As in developmental and regenerative processes, cell survival is of fundamental importance in cancer. Thus, a tremendous effort has been devoted to dissecting the molecular mechanisms involved in understanding the resistance of tumor cells to programmed cell death. Recently, the importance of stromal fibroblasts in tumor initiation and progression has been elucidated. Here, we show that stromal cell apoptosis occurs in human breast carcinoma but is only rarely seen in nonmalignant breast lesions. Furthermore, we show that ADAM12, a disintegrin and metalloprotease up-regulated in human breast cancer, accelerates tumor progression in a mouse breast cancer model. ADAM12 does not influence tumor cell proliferation but rather confers both decreased tumor cell apoptosis and increased stromal cell apoptosis. This dual role of ADAM12 in governing cell survival is underscored by the finding that ADAM12 increases the apoptotic sensitivity of nonneoplastic cells in vitro while rendering tumor cells more resistant to apoptosis. Together, these results show that the ability of ADAM12 to influence apoptosis may contribute to tumor progression. (Cancer Res 2005; 65(11): 4754-61)

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IntroductionAdult stem cells and progenitor cells can integrate and respond to appropriate extracellular stimuli in the form of hormones and growth factors, or contact with the extracellular matrix (ECM) and neighboring cells. The delicate balance between the dormancy of progenitor cells and their timely proliferation and differentiation is a crucial parameter in tissue homeostasis that is often perturbed in disease. In vitro studies and animal implant experiments have revealed the multipotential nature of mesenchymal stem cells, which contribute to the regeneration of mesenchymal tissues such as bone, cartilage, muscle, tendon, stroma and adipose tissues (Pittenger et al., 1999;Prockop, 1997).Adipogenesis is a complex process characterized by the strict temporal regulation of multiple and interacting signaling events that ultimately lead to the expression of adipocytespecific genes (Gregoire et al., 1998;Rosen and Spiegelman, 2000). A cascade of transcription factors is induced that involves the sequential activation of the CCAAT/enhancerbinding proteins (C/EBPs) and the peroxisome proliferatoractivated receptor γ (PPARγ), which leads to the activation of several genes, such as those responsible for lipid transport and metabolism. Initially, fibroblastic preadipocytes stop dividing and acquire a rounded morphology. The change of shape from fibroblastic preadipocytes to rounded, mature adipocytes is accompanied by changes in cytoskeletal organization and contacts with the ECM. The expression of fibronectin, integrins, actin and several cytoskeletal proteins is downregulated during adipogenesis (Rodriguez Fernandez and Ben-Ze'ev, 1989;Spiegelman and Farmer, 1982). In fact, the disruption of contacts with the ECM is a requirement for adipocyte differentiation (Spiegelman and Ginty, 1983).Our understanding of the molecular events involved in adipogenesis is based mainly on in vitro, cell culture models, 3893Changes in cell shape are a morphological hallmark of differentiation. In this study we report that the expression of ADAM12, a disintegrin and metalloprotease, dramatically affects cell morphology in preadipocytes, changing them from a flattened, fibroblastic appearance to a more rounded shape. We showed that the highest levels of ADAM12 mRNA were detected in preadipocytes at the critical stage when preadipocytes become permissive for adipogenic differentiation. Furthermore, as assessed by immunostaining, ADAM12 was transiently expressed at the cell surface concomitant with the reduced activity of β1 integrin. Co-immunoprecipitation studies indicated the formation of ADAM12/β1 integrin complexes in these preadipocytes. Overexpression of ADAM12 at the cell surface of 3T3-L1 preadipocytes achieved by transient transfection or retroviral transduction led to the disappearance of the extensive network of actin stress fibers that are characteristic of these cells, and its reorganization into a cortical network located beneath the cell membrane. The cells became more rounded, exhibited fewer vinculin-positive focal ad...

PP2A is an essential protein phosphatase that regulates most cellular processes through the formation of holoenzymes containing distinct regulatory B‐subunits. Only a limited number of PP2A‐regulated phosphorylation sites are known. This hampers our understanding of the mechanisms of site‐specific dephosphorylation and of its tumor suppressor functions. Here, we develop phosphoproteomic strategies for global substrate identification of PP2A‐B56 and PP2A‐B55 holoenzymes. Strikingly, we find that B‐subunits directly affect the dephosphorylation site preference of the PP2A catalytic subunit, resulting in unique patterns of kinase opposition. For PP2A‐B56, these patterns are further modulated by affinity and position of B56 binding motifs. Our screens identify phosphorylation sites in the cancer target ADAM17 that are regulated through a conserved B56 binding site. Binding of PP2A‐B56 to ADAM17 protease decreases growth factor signaling and tumor development in mice. This work provides a roadmap for the identification of phosphatase substrates and reveals unexpected mechanisms governing PP2A dephosphorylation site specificity and tumor suppressor function.

