Basement membrane (BM) supports and regulates the structural integrity, function and differentiation of epithelial tissues and protects against stromal invasion of breast cancer cells. Here we show that LAMA5, a central BM component, is critical for luminal mammary tumour initiation, and controls tumor progression and phenotype development. LAMA5 is overexpressed in both human and mouse luminal mammary tumours and LAMA5 downregulation attenuates growth of human breast cancer cells. Prepubertal luminal deletion of Lama5 in MMTV-PyMT mice results in marked reduction in emergence of early hyperplasias, along with a shift from mature hormone receptor (HR+) positive luminal epithelial phenotype towards a HR+ progenitor phenotype, and widespread alterations in extracellular matrix and Fibroblast growth factor (Fgf) signalling genes, including overexpression of Fgf receptor 2 (Fgfr2) in the HR+ progenitor cells. Single allele deletion, but not biallelic deletion of Lama5 additionally inhibits growth and progression towards advanced mammary carcinomas. However, inhibition of Fgf receptors restores growth decrease and induces apoptosis in biallelic-Lama5-deleted organoids without affecting wildtype organoids. Our analyses demonstrate a critical role for a BM component LAMA5 in mammary tumour initiation and reveal mechanisms of extracellular matrix-epithelial interplay in breast tumour progression and phenotype maintenance. ### Competing Interest Statement The authors have declared no competing interest.

Epithelial attachment to the basement membrane (BM) is essential for mammary gland development, yet the exact roles of specific BM components remain unclear. Here, we show that Laminin α5 (Lama5) expression specifically in the luminal epithelial cells is necessary for normal mammary gland growth during puberty, and for alveologenesis during pregnancy. Lama5 loss in the keratin 8-expressing cells results in reduced frequency and differentiation of hormone receptor expressing (HR+) luminal cells. Consequently, Wnt4-mediated crosstalk between HR+ luminal cells and basal epithelial cells is compromised during gland remodeling, and results in defective epithelial growth. The effects of Lama5 deletion on gland growth and branching can be rescued by Wnt4 supplementation in the in vitro model of branching morphogenesis. Our results reveal a surprising role for BM-protein expression in the luminal mammary epithelial cells, and highlight the function of Lama5 in mammary gland remodeling and luminal differentiation.

Epithelial attachment to the basement membrane (BM) is essential for mammary gland development, yet the exact roles of specific BM components remain unclear. Here, we demonstrate that expression of distinct laminin α-isoforms by luminal and basal mammary epithelial cells enforces lineage identity that is necessary for normal mammary gland growth and function. Laminin α5 (LMα5) is mainly expressed by the luminal epithelial cells, and it is necessary for pubertal mammary gland growth, pregnancy induced gland remodeling, and for alveolar function. Adhesion to LMα5 containing laminin promotes luminal traits in both luminal and basal epithelial cells, and reduces progenitor activity of basal epithelial cells. Mechanistically, we show that loss interferes with differentiation of hormone receptor positive luminal cells, which results in reduced Wnt4 expression and defective crosstalk between luminal and basal epithelial cells during gland remodeling. Our results reveal a novel BM-mediated mechanism, which regulates mammary gland remodeling and function via specification of luminal epithelial cells.

Genetically-engineered mouse models (GEMMs) lacking diseased-associated gene(s) globally or in a tissue-specific manner represent an attractive tool with which to assess the efficacy and toxicity of targeted pharmacological inhibitors. Stat3 and Stat5a/b transcription factors have been implicated in several pathophysiological conditions, and pharmacological inhibition of both transcription factors has been proposed to treat certain diseases, such as malignancies. To model combined inhibition of Stat3 and Stat5a/b we have developed a GEMM harboring a flox Stat3-Stat5a/b allele (Stat5/3loxP/loxP mice) and generated mice lacking Stat3 and Stat5a/b in hepatocytes (Stat5/3Δhep/Δhep). Stat5/3Δhep/Δhep mice exhibited a marked reduction of STAT3, STAT5A and STAT5B proteins in the liver and developed steatosis, a phenotype that resembles mice lacking Stat5a/b in hepatocytes. In addition, embryonic deletion of Stat3 and Stat5a/b (Stat5/3Δ/Δ mice) resulted in lethality, similar to Stat3Δ/Δ mice. This data illustrates that Stat5/3loxP/loxP mice are functional and can be used as a valuable tool to model the combined inhibition of Stat3 and Stat5a/b in tumorigenesis and other diseases.

