic mice (expressing only the IGF-IR transgene) developed mammary tumors. RNA sequencing, qRT-PCR, and immunohistochemistry of mammary tissue from 55-day old mice found Spp1, Saa1, and Saa2 to be elevated in mammary tumors and inhibited by miR-200b/200a/429 overexpression. This study suggests that miR-200s could be used as a preventative strategy to protect women from developing breast cancer. One concern with this approach is the potential negative impact miR-200 overexpression may have on mammary function. However, transgenic overexpression of miR-200s, on their own, did not significantly impact mammary ductal development indicating the miR-200 overexpression should not significantly impact mammary function. Thus, this study provides the initial foundation for using miR-200s for breast cancer prevention and additional studies should be performed to identify strategies for increasing mammary miR-200 expression and determine whether miR-200s can prevent mammary tumor initiation by other genetic alterations.

Isolated Soy Protein Promotes Mammary Tumor Development Induced by the Type I Insulin-like Growth Factor Receptor in Transgenic Mice.

Studies suggest consuming soy may protect women from breast cancer. In this study, lifetime exposure to 20%, 5% and 1% ISP in MTB-IGFIR mice (mammary-specific expression of IGF-IR) were evaluated to determine whether ISP could protect against mammary tumorigenesis. MTB-IGFIR mice fed ISP diets displayed increased mammary tumor incidence and reduced tumor latency compared to mice fed 20% casein. To evaluate whether a diet containing a less refined form of soy could protect against mammary tumor development MTB-IGFIR mice were fed Teklad 2018 (contains soybean meal). MTB-IGFIR mice fed the Teklad 2018 diet were completely protected against mammary tumor development. To determine whether dietary ISP was sufficient to induce mammary tumorigenesis, MTB-IGFIR mice were fed Teklad 2018ISP (soybean meal of Teklad 2018 was replaced with an equivalent amount of ISP). Only two of 10 MTB-IGFIR mice fed Teklad 2018ISP developed mammary tumors. This study demonstrates the complex interaction between soy and other dietary components in modifying mammary tumor development.

The miR-200b/200a/429 cluster prevents metastasis and induces dormancy in a murine claudin-low mammary tumor cell line.

The miR-200 family of microRNAs consisting of miR-141, miR-200a, miR-200b, miR-200c and miR-429 are emerging as important regulators of breast cancer progression. This family of microRNAs maintain mammary epithelial identity and downregulation of miR-200 expression has been associated with epithelial-to-mesenchymal transition in mammary tumors. Therefore, re-expression of one or more miR-200 family members in mammary tumor cells with mesenchymal characteristics may restore an epithelial phenotype including growth and metastasis suppression. To test this hypothesis, the miR-200b/200a/429 cluster was re-expressed in a murine claudin-low cell line, RJ423. Re-expression of the miR-200b/200a/429 cluster in RJ423 cells significantly suppressed the expression of Vim, Snai1, Twist1, Twist2 and Zeb1, reverted RJ423 cells to a more epithelial morphology and significantly inhibited proliferation in vitro. Moreover, the miR-200b/200a/429 cluster prevented lung metastasis in an experimental metastasis model and although tumor initiation was not prevented, re-expression of the miR-200b/200a/429 cluster induced a dormancy-like state where mammary tumors failed to grow beyond ~150 mm3 or grew extremely slowly following intra-mammary injection. These dormant tumors contained elevated levels of collagen and were highly vascularized. Therefore, re-expression of the miR-200b/200a/429 cluster in the claudin-low mammary tumor cell line, RJ423, is sufficient to alter cell morphology, impair metastasis and induce tumor dormancy.

Re-expression of miR-200c suppresses proliferation, colony formation and in vivo tumor growth of murine claudin-low mammary tumor cells.

