Cell-cycle exit and differentiation of suprabasal epidermal keratinocytes require nuclear IB kinase ␣ (IKK␣), but not its protein kinase activity. IKK␣ also is a suppressor of squamous cell carcinoma (SCC), but its mode of action remains elusive. Postulating that IKK␣ may serve as a transcriptional regulator in keratinocytes, we searched for cell-cycle-related genes that could illuminate this function. IKK␣ was found to control several Myc antagonists, including Mad1, Mad2, and Ovol1, through the association with TGF␤-regulated Smad2/3 transcription factors and is required for Smad3 recruitment to at least one of these targets. Surprisingly, Smad2/3-dependent Mad1 induction and keratinocyte differentiation are independent of Smad4, the almost universal coregulator of canonical TGF␤ signaling. IKK␣ also is needed for nuclear accumulation of activated Smad2/3 in the epidermis, and Smad2/3 are required for epidermal differentiation. We suggest that a TGF␤-Smad2/3-IKK␣ axis is a critical Smad4-independent regulator of keratinocyte proliferation and differentiation.epidermis ͉ cornification ͉ terminal differentiation A critical mediator of NF-B activation (1), IB kinase (IKK) consists of two catalytic subunits, IKK␣ and IKK␤ (2-5), and a regulatory subunit, IKK␥/NEMO (6, 7). Despite structural similarity, IKK␣ and IKK␤ have nonredundant functions, with IKK␤ being the predominant IKK (1, 8) and IKK␣ being a critical regulator of keratinocyte differentiation (9, 10). Without IKK␣, epidermal keratinocytes exhibit enhanced proliferation and failure to differentiate. Consequently, Ikk␣ Ϫ/Ϫ mice are born enshrouded in a taut and thickened, nonstratified, epidermal sheet devoid of barrier function.The mammalian epidermis is a stratified squamous epithelium in which basal keratinocytes undergo asymmetric cell divisions, giving rise to nonproliferative progeny that embark on a differentiation program as they delaminate and move upward through the spinous and granular layers before generating the cornified layer, which provides the crucial barrier function (11, 12). Without IKK␣, this process is blocked, and basal keratinocytes fail to exit the cell cycle (9, 10, 13). Isolated Ikk␣ Ϫ/Ϫ keratinocytes proliferate uncontrollably and do not respond to differentiation-inducing signals such as high Ca 2ϩ (9, 13). The reexpression of IKK␣ in Ikk␣ Ϫ/Ϫ keratinocytes induces growth arrest and allows terminal differentiation, but this function depends neither on IKK␣'s protein kinase activity nor on NF-B. Instead, it requires nuclear accumulation of IKK␣ (14).Recently, IKK␣ was identified as a tumor suppressor in squamous cell carcinoma (SCC), a type of cancer derived from squamous epithelia of the skin, oral and nasal cavities, esophagus, and other sites (15). Decreased nuclear IKK␣ expression was found in about one third of oral SCCs, mainly those that exhibit poorly differentiated phenotype and poor prognosis (16). These results strongly suggest that loss of nuclear IKK␣ contributes to malignant conversion of keratinocytes to less dif...

