Parity-induced mammary epithelial cells are multipotent and express cell surface markers associated with stem cells

AbstractParity-induced mammary epithelial cells (PI-MECs) are defined as a pregnancy hormone-responsive cell population that activates the promoter of late milk protein genes during the second half of pregnancy and lactation. However, unlike their terminally differentiated counterparts, these cells do not undergo programmed cell death during post-lactational remodeling of the gland. We previously demonstrated that upon transplantation into an epithelial-free mammary fat pad, PI-MECs exhibited two important features of multipotent mammary epithelial progenitors: a) self-renewal, and b) contribution to ductal and alveolar morphogenesis. In this new report, we introduce a new method to viably label PI-MECs. Using this methodology, we analyzed the requirement of ovarian hormones for the maintenance of this epithelial subtype in the involuted mammary gland. Furthermore, we examined the expression of putative stem cell markers and found that a portion of GFP-labeled PI-MECs were part of the CD24+/CD49fhigh mammary epithelial subtype, which has recently been suggested to contain multipotent stem cells. Subsequently, we demonstrated that isolated PI-MECs were able to form mammospheres in culture, and upon transplantation, these purified epithelial cells were capable of establishing a fully functional mammary gland. These observations suggest that PI-MECs contain multipotent progenitors that are able to self renew and generate diverse epithelial lineages present in the murine mammary gland

A Mammary-Specific, Long-range Deletion on Mouse Chromosome 11 Accelerates Brca1-Associated Mammary Tumorigenesis12

We engineered a mammary-specific knockout model for Brca1 deficiency that also lacks the majority of one chromosome 11 to determine whether tumor susceptibility loci reside on this chromosome that cooperate with the loss of Brca1 during mammary cancer formation. Brca1-deficient females that are haploinsufficient in 60 cM of chromosome 11 exhibited accelerated mammary tumorigenesis in comparison to Brca1 conditional knockout mice. On the histopathologic level, these tumors were either adenocarcinomas or benign, inflammatory lesions. Like human BRCA1-associated breast cancers, mammary carcinomas in this new mouse model were ERα-negative and of basal epithelial origin. Brca1 deficiency and haploinsufficiency in 60 cM of chromosome 11 caused widespread genome instability as determined by spectral karyotyping analysis. In addition to the verification of the long-range deletion event, the spectral karyotyping analysis revealed that the duplication of the genome and higher degree of aneuploidy occur rather late in tumor progression. Despite chromosomal rearrangements near the Trp53 locus as determined by fluorescence in situ hybridization, the Trp53 gene was transcriptionally active. The analysis of the coding sequence and expression pattern of p53 and p21 suggests that loss-of-heterozygosity of Trp53 caused by somatic mutations contributes to accelerated mammary tumorigenesis in this model

Autocrine IGF1 Signaling Mediates Pancreatic Tumor Cell Dormancy in the Absence of Oncogenic Drivers

Summary: Mutant KRAS and c-MYC are oncogenic drivers and rational therapeutic targets for the treatment of pancreatic cancer. Although tumor growth and homeostasis are largely dependent on these oncogenes, a few residual cancer cells are able to survive the ablation of mutant KRAS and c-MYC. By performing a genome-wide gene expression analysis of in vivo-derived bulk tumor cells and residual cancer cells lacking the expression of mutant KRAS or c-MYC, we have identified an increase in autocrine IGF1/AKT signaling as a common survival mechanism in dormant cancer cells. The pharmacological inhibition of IGF-1R reduces residual disease burden and cancer recurrence, suggesting that this molecular pathway is crucial for the survival of cancer cells in the absence of the primary oncogenic drivers. : Rajbhandari et al. demonstrate that an increase in autocrine IGF1 signaling mediates the survival of residual pancreatic cancer cells following the ablation of oncogenic drivers (mutant KRAS and c-MYC). They provide experimental evidence that inhibiting IGF-1R can eradicate minimal residual disease and reduce cancer recurrence in vivo. Keywords: pancreatic cancer, oncogenes, KRAS, c-MYC, cancer dormancy, mouse models, genetic engineering, IGF1 signaling, AK

The Multifaceted Roles of the Tumor Susceptibility Gene 101 (TSG101) in Normal Development and Disease

The multidomain protein encoded by the Tumor Susceptibility Gene 101 (TSG101) is ubiquitously expressed and is suggested to function in diverse intracellular processes. In this review, we provide a succinct overview of the main structural features of the protein and their suggested roles in molecular and cellular functions. We then summarize, in more detail, key findings from studies using genetically engineered animal models that demonstrate essential functions of TSG101 in cell proliferation and survival, normal tissue homeostasis, and tumorigenesis. Despite studies on cell lines that provide insight into the molecular underpinnings by which TSG101 might function as a negative growth regulator, a biologically significant role of TSG101 as a tumor suppressor has yet to be confirmed using genuine in vivo cancer models. More recent observations from several cancer research teams suggest that TSG101 might function as an oncoprotein. A potential role of post-translational mechanisms that control the expression of the TSG101 protein in cancer is being discussed. In the final section of the review, we summarize critical issues that need to be addressed to gain a better understanding of biologically significant roles of TSG101 in cancer