Claudin-low breast cancer is a relatively rare breast cancer subtype. These cancers are typically ER-/PR-/HER2- and express high levels of mesenchymal genes as well as genes associated with inflammation, angiogenesis and stem cell function. In addition to alterations in gene expression, it was recently demonstrated that claudin-low breast cancers express very low levels of the miR-200 family of miRNAs. Given that each miRNA can regulate tens, hundreds or even thousands of genes, miRNAs are being evaluated as therapeutic targets. In this study we show that mammary tumors from MTB-IGFIR transgenic mice and cell lines derived from these tumors represent a model of human claudin-low breast cancer and murine claudin-low mammary tumors and cell lines express only very low levels of all five members of the miR-200 family. Reduced miR-200 family expression appears to be regulated via methylation as cells and tumors expressing low levels of miR-200 family members had higher levels of CpG methylation in a putative promoter region than tumors and cells expressing high levels of miR-200 family members. Re-expression of miR-200c in murine claudin-low mammary tumor cells inhibited tumor cell proliferation and colony formation in vitro and tumor growth in vivo. With respect to tumor growth in vivo, re-expression of miR-200c was associated with a reduction in tumor vasculature and expression of Flt1 and Vegfc. Therefore, miR-200c is an important regulator of mesenchymal tumor cell growth.

High levels of dietary soy decrease mammary tumor latency and increase incidence in MTB-IGFIR transgenic mice.

BACKGROUND: Epidemiologic data indicates that Asian diets, which are high in soy protein, reduce a women's risk of developing breast cancer. However, it has been difficult to dissociate the benefits of soy from other variables including environmental and lifestyle factors. Since prospective studies in humans would take decades to complete, rodent models provide a valuable research alternative. METHODS: In this study, MTB-IGFIR transgenic mice, which develop mammary tumors resulting from overexpression of the type I insulin-like growth factor receptor (IGF-IR), were utilized. MTB-IGFIR mice were fed a soy-based or casein-based diet throughout all stages of development to reflect soy exposure in Asian cultures. Mammary tumors were initiated at 2 different developmental stages by commencing IGF-IR transgene expression either during puberty or in adult mice. RESULTS: MTB-IGFIR mice fed a soy-based diet displayed increased tumor incidence and accelerated tumor onset compared to MTB-IGFIR mice fed a casein diet. Two markers of estrogen receptor signaling, Pgr and Areg, were elevated in mammary tissue from mice fed the soy diet compared to mice fed the casein diet suggesting that high levels of soy may promote mammary tumor development through acting as an estrogen receptor agonist. Mammary tumors from mice fed a soy diet more frequently expressed metaplastic markers such as cytokeratins 5 and 14 as well as p63 and displayed reduced lung metastases compared to mammary tumors from mice fed a casein diet. CONCLUSIONS: Diets consisting of very high levels of soy protein promote mammary tumor development and decrease tumor latency possibly through activating estrogen receptor signaling. Additional studies are required to determine whether a more moderate amount of dietary soy can inhibit oncogene-induced mammary tumorigenesis.

IGF-IR mediated mammary tumorigenesis is enhanced during pubertal development.

Although breast cancer typically develops in women over the age of 40, it remains unclear when breast cancer initiating events occur or whether the mammary gland is particularly susceptible to oncogenic transformation at a particular developmental stage. Using MTB-IGFIR transgenic mice that overexpress type I insulin-like growth factor receptor (IGF-IR) in a doxycycline-inducible manner, mammary tumorigenesis was initiated at different developmental stages. Tumor multiplicity was significantly increased while tumor latency was significantly decreased when the IGF-IR transgene was expressed during pubertal development compared to post-pubertal transgene expression. Moreover, metastatic spread of mammary tumors to the lungs was approximately twice as likely when IGF-IR was overexpressed in pubertal mice compared to post-pubertal mice. In addition, engraftment of pubertal MTB-IGFIR mammary tissue into cleared mammary fat pads of pubertal hosts produced tumors more frequently and faster than engraftment into adult hosts. These experiments show that the mammary microenvironment created during puberty renders mammary epithelial cells particularly susceptible to transformation.

Loss of Akt1 or Akt2 delays mammary tumor onset and suppresses tumor growth rate in MTB-IGFIR transgenic mice.