HIGHLIGHTS Outline of EGF dependent in-vivo phosphotyrosine signaling in lung tissue  A peptide based screen identifies proteins recruited to regulated phosphotyrosine sites in lung tissue  Several somatic mutations affect proline residues within position +3 of regulated phosphotyrosine sites and introduce molecular switches  A lung cancer oncogenic mutation in EGFR causes aberrant activation of phosphorylation signaling pathways by switching of a recruited protein complex  Cancer mutations in vicinity of phosphotyrosine sites induce molecular switches that alter protein signaling networks  Introduction of cutting-edge LC-MS instrumentation and DIA enables scalable, rapid and highthroughput analysis of phosphotyrosine site interactions

Osteoblasts and adipocytes may develop from common bone marrow mesenchymal precursors. Transgenic mice overexpressing ⌬FosB, an AP-1 transcription factor, under the control of the neuron-specific enolase (NSE) promoter show both markedly increased bone formation and decreased adipogenesis. To determine whether the two phenotypes were linked, we targeted overexpression of ⌬FosB in mice to the osteoblast by using the osteocalcin (OG2) promoter. OG2-⌬FosB mice demonstrated increased osteoblast numbers and an osteosclerotic phenotype but normal adipocyte differentiation. This result firmly establishes that the skeletal phenotype is cell autonomous to the osteoblast lineage and independent of adipocyte formation. It also strongly suggests that the decreased fat phenotype of NSE-⌬FosB mice is independent of the changes in the osteoblast lineage. In vitro, overexpression of ⌬FosB in the preadipocytic 3T3-L1 cell line had little effect on adipocyte differentiation, whereas it prevented the induction of adipogenic transcription factors in the multipotential stromal cell line ST2. Also, ⌬FosB isoforms bound to and altered the DNA-binding capacity of C/EBP␤. Thus, the inhibitory effect of ⌬FosB on adipocyte differentiation appears to occur at early stages of stem cell commitment, affecting C/EBP␤ functions. It is concluded that the changes in osteoblast and adipocyte differentiation in ⌬FosB transgenic mice result from independent cell-autonomous mechanisms.Although osteoblasts and adipocytes represent two morphologically and functionally distinct cell types, it has been proposed that they may develop from a common mesenchymal precursor in the bone marrow (36,40,43). Indeed, an inverse relationship between adipocyte and osteoblast differentiation has been suggested, as exemplified by increased bone marrow adipocytes in age-related bone loss (4,6,29,32,60) or after treatment with glucocorticoids (57). Several regulatory factors involved in osteoblast and adipocyte differentiation have been identified (27,42,45). However, the identities of the factors that control commitment at the branching point between the osteoblast and adipocyte lineages and the degree of plasticity between the two cell types are still uncertain (33,38).We have recently reported that transgenic mice overexpressing ⌬FosB, a member of the activator protein 1 (AP-1) family of transcription factors, under the control of the neuron-specific enolase (NSE) promoter develop not only a severe and progressive osteosclerotic phenotype, characterized as increased bone formation, but also a pronounced decrease in adipogenesis and fat levels (25, 47, 53). The AP-1 family of basic leucine zipper transcription factors comprises various combinations of Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and Fra-2) proteins, which upon dimer formation regulate gene transcription by binding to consensus response elements present in the promoter region of target genes (23). Several studies have demonstrated an important regulatory role of AP-1 factors, especially the ...

High metabolic and proliferative rates in cancer cells lead to production of large amounts of H and CO , and as a result, net acid extruding transporters are essential for the function and survival of cancer cells. We assessed protein expression of the Na /H exchanger NHE1, the Na - HCO3- cotransporter NBCn1, and the lactate-H cotransporters MCT1 and -4 by immunohistochemical analysis of a large cohort of breast cancer samples. We found robust expression of these transporters in 20, 10, 4 and 11% of samples, respectively. NHE1 and NBCn1 expression both correlated positively with progesterone receptor status, NHE1 correlated negatively and NBCn1 positively with HER2 status, whereas MCT4 expression correlated with lymph node status. Stable shRNA-mediated knockdown (KD) of either NHE1 or NBCn1 in the MDA-MB-231 triple-negative breast cancer (TNBC) cell line significantly reduced steady-state intracellular pH (pH ) and capacity for pH recovery after an acid load. Importantly, KD of any of the three transporters reduced in vivo primary tumor growth of MDA-MB-231 xenografts. However, whereas KD of NBCn1 or MCT4 increased tumor-free survival and decreased in vitro proliferation rate and colony growth in soft agar, KD of NHE1 did not have these effects. Moreover, only MCT4 KD reduced Akt kinase activity, PARP and CD147 expression and cell motility. This work reveals that different types of net acid extruding transporters, NHE1, NBCn1 and MCT4, are frequently expressed in patient mammary tumor tissue and demonstrates for the first time that they promote growth of TNBC human mammary tumors in vivo via distinct but overlapping mechanisms.

TGF-beta increases osteoblastic cell proliferation irrespective of the differentiation state. In presence of calcitriol, it initiates osteoblast cell differentiation and matrix formation. As TGF-beta inhibits osteocalcin production, other factors are necessary for inducing terminal differentiation of osteoblasts. The observed effects of TGF-beta on human osteoblasts in vitro may represent important regulatory steps in controlling osteoblast cell proliferation and differentiation in vivo.