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During mammary gland development, an epithelial bud undergoes branching morphogenesis to expand into a continuous tree-like network of branched ducts [1]. The process involves multiple cell types that are coordinated by hormones and growth factors coupled with signaling events including Wnt and Hedgehog [2-5]. Primary cilia play key roles in the development of many organs by coordinating extracellular signaling (of Wnt and Hedgehog) with cellular physiology [6-8]. During mammary development, we find cilia on luminal epithelial, myoepithelial, and stromal cells during early branching morphogenesis when epithelial ducts extend into the fat pad and undergo branching morphogenesis. When branching is complete, cilia disappear from luminal epithelial cells but remain on myoepithelial and stromal cells. Ciliary dysfunction caused by intraflagellar transport defects results in branching defects. These include decreased ductal extension and decreased secondary and tertiary branching, along with reduced lobular-alveolar development during pregnancy and lactation. We find increased canonical Wnt and decreased Hedgehog signaling in the mutant glands, which is consistent with the role of cilia in regulating these pathways [6-11]. In mammary gland and other organs, increased canonical Wnt [12-14] and decreased Hedgehog [15, 16] signaling decrease branching morphogenesis, suggesting that Wnt and Hedgehog signaling connect ciliary dysfunction to branching defects.

Abstract Introduction We have previously identified a rare subpopulation of variant human mammary epithelial cells (vHMEC) with repressed p16INK4A that exist in disease-free women yet display premalignant properties, suggesting that they have engaged the process of malignant transformation. In order to gain insight into the molecular alterations required for vHMEC to progress to malignancy, and to characterize the epigenetic events associated with early progression, we examined the effect of oncogenic stress on the behavior of these cells. Methods HMEC that express p16INK4A and vHMEC that do not, were transduced with constitutively active Ha-rasV12 and subsequently exposed to serum to determine whether signals from the cellular microenvironment could cooperate with ras to promote the malignant transformation of vHMEC. Epigenetic alterations were assessed using methylation-specific polymerase chain reaction (PCR). Results vHMEC expressing Ha-rasV12 (vHMEC-ras) bypassed the classic proliferative arrest that has been previously documented in normal fibroblasts following oncogenic stress, and that we also observe here in normal HMEC. Moreover, vHMEC-ras cells exhibited many additional alterations that are observed during progression to malignancy such as the generation of chromosomal abnormalities, upregulation of telomerase activity, immortalization following exposure to serum, and anchorage-independent growth, but they did not form tumors following orthotopic injection in vivo. Associated with their early progression to malignancy was an increase in the number of genes methylated, two of which (RASSF1A and SFRP1) were also methylated in other immortalized mammary cell lines as well as in breast cancer cells and tissues. Conclusions We have characterized a mammary progression model that recapitulates molecular and methylation alterations observed in many breast cancers. Our data suggest that concomitant methylation of RASSF1A and SFRP1 marks an early event in mammary transformation and may thus have prognostic potential.

Aneuploidy, frequently observed in premalignant lesions, disrupts gene dosage and contributes to neoplastic progression. Theodor Boveri hypothesized nearly 100 years ago that aneuploidy was due to an increase in centrosome number (multipolar mitoses) and the resultant abnormal segregation of chromosomes. We performed immunocytochemistry, quantitative immunofluorescence, karyotypic analysis, and time-lapse microscopy on primary human diploid epithelial cells and fibroblasts to better understand the mechanism involved in the production of supernumerary centrosomes (more than two microtubule nucleating bodies) to directly demonstrate that the presence of supernumerary centrosomes in genomically intact cells generates aneuploid daughter cells. We show that loss of p16(INK4a) generates supernumerary centrosomes through centriole pair splitting. Generation of supernumerary centrosomes in human diploid epithelial cells was shown to nucleate multipolar spindles and directly drive production of aneuploid daughter cells as a result