BACKGROUND: Akt is a serine/threonine kinase that mediates signaling downstream of tyrosine kinase receptors like the type I insulin-like growth factor receptor (IGF-IR). In fact, we have previously shown that mammary tumors induced by elevated expression of the IGF-IR are associated with hyperactivation of Akt. However, there are three mammalian isoforms of Akt (Akt1, Akt2 and Akt3) and these isoforms regulate distinct physiologic properties within cells. In this manuscript, the impact of disrupting Akt1 or Akt2 in mammary tumors induced by IGF-IR overexpression were examined to determine whether specific Akt isoforms regulate different aspects of mammary tumorigenesis. METHODS: Akt1 and Akt2 levels were stably ablated in mammary tumors of MTB-IGFIR transgenic mice by crossing MTB-IGFIR transgenic mice with either Akt1(-/-) or Akt2(-/-) mice. Tumor onset, growth rate, and metastasis were determined. RESULTS: Ablation of Akt1 or Akt2 significantly delayed tumor onset and tumor growth rate but did not significantly alter lung metastasis. Despite the absence of Akt1 or Akt2, mammary tumors that developed in the MTB-IGFIR mice maintained detectable levels of phosphorylated Akt. Disruption of Akt1 or Akt2 did not affect cell morphology or the expression of luminal or basal cytokeratins in mammary tumors. CONCLUSIONS: Although loss of Akt1 or Akt2 significantly inhibited mammary tumor onset and growth rates the effects were less dramatic than anticipated. Despite the complete loss of Akt1 or Akt2, the level of total phosphorylated Akt remained largely unaffected in the mammary tumors suggesting that loss of one Akt isoform is compensated by enhanced activation of the remaining Akt isoforms. These findings indicate that therapeutic strategies targeting the activation of individual Akt isoforms will prove less effective than simultaneously inhibiting the activity of all three Akt isoforms for the treatment of breast cancer.

Type I insulin-like growth factor receptor induces pulmonary tumorigenesis.

Despite the type I insulin-like growth factor receptor (IGF-IR) being highly expressed in more than 80% of human lung tumors, a transgenic model of IGF-IR overexpression in the lung has not been created. We produced two novel transgenic mouse models in which IGF-IR is overexpressed in either lung type II alveolar cells (surfactant protein C [SPC]-IGFIR) or Clara cells (CCSP-IGFIR) in a doxycycline-inducible manner. Overexpression of IGF-IR in either cell type caused multifocal adenomatous alveolar hyperplasia with papillary and solid adenomas. These tumors expressed thyroid transcription factor 1 and Kruppel-like factor 5 in most tumor cells. Similar to our previous work with lung tumors that developed in the mouse mammary tumor virus-IGF-II transgenic mice, the lung tumors that develop in the SPC-IGFIR and CCSP-IGFIR transgenic mice expressed high levels of the cyclic adenosine monophosphate response element binding protein that was localized primarily to the nucleus. Although elevated IGF-IR expression can initiate lung tumor development, tumors can become independent of IGF-IR signaling as IGF-IR down-regulation in established tumors produced tumor regression in some, but not all, of the tumors. These findings implicate IGF-IR as an important initiator of lung tumorigenesis and suggest that the SPC-IGFIR and CCSP-IGFIR transgenic mice can be used to further our understanding of human lung cancer and the role IGF-IR plays in this disease.

Predator detection in Lymnaea stagnalis.

Laboratory-reared Lymnaea are capable of detecting and responding to the scent of a crayfish predator. The present investigation is a first attempt to characterize multiple stress-related behavioural responses resulting from predator detection and to depict the neurophysiological correlates of one of these illustrated behaviours. Snails respond to crayfish effluent (CE) by increasing the following behaviours: aerial respiration, exploratory/searching phase and sensitivity to the shadow-elicited full-body withdrawal response. In contrast, when snails detect CE they decrease both their righting response time when dislodged from the substratum and their basal cutaneous oxygen consumption. Interestingly, basal heart rate does not change in response to CE exposure. Finally, we directly measured the activity of the neuron that initiates aerial respiratory behaviour, RPeD1, in semi-intact preparations. Naïve snails exposed to CE prior to recording demonstrated both a significantly reduced spontaneous firing rate and fewer bouts of bursting activity compared with non-exposed snails. These data show that laboratory-reared Lymnaea that have never experienced a natural predator are still capable of detecting and responding to the presence of a historically sympatric predator. These data open a new avenue of research, which may allow a direct investigation from the behavioural to the neuronal level as to how an ecologically relevant stressful stimulus alters behaviour.