The gene desert upstream of the MYC oncogene on chromosome 8q24 contains susceptibility loci for several major forms of human cancer. The region shows high conservation between human and mouse and contains multiple MYC enhancers that are activated in tumor cells. However, the role of this region in normal development has not been addressed. Here we show that a 538 kb deletion of the entire MYC upstream super-enhancer region in mice results in 50% to 80% decrease in Myc expression in multiple tissues. The mice are viable and show no overt phenotype. However, they are resistant to tumorigenesis, and most normal cells isolated from them grow slowly in culture. These results reveal that only cells whose MYC activity is increased by serum or oncogenic driver mutations depend on the 8q24 super-enhancer region, and indicate that targeting the activity of this element is a promising strategy of cancer chemoprevention and therapy.

Nature Communications 7:12321 doi: (2016); Published 5 Aug 2016; Updated 13 Sep 2016 In Fig. 6f of this Article, the labelling of the two immunohistochemistry images was inadvertently changed from ‘Haematoxylin, ki67’ to ‘Haematoxylin, Casp-3’ during the production process. The correct version of Fig.

The mammary gland is composed of a complex cellular hierarchy with unusual postnatal plasticity. The identities of stem/progenitor cell populations, as well as tumour-initiating cells that give rise to breast cancer, are incompletely understood. Here we show that Lgr6 marks rare populations of cells in both basal and luminal mammary gland compartments in mice. Lineage tracing analysis showed that Lgr6 + cells are unipotent progenitors, which expand clonally during puberty but diminish in adulthood. In pregnancy or following stimulation with ovarian hormones, adult Lgr6 + cells regained proliferative potency and their progeny formed alveoli over repeated pregnancies. Oncogenic mutations in Lgr6 + cells resulted in expansion of luminal cells, culminating in mammary gland tumours. Conversely, depletion of Lgr6 + cells in the MMTV-PyMT model of mammary tumorigenesis significantly impaired tumour growth. Thus, Lgr6 marks mammary gland progenitor cells that can initiate tumours, and cells of luminal breast tumours required for efficient tumour maintenance.

Growth hormone (GH) resistance has been associated with liver cirrhosis in humans but its contribution to the disease remains controversial. In order to elucidate whether GH resistance plays a causal role in the establishment and development of liver fibrosis, or rather represents a major consequence thereof, we challenged mice lacking the GH receptor gene (Ghr –/– , a model for GH resistance) by crossing them with Mdr2 knockout mice (Mdr2 –/– ), a mouse model of inflammatory cholestasis and liver fibrosis. Ghr –/– ;Mdr2 –/– mice showed elevated serum markers associated with liver damage and cholestasis, extensive bile duct proliferation, and increased collagen deposition relative to Mdr2 –/– mice, thus suggesting a more severe liver fibrosis phenotype. Additionally, Ghr –/– ;Mdr2 –/– mice had a pronounced down‐regulation of hepatoprotective genes Hnf6 , Egfr , and Igf‐1 , and significantly increased levels of reactive oxygen species (ROS) and apoptosis in hepatocytes, compared to control mice. Moreover, single knockout mice (Ghr –/– ) fed with a diet containing 1% cholic acid displayed an increase in hepatocyte ROS production, hepatocyte apoptosis, and bile infarcts compared to their wild‐type littermates, indicating that loss of Ghr renders hepatocytes more susceptible to toxic bile acid accumulation. Surprisingly, and despite their severe fibrotic phenotype, Ghr –/– ;Mdr2 –/– mice displayed a significant decrease in tumor incidence compared to Mdr2 –/– mice, indicating that loss of Ghr signaling may slow the progression from fibrosis/cirrhosis to cancer in the liver. Conclusion : GH resistance dramatically exacerbates liver fibrosis in a mouse model of inflammatory cholestasis, therefore suggesting that GH resistance plays a causal role in the disease and provides a novel target for the development of liver fibrosis treatments. (H epatology 2015;61:613‐626)