qRT-PCR. Whole-tumor RNA was harvested with an RNeasy kit (QIAGEN), and cDNA was synthesized (High Capacity; Applied Biosystems) and amplified using the murine cDNA-specific primers (Integrated DNA Technologies) listed in Supplemental Methods, along with SYBR Green Supermix (Bio-Rad). The following primers were used: MRC1 (forward: 5′ CCCTCAGCAAGCGATGTGC 3′; reverse: 5′-GGATACTTGCCAGGT CCCCA-3′); iNOS (forward: 5′-GGAGCATCCCAAGTACGAGTGG-3′; reverse: 5′-CGGCC-CACTTCCTCCAG); IL10 (forward: 5′-GGCGCTGTCATC-GATTTCTCC; reverse: 5′-GGCCTTGTAGACACCTTGGTC); Tgfb1 (forward: 5′-CGCAACAACGCCATCTATGAG; reverse: 5′-CGG-GACAGCAATGGGGGTTC); IL4 (forward: 5′-GGTCACAGGAGAAGG-GACG; reverse: 5′-GCGAAGCACCTTGGAAGCC);, IL12b (forward: 5′-GGAGTGGGATGTGTCCTCAG; reverse: 5′-CGGGAGTCCAGTC-CACCTCT); CCL3 (forward: 5′-CCACTGCCCTTGCTGTTCTTCTCT; reverse: 5′-GGGTGTCAGCTCCATATGGCG); and Rplp0 (forward: 5′-TCCTATAAAAGGCACACGCGGGC; reverse: 5′-AGACGATGT-CACTCCAACGAGGACG). Target To generate apoptotic MCF7, cells were treated in suspension with 1 μm BKM120 plus 2 μm ABT-263 (both inhibitors from Selleck Chemicals) for 4 hours, washed 5 times with PBS to remove residual drug, and used directly for efferocytosis assays or for annexin V staining. For efferocytosis coculture assays, Raw264.7-GFP cells (10 4 /well) and PyVmT or MCF7 cells (72 hours after infection with Ad.mCherry and Ad.HS-V-TK) were seeded together in a monolayer in 24-well plates in 2% FBS and cultured for 24 hours prior to the addition PBS or gancyclovir. Cells were imaged at 8, 16, and 32 h after addition of gancyclovir. Cells were collected and counted under fluorescence after 32 hours of coculture. In some experiments, Raw264.7-GFP cells (10 4 / well) were seeded in a monolayer in 24-well plates and cultured for 24 hours prior to the addition of 10 3 live MCF7-mCherry or 10 3 dead MCF7-mCherry cells in serum-free media. Where indicated in the figures, BMS-777607 (1 μm) or a neutralizing goat anti-mouse MerTK antibody (AF591, 25 μg/ml; R&D Systems)(44) was added 2 hours prior to the addition of gancyclovir or 2 hours prior to the addition of dead MCF7 cells to macrophage monolayers. Live and dead MCF7 cells were similarly seeded without Raw264.7 cells as single cultures. Media were collected after 16 hours of coculture, passed through a 0.2-μm filter, and used neat (250 μl) to quantify murine IL-10 and IL-4 by ELISA (BioLegend) according to the manufacturer's protocol. Total remaining cells were collected after 16 hours of coculture, lysed, and RNA was collected using an RNeasy kit (QIAGEN). MethodsMice. All mice were inbred on an FVB background for more than 10 generations. WT FVB, MMTV PyVmT and MerTK -/-mice (67), originally referred to as Mer KD , were purchased from The Jackson Laboratory. Mice were genotyped by PCR of genomic DNA as previously described(30). Female virgin mice were randomized into 2 groups: (a) 1 group that remained virgin, and (b) 1 group that was bred from 42 to 44 days of age with WT male mice. Pregnancies were timed according to identification of a va...

There is growing evidence that generation of adenosine from ATP, which is mediated by the CD39/CD73 enzyme pair, predetermines immunosuppressive and pro-angiogenic properties of myeloid cells. We have previously shown that the deletion of the TGFβ type II receptor gene (Tgfbr2) expression in myeloid cells is associated with decreased tumor growth suggesting pro-tumorigenic effect of TGFβ signaling. In this study, we tested the hypothesis that TGFβ drives differentiation of myeloid-derived suppressor cells (MDSCs) into pro-tumorigenic terminally differentiated myeloid mononuclear cells (TDMMCs) characterized by high levels of cell surface CD39/CD73 expression. We found that TDMMCs represent a major cell subpopulation expressing high levels of both CD39 and CD73 in the tumor microenvironment. In tumors isolated from MMTV-PyMT/TGFRIIKO mice, an increased level of TGFβ protein was associated with further increase in number of CD39+CD73+ TDMMCs compared to MMTV-PyMT/TGFRIIWT control tumors with intact TGFβ signaling. Using genetic and pharmacological approaches, we demonstrated that the TGFβ signaling mediates maturation of MDSCs into TDMMCs with high levels of cell surface CD39/CD73 expression and adenosine-generating capacity. Disruption of TGFβ signaling in myeloid cells resulted in decreased accumulation of TDMMCs, expressing CD39 and CD73, and was accompanied by increased infiltration of T lymphocytes, reduced density of blood vessels and diminished progression of both Lewis Lung carcinoma and spontaneous mammary carcinomas. We propose that TGFβ signaling can directly induce the generation of CD39+CD73+ TDMMCs, thus contributing to the immunosuppressive, pro-angiogenic, and tumor-promoting effects of this pleiotropic effector in the tumor microenvironment.