Casitas B-cell lymphoma (Cbl) proteins protect mammary epithelial cells from proteotoxicity of active c-Src accumulation

Casitas B-cell lymphoma (Cbl) family ubiquitin ligases negatively regulate tyrosine kinase-dependent signal transduction by promoting degradation of active kinases. We and others previously reported that loss of Cbl functions caused hyperproliferation in lymphoid and hematopoietic systems. Unexpectedly, Cbl deletion in Cbl-b–null, Cbl-c–null primary mouse mammary epithelial cells (MECs) (Cbl triple-deficiency) induced rapid cell death despite enhanced MAP kinase and AKT activation. Acute Cbl triple-deficiency elicited distinct transcriptional and biochemical responses with partial overlap with previously described cellular reactions to unfolded proteins and oxidative stress. Although the levels of reactive oxygen species were comparable, detergent-insoluble protein aggregates containing phosphorylated c-Src accumulated in Cbl triple-deficient MECs. Treatment with a broad-spectrum kinase inhibitor dasatinib blocked protein aggregate accumulation and restored in vitro organoid formation. This effect is most likely mediated through c-Src because Cbl triple-deficient MECs were able to form organoids upon shRNA-mediated c-Src knockdown. Taking these data together, the present study demonstrates that Cbl family proteins are required to protect MECs from proteotoxic stress-induced cell death by promoting turnover of active c-Src

Stat5 Promotes Survival of Mammary Epithelial Cells through Transcriptional Activation of a Distinct Promoter in Akt1▿

The signal transducer and activator of transcription 5 (Stat5) plays a pivotal role in the proliferation, secretory differentiation, and survival of mammary epithelial cells. However, there is little information about Stat5 target genes that facilitate these biological processes. We provide here experimental evidence that the prolactin-mediated phosphorylation of Stat5 regulates the transcriptional activation of the Akt1 gene. Stat5 binds to consensus sequences within the Akt1 locus in a growth factor-dependent manner to initiate transcription of a unique Akt1 mRNA from a distinct promoter, which is only active in the mammary gland. Elevating the levels of active Akt1 restores the expression of cyclin D1 and proliferation of Jak2-deficient mammary epithelial cells, which provides evidence that Akt1 acts downstream of Jak/Stat signaling. The ligand-inducible expression of Stat5 in transgenic females mediates a sustained upregulation of Akt1 in mammary epithelial cells during the onset of postlactational involution. Stat5-expressing mammary glands exhibit a delay in involution despite induction of proapoptotic signaling events. Collectively, the results of the present study elucidate an underlying mechanism by which active Stat5 mediates evasion from apoptosis and self-sufficiency in growth signals

Janus Kinase 1 Plays a Critical Role in Mammary Cancer Progression

Summary: Janus kinases (JAKs) and their downstream STAT proteins play key roles in cytokine signaling, tissue homeostasis, and cancer development. Using a breast cancer model that conditionally lacks Janus kinase 1, we show here that JAK1 is essential for IL-6-class inflammatory cytokine signaling and plays a critical role in metastatic cancer progression. JAK1 is indispensable for the oncogenic activation of STAT1, STAT3, and STAT6 in ERBB2-expressing cancer cells, suggesting that ERBB2 receptor tyrosine kinase complexes do not directly activate these STAT proteins in vivo. A genome-wide gene expression analysis revealed that JAK1 signaling has pleiotropic effects on several pathways associated with cancer progression. We established that FOS and MAP3K8 are targets of JAK1/STAT3 signaling, which promotes tumorsphere formation and cell migration. The results highlight the significance of JAK1 as a rational therapeutic target to block IL-6-class cytokines, which are master regulators of cancer-associated inflammation. : Wehde et al. demonstrate that JAK1 is the pivotal kinase that controls cytokine-mediated activation of three STAT proteins in ERBB2-driven mammary cancer cells. They provide experimental evidence that deficiency in JAK1/STAT3 signaling and consequential downregulation of oncogenic targets inhibit migration, tumorsphere formation, and metastatic dissemination of mammary cancer cells. Keywords: breast cancer, mammary neoplasms, Janus kinase 1, Stat transcription factor, gene targeting, signal transduction, Cre recombinase, RNA sequencing, c-Fos, MAP3K

The majority of lung-derived hematopoietic cells expressing exogenous Stat5 that can be maintained for a prolonged period in culture are immature granulocytes.

<p>Flow cytometric analysis of various lineage markers on lung-derived hematopoietic cells that were maintained <i>ex vivo</i> together with pulmonary fibroblasts for 12 weeks.</p