STAT3 is considered to play an oncogenic role in several malignancies including lung cancer; consequently, targeting STAT3 is currently proposed as therapeutic intervention. Here we demonstrate that STAT3 plays an unexpected tumour-suppressive role in KRAS mutant lung adenocarcinoma (AC). Indeed, lung tissue-specific inactivation of Stat3 in mice results in increased Kras G12D -driven AC initiation and malignant progression leading to markedly reduced survival. Knockdown of STAT3 in xenografted human AC cells increases tumour growth. Clinically, low STAT3 expression levels correlate with poor survival and advanced malignancy in human lung AC patients with smoking history, which are prone to KRAS mutations. Consistently, KRAS mutant lung tumours exhibit reduced STAT3 levels. Mechanistically, we demonstrate that STAT3 controls NF-κB-induced IL-8 expression by sequestering NF-κB within the cytoplasm, thereby inhibiting IL-8-mediated myeloid tumour infiltration and tumour vascularization and hence tumour progression. These results elucidate a novel STAT3–NF-κB–IL-8 axis in KRAS mutant AC with therapeutic and prognostic relevance.

We here establish a mouse cancer model called Multi-Hit that allows for the evaluation of oncogene cooperativities in tumor development. The model is based on the stochastic expression of oncogene combinations ('hits') that are mediated by Cre in a given tissue. Cells with cooperating hits are positively selected and give rise to tumors. We used this approach to evaluate the requirement of Ras downstream effector pathways in tumorigenesis.

Chronic liver injury, liver fibrosis and formation of hepatocellular carcinoma are intimately linked and represent a major medical challenge since treatment options are limited. Therefore, it is important to identify cellular and molecular pathways that promote liver damage or provide hepatoprotection for development of therapeutic approaches. Recently, the transcription factors STAT3 and STAT5 have been implicated in liver fibrosis induced by cholestatic liver damage. In this review, we summarize our current knowledge about STAT proteins in liver fibrosis and focus on common activities that underlie the hepatoprotective mechanisms regulated by IL-6/gp130/STAT3 and GH/STAT5/IGF-1 signaling pathways.

Growth hormone (GH)‐activated signal transducer and activator of transcription 5 (STAT5) and the glucocorticoid (GC)‐responsive glucocorticoid receptor (GR) are important signal integrators in the liver during metabolic and physiologic stress. Their deregulation has been implicated in the development of metabolic liver diseases, such as steatosis and progression to fibrosis. Using liver‐specific STAT5 and GR knockout mice, we addressed their role in metabolism and liver cancer onset. STAT5 single and STAT5/GR double mutants developed steatosis, but only double‐mutant mice progressed to liver cancer. Mechanistically, STAT5 deficiency led to the up‐regulation of prolipogenic sterol regulatory element binding protein 1 (SREBP‐1) and peroxisome proliferator activated receptor gamma (PPAR‐γ) signaling. Combined loss of STAT5/GR resulted in GH resistance and hypercortisolism. The combination of both induced expression of adipose tissue lipases, adipose tissue lipid mobilization, and lipid flux to the liver, thereby aggravating STAT5‐dependent steatosis. The metabolic dysfunctions in STAT5/GR compound knockout animals led to the development of hepatic dysplasia at 9 months of age. At 12 months, 35% of STAT5/GR‐deficient livers harbored dysplastic nodules and ∼60% hepatocellular carcinomas (HCCs). HCC development was associated with GH and insulin resistance, enhanced tumor necrosis factor alpha (TNF‐α) expression, high reactive oxygen species levels, and augmented liver and DNA damage parameters. Moreover, activation of the c‐Jun N‐terminal kinase 1 (JNK1) and STAT3 was prominent. Conclusion: Hepatic STAT5/GR signaling is crucial for the maintenance of systemic lipid homeostasis. Impairment of both signaling cascades causes severe metabolic liver disease and promotes spontaneous hepatic tumorigenesis. (HEPATOLOGY 2011;54:1398–1409)

Growth hormone (GH) resistance and low serum levels of insulinlike growth factor 1 (IGF‐1) are common features in human liver fibrosis and cirrhosis. Signal transducer and activator of transcription 5 (STAT5) controls several vital functions in the liver, including GH‐mediated transcription of IGF‐1. To investigate the role of STAT5 in liver fibrogenesis, we specifically deleted the Stat5a/b locus both in hepatocytes and cholangiocytes in the multidrug resistance gene 2 knockout (Mdr2−/−) mouse model of cholestasis. Double knockout mice develop an early and severe liver fibrosis phenotype, accompanied by perturbed expression of key regulators of bile acid homeostasis. Deletion of Stat5 resulted in GH resistance, and IGF‐1 levels in serum were undetectable. We could observe reduced expression of important hepatoprotective genes, such as epidermal growth factor receptor (Egfr), hepatocyte nuclear factor 6 (Hnf6), prolactin receptor (Prlr), and leukemia inhibitory factor receptor (Lifr) as well as increased numbers of apoptotic hepatocytes. Conclusion: Our data suggest that loss of STAT5 sensitizes hepatocytes to bile acid–induced damage and apoptosis caused by disruption of GH‐induced transcription of Igf‐1 and down‐regulation of hepatoprotective genes. These findings could contribute to the understanding of liver fibrosis and future treatment strategies for liver fibrosis. (HEPATOLOGY 2010.)