Purpose Metastatic breast cancers continue to elude current therapeutic strategies, including those utilizing PI3K inhibitors. Given the prominent role of PI3Kα,β in tumor growth and PI3Kγ,δ in immune cell function, we sought to determine whether PI3K inhibition altered anti-tumor immunity. Experimental Design The effect of PI3K inhibition on tumor growth, metastasis, and anti-tumor immune response was characterized in mouse models utilizing orthotopic implants of 4T1 or PyMT mammary tumors into syngeneic or PI3Kγ null mice, and patient-derived breast cancer xenografts in humanized mice. Tumor infiltrating leukocytes were characterized by IHC and FACS analysis in BKM120 (30mg/kg, QD) or vehicle treated mice and PI3Kγnull versus PI3KγWT mice. Based on the finding that PI3K inhibition resulted in a more inflammatory tumor leukocyte infiltrate, the therapeutic efficacy of BKM120 (30mg/kg, QD) and anti-PD1 (100µg, twice weekly) was evaluated in PyMT tumor bearing mice. Results Our findings show that PI3K activity facilitates tumor growth and surprisingly, restrains tumor immune surveillance. These activities could be partially suppressed by BKM120 or by genetic deletion of PI3Kγ in the host. The anti-tumor effect of PI3Kγ loss in host, but not tumor, was partially reversed by CD8+T cell depletion. Treatment with therapeutic doses of both BKM120 and antibody to PD-1 resulted in consistent inhibition of tumor growth compared to either agent alone. Conclusions PI3K inhibition slows tumor growth, enhances anti-tumor immunity, and heightens susceptibility to immune checkpoint inhibitors. We propose that combining PI3K inhibition with anti-PD1 may be a viable therapeutic approach for triple negative breast cancer.

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The effect of moderate food restriction on pregnancy-associated changes in weight gain, body composition, and circulating insulin-like growth factors (IGF) I and II and IGF-binding proteins (IGFBP)-1 through -4 and their relationship was determined in the guinea pig. Pregnancy did not stimulate weight gain but reduced fat deposition in ad libitum-fed animals and increased weight gain and fat deposition in food-restricted animals relative to their respective virginal group. Pregnancy increased the abundance of circulating IGF-I regardless of food intake and increased that of IGF-II in food-restricted animals only. Pregnancy also increased circulating IGFBP-1 and -2 in ad libitum-fed and food-restricted animals and IGFBP-4 in ad libitum-fed animals. Multiple regression analysis showed that maternal weight gain was negatively associated with circulating IGF-II and IGFBP-2. Fetal weight was positively associated with maternal circulating IGF-II and negatively associated with maternal circulating IGFBP-1 and -2. Significant interactions indicate, however, that the role of IGF-II and IGFBP-1 on fetal growth is dependent on the nutritional status of the mother.

The effects of cortisol on hepatic GH receptor and insulin-like growth factor-I (IGF-I) gene expression were investigated in sheep fetuses during late gestation and after experimental manipulation of plasma cortisol levels by fetal adrenalectomy and exogenous infusion of cortisol. Hepatic GH receptor and IGF-I messenger RNA (mRNA) levels increased with increasing gestational age in parallel with the normal rise in fetal cortisol levels toward term (145 +/- 2 days). These increases in mRNA abundance toward term were prevented when the prepartum cortisol surge was abolished by fetal adrenalectomy and were stimulated prematurely in fetuses younger than 130 days by exogenous infusion of cortisol. Both the class 1 and class 2 transcripts of the IGF-I gene were increased when cortisol levels were elevated either endogenously or exogenously. However, there were no significant changes in fetal plasma IGF-I levels either with increasing gestational age or in response to experimental manipulation of the fetal cortisol level. When the data from all the fetuses were combined irrespective of treatment or gestational age, there were significant positive correlations between the log plasma cortisol concentration in utero and the abundance of GH receptor and IGF-I mRNA in the fetal liver. There was also a significant inverse relationship between log plasma cortisol and the ratio of class 1 to class 2 transcript abundance in the fetal liver. These findings show that cortisol is a physiological regulator of hepatic GH receptor and IGF-I gene expression in fetal sheep during late gestation and indicate that it preferentially increases the class 2 transcript of the IGF-I gene. The prepartum cortisol surge therefore appears to have an important maturational role in initiating the perinatal switch from the fetal to adult modes of somatotrophic regulation.

Leptin mRNA was measured in adipose tissue of fetal sheep by reverse transcription polymerase chain reaction (RTPCR). Thus the leptin synthetic capacity of fetal adipose tissue appears to increase in late gestation but this is accompanied by constraint of its sensitivity to fetal body weight. We hypothesise that leptin synthesis in fetal adipose tissue is related to fetal nutrient supply and growth rate.