Circulating hormones and estrous stage predict cellular and stromal remodeling in murine uterus.

The understanding of how estrogen and progesterone (P(4)) drive uterine remodeling in rodents has largely been based on studies involving administration of exogenous hormones, using steroid receptor-deficient mice, or relying on vaginal smears. In all these cases, the actual serum levels of 17beta-estradiol (E(2)) and P(4) are not directly measured, and the relationship between physiological levels of female sex hormones and uterine remodeling in cycling mice has not been fully explored. Here, we measured the circulating levels of E(2) and P(4) in cycling mice and performed correlation analysis between hormone levels and epithelial and stromal uterine parameters, irrespective of the estrous stage. In parallel, these parameters were analyzed in relation to the more conventional method of vaginal smear classification of estrous stage. We found that circulating P(4) inversely correlated with uterine width, luminal epithelial proliferation, stromal apoptosis, and degradation of luminal epithelial basement membrane collagen type-IV. Circulating E(2) positively correlated with uterine width, stromal cell proliferation, and collagen type-I content, while it negatively correlated with glandular epithelial proliferation, loss of collagen type-IV surrounding glandular epithelium, and apoptosis in luminal, glandular, and stromal compartments. Our findings indicate that measuring P(4) or E(2) levels can predict many concurrent cellular and stromal events in the mouse uterus, suggesting that in naturally cycling mice cellular responses to hormone changes are not delayed, but occur very rapidly.

Lamivudine resistance in patients with chronic hepatitis B: role of clinical and virological factors.

BACKGROUND: Lamivudine resistance is associated with long-term monotherapy for chronic hepatitis B and can lead to potentially serious clinical consequences. Scant information exists regarding the influence of hepatitis B virus variants in the development of resistance. The present study was designed to identify factors predictive of lamivudine resistance, with a particular focus on the role of precore and basal core promoter variants in the setting of hepatitis B e antigen-negative disease. METHODS: Eighty-five patients, representing four major genotypes, were followed prospectively on lamivudine therapy. Resistance was defined as an increase in viral load, with polymerase gene sequencing confirming a lamivudine resistance mutation. Median follow up was 19 months (6-54 months). The Cox proportional hazards model was used to determine variables independently predicting for the early onset of lamivudine resistance. RESULTS: The rate of lamivudine resistance was 6%, 31% and 51% at 12, 24 and 48 months, respectively. Multivariate analysis identified the precore variant, high baseline alanine aminotransferase (ALT), and persistent viremia (at 6 months) as independent predictors of the early development of lamivudine resistance, with rate ratios of 4.93 (95% confidence interval [CI]: 1.32-18.5), 1.22 (95%CI: 1.08-1.49), and 4.73 (95%CI: 1.49-15.0), respectively (P < 0.05). Female sex predicted early resistance (rate ratio 5.27 [95%CI: 1.23-22.5, P < 0.05]) although numbers were small (n = 12). Genotype did not influence treatment response nor time to onset of resistance. CONCLUSION: Patients with precore variant hepatitis B virus are likely to develop lamivudine resistance early and should be considered for alternate first-line monotherapy. In the future, combination antiviral therapy may limit the development of resistance.

Surgeon, test (and heal) thyself: sharps injuries and hepatitis C risk.

Sharps injuries experienced by surgeons are common, but are under-recognised and under-reported. The overall risks of transmission of blood-borne viruses to surgeons are low, with hepatitis C posing the greatest transmission risk. Recent trials show that early treatment of acute hepatitis C results in a cure rate approaching 100%. Surgeons and theatre staff should be encouraged to report and follow up sharps injuries to allow early detection and treatment. Additionally, because exposures to blood-borne viruses may be unrecognised, surgeons should have regular tests for blood-borne viruses. There should be no restriction of practice in the "window period" between potential exposure and obtaining results of testing, because of the overall low risk of transmission.