Here we describe the generation of an inducible Cre transgenic line allowing conditional mutagenesis in ovarian granulosa cells. We have expressed the tamoxifen inducible CreER(T)² fusion protein from a Bacterial Artificial Chromosome (BAC) containing the regulatory elements of the hydroxysteroid (17-beta) dehydrogenase 1 (Hsd17b1) gene. Hsd17b1-iCreER(T)² transgenic mice express the iCreER(T)² fusion protein exclusively in ovarian granulosa cells. Recombination analysis at the genomic DNA level using mice with "floxed" Stat3 alleles showed no Cre activity in absence of tamoxifen whereas tamoxifen treatment induced Cre activity solely in the ovaries. Further characterization of Hsd17b1-iCreER(T)² mice using a Cre reporter line demonstrated that Cre-mediated recombination was restricted to ovarian granulosa cells. Therefore, Hsd17b1-iCreER(T)² mice should be a useful tool to analyze the gene functions in ovarian granulosa cells.

BACKGROUND AND AIMSThe transcription factor signal transducer and activator of transcription 3 (Stat3) has been considered to promote progression and metastasis of intestinal cancers.METHODSWe investigated the role of Stat3 in intestinal tumors using mice with conditional ablation of Stat3 in intestinal epithelial cells (Stat3(DeltaIEC)).RESULTSIn the Apc(Min) mouse model of intestinal cancer, genetic ablation of Stat3 reduced the multiplicity of early adenomas. However, loss of Stat3 promoted tumor progression at later stages, leading to formation of invasive carcinomas, which significantly shortened the lifespan of Stat3(DeltaIEC)Apc(Min/+) mice. Interestingly, loss of Stat3 in tumors of Apc(Min/+) mice had no significant impact on cell survival and angiogenesis, but promoted cell proliferation. A genome-wide expression analysis of Stat3-deficient tumors suggested that Stat3 might negatively regulate intestinal cancer progression via the cell adhesion molecule CEACAM1.CONCLUSIONSOur data suggest that Stat3 impairs invasiveness of intestinal tumors. Therefore, therapeutic targeting of the Stat3 signaling pathway in intestinal cancer should be evaluated for adverse effects on tumor progression.

BACKGROUND & AIMS:Signal transducer and activator of transcription 3 (Stat3) is the main mediator of interleukin-6-type cytokine signaling required for hepatocyte proliferation and hepatoprotection, but its role in sclerosing cholangitis and other cholestatic liver diseases remains unresolved.METHODS:We investigated the role of Stat3 in inflammation-induced cholestatic liver injury and used mice lacking the multidrug resistance gene 2 (mdr2(-/-)) as a model for SC.RESULTS:We show that conditional inactivation of Stat3 in hepatocytes and cholangiocytes (stat3(Deltahc)) of mdr2(-/-) mice strongly aggravated bile acid-induced liver injury and fibrosis. A similar phenotype was observed in mdr2(-/-) mice lacking interleukin-6 production. Biochemical and molecular characterization suggested that Stat3 exerts hepatoprotective functions in both hepatocytes and cholangiocytes. Loss of Stat3 led to increased expression of tumor necrosis factor alpha, which might reduce the barrier function of bile ducts. Moreover, Stat3-deficient hepatocytes displayed up-regulation of bile acid biosynthesis genes and down-regulation of hepatoprotective epidermal growth factor receptor and insulin-like growth factor 1 signaling pathways. Consistently, stat3(Deltahc) mice were more sensitive to cholic acid-induced liver damage than control mice.CONCLUSIONS:Our data suggest that Stat3 prevents cholestasis and liver damage in sclerosing cholangitis via regulation of pivotal functions in hepatocytes and cholangiocytes.