Robert B. Boxer

c-MYC induces mammary tumorigenesis by means of a preferred pathway involving spontaneous Kras2 mutations

Although the process of mammary tumorigenesis requires multiple genetic events, it is unclear to what extent carcinogenesis proceeds through preferred secondary pathways following a specific initiating oncogenic event. Similarly, the extent to which established mammary tumors remain dependent on individual mutations for maintenance of the transformed state is unknown. Here we use the tetracycline regulatory system to conditionally express the human c-MYC oncogene in the mammary epithelium of transgenic mice. MYC encodes a transcription factor implicated in multiple human cancers. In particular, amplification and overexpression of c-MYC in human breast cancers is associated with poor prognosis, although the genetic mechanisms by which c-MYC promotes tumor progression are poorly understood. We show that deregulated c-MYC expression in this inducible system results in the formation of invasive mammary adenocarcinomas, many of which fully regress following c-MYC deinduction. Approximately half of these tumors harbor spontaneous activating point mutations in the ras family of proto-oncogenes with a strong preference for Kras2 compared with Hras1. Nearly all tumors lacking activating ras mutations fully regressed following c-MYC deinduction, whereas tumors bearing ras mutations did not, suggesting that secondary mutations in ras contribute to tumor progression. These findings demonstrate that c-MYC-induced mammary tumorigenesis proceeds through a preferred secondary oncogenic pathway involving Kras2.

Dose-dependent oncogene-induced senescence in vivo and its evasion during mammary tumorigenesis

Activating Ras mutations can induce either proliferation or senescence depending on the cellular context. To determine whether Ras activation has context-dependent effects in the mammary gland, we generated doxycycline-inducible transgenic mice that permit Ras activation to be titrated. Low levels of Ras activation — similar to those found in non-transformed mouse tissues expressing endogenous oncogenic Kras2 — stimulate cellular proliferation and mammary epithelial hyperplasias. In contrast, high levels of Ras activation — similar to those found in tumours bearing endogenous Kras2 mutations — induce cellular senescence that is Ink4a–Arf- dependent and irreversible following Ras downregulation. Chronic low-level Ras induction results in tumour formation, but only after the spontaneous upregulation of activated Ras and evasion of senescence checkpoints. Thus, high-level, but not low-level, Ras activation activates tumour suppressor pathways and triggers an irreversible senescent growth arrest in vivo. We suggest a three-stage model for Ras-induced tumorigenesis consisting of an initial activating Ras mutation, overexpression of the activated Ras allele and, finally, evasion of p53–Ink4a–Arf-dependent senescence checkpoints.

A novel doxycycline-inducible system for the transgenic analysis of mammary gland biology

Normal developmental events such as puberty, pregnancy, and parity influence the susceptibility of the mammary gland to tumorigenesis in both humans and rodent model systems. Unfortunately, constitutive transgenic mouse models that rely on mammary‐specific promoters to control transgene expression have limited utility for studying the effect of developmental events on breast cancer risk since the hormonal signals governing these events also markedly influence transgene expression levels. A novel transgenic mouse system is described that uses the MMTV‐LTR to drive expression of the reverse tetracycline‐dependent transactivator rtTA. Transgenic mice expressing rtTA in the mammary epithelium were crossed with reporter lines bearing tet operator‐controlled transgenes. We tested the ability to spatially, temporally, and quantitatively control reporter gene expression after administration of doxycycline to bitransgenic mice. Transgene expression using this system can be rapidly induced and deinduced, is highly mammary specific, can be reproducibly titrated over a wide range of expression levels, and is essentially undetectable in the uninduced state. Homogeneous transgene expression throughout the mammary epithelium can be achieved. This system permits transgene expression to be restricted to any desired stage of postnatal mammary gland development. We have developed a mammary‐specific, doxycycline‐inducible transgenic mouse model for studying the effect of mammary gland development on transgene‐mediated phenotypes. Unlike other mammary‐specific, transgenic systems that have been described, this system combines spatially homogeneous transgene expression in the mammary epithelium during puberty, pregnancy, lactation, and involution with the use of an orally administered, inexpensive, and widely available inducing agent. This system offers new opportunities for the transgenic analysis of mammary gland biology in vivo.—Gunther, E. J., Belka, G. K., Wertheim, G. B. W., Wang, J., Hartman, J. L., Boxer, R. B., Chodosh, L. A. A novel doxycycline‐inducible system for the transgenic analysis of mammary gland biology. FASEB J. 16, 283–292 (2002)

Autocrine prolactin induced by the Pten–Akt pathway is required for lactation initiation and provides a direct link between the Akt and Stat5 pathways

Extrapituitary prolactin (Prl) is produced in humans and rodents; however, little is known about its in vivo regulation or physiological function. We now report that autocrine prolactin is required for terminal mammary epithelial differentiation during pregnancy and that its production is regulated by the Pten-PI3K-Akt pathway. Conditional activation of the PI3K-Akt pathway in the mammary glands of virgin mice by either Akt1 expression or Pten deletion rapidly induced terminal mammary epithelial differentiation accompanied by the synthesis of milk despite the absence of lobuloalveolar development. Surprisingly, we found that mammary differentiation was due to the PI3K-Akt-dependent synthesis and secretion of autocrine prolactin and downstream activation of the prolactin receptor (Prlr)-Jak-Stat5 pathway. Consistent with this, Akt-induced mammary differentiation was abrogated in Prl(-/-), Prlr(-/-), and Stat5(-/-) mice. Furthermore, cells treated with conditioned medium from mammary glands in which Akt had been activated underwent rapid Stat5 phosphorylation in a manner that was blocked by inhibition of Jak2, treatment with an anti-Prl antibody, or deletion of the prolactin gene. Demonstrating a physiological requirement for autocrine prolactin, mammary glands from lactation-defective Akt1(-/-);Akt2(+/-) mice failed to express autocrine prolactin or activate Stat5 during late pregnancy despite normal levels of circulating serum prolactin and pituitary prolactin production. Our findings reveal that PI3K-Akt pathway activation is necessary and sufficient to induce autocrine prolactin production in the mammary gland, Stat5 activation, and terminal mammary epithelial differentiation, even in the absence of the normal developmental program that prepares the mammary gland for lactation. Together, these findings identify a function for autocrine prolactin during normal development and demonstrate its endogenous regulation by the PI3K-Akt pathway.

Akt is required for Stat5 activation and mammary differentiation.

IntroductionThe Akt pathway plays a central role in regulating cell survival, proliferation and metabolism, and is one of the most commonly activated pathways in human cancer. A role for Akt in epithelial differentiation, however, has not been established. We previously reported that mice lacking Akt1, but not Akt2, exhibit a pronounced metabolic defect during late pregnancy and lactation that results from a failure to upregulate Glut1 as well as several lipid synthetic enzymes. Despite this metabolic defect, however, both Akt1-deficient and Akt2-deficient mice exhibit normal mammary epithelial differentiation and Stat5 activation.MethodsIn light of the overlapping functions of Akt family members, we considered the possibility that Akt may play an essential role in regulating mammary epithelial development that is not evident in Akt1-deficient mice due to compensation by other Akt isoforms. To address this possibility, we interbred mice bearing targeted deletions in Akt1 and Akt2 and determined the effect on mammary differentiation during pregnancy and lactation.ResultsDeletion of one allele of Akt2 in Akt1-deficient mice resulted in a severe defect in Stat5 activation during late pregnancy that was accompanied by a global failure of terminal mammary epithelial cell differentiation, as manifested by the near-complete loss in production of the three principal components of milk: lactose, lipid, and milk proteins. This defect was due, in part, to a failure of pregnant Akt1-/- ;Akt2+/- mice to upregulate the positive regulator of Prlr-Jak-Stat5 signaling, Id2, or to downregulate the negative regulators of Prlr-Jak-Stat5 signaling, caveolin-1 and Socs2.ConclusionsOur findings demonstrate an unexpected requirement for Akt in Prlr-Jak-Stat5 signaling and establish Akt as an essential central regulator of mammary epithelial differentiation and lactation.

Therapeutic efficacy of chloroquine and sequence variation in pfcrt gene among patients with falciparum malaria in central India.

Objectives  To assess the therapeutic efficacy of chloroquine (CQ) treatment against uncomplicated Plasmodium falciparum infections in a tribal population of central India (Madhya Pradesh) and to investigate the prevalence of mutant P. falciparum chloroquine‐resistant transporter (pfcrt) gene in the parasite population.

All together now: Impact of a regionalization and bedside rounding initiative on the efficiency and inclusiveness of clinical rounds

INTERVENTIONS: General medical teams were regionalized to specific units, the admitting structure was changed to facilitate regionalization, and teams were encouraged to round bedside. MEASUREMENTS: Primary outcomes included proportion of time each team member was present on rounds and proportion of bedside rounding time. Secondary outcomes included round duration and non-patient time during rounds.

Impact of regionalized care on concordance of plan and preventable adverse events on general medicine services

BACKGROUND Dispersion of inpatient care teams across different medical units impedes effective team communication, potentially leading to adverse events (AEs). OBJECTIVE To regionalize 3 inpatient general medical teams to nursing units and examine the association with communication and preventable AEs. DESIGN Pre-post cohort analysis. SETTING A 700-bed academic medical center. PATIENTS General medicine patients on any of the participating nursing units before and after implementation of regionalized care. INTERVENTION Regionalizing 3 general medical physician teams to 3 corresponding nursing units. MEASUREMENTS Concordance of patient care plan between nurse and intern, and adjusted odds of preventable AEs. RESULTS Of the 414 included nurse and intern paired surveys, there were no significant differences pre- versus postregionalization in total mean concordance scores (0.65 vs 0.67, P = 0.26), but there was significant improvement in agreement on expected discharge date (0.56 vs 0.68, P = 0.003), knowledge of the other providers name (0.56 vs 0.86,P < 0.001), and daily care plan discussions (0.73 vs 0.88, P < 0.001). Of the 392 reviewed patient medical records, there was no significant difference in the adjusted odds of preventable AEs pre- versus postregionalization (adjusted odds ratio: 1.37, 95% confidence interval: 0.69, 2.69). CONCLUSIONS We found that regionalization of care teams improved recognition of care team members, discussion of daily care plan, and agreement on estimated discharge date, but did not significantly improve nurse and physician concordance of the care plan or reduce the odds of preventable AEs. Our findings suggest that regionalization alone may be insufficient to effectively promote communication and lead to patient safety improvements. Journal of Hospital Medicine 2016;11:620-627.

User-Centered Collaborative Design and Development of an Inpatient Safety Dashboard

Patient safety remains a key concern in hospital care. This article summarizes the iterative participatory development, features, functions, and preliminary evaluation of a patient safety dashboard for interdisciplinary rounding teams on inpatient medical services. This electronic health record (EHR)-embedded dashboard collects real-time data covering 13 safety domains through web services and applies logic to generate stratified alerts with an interactive check-box function. The technological infrastructure is adaptable to other EHR environments. Surveyed users perceived the tool as highly usable and useful. Integration of the dashboard into clinical care is intended to promote communication about patient safety and facilitate identification and management of safety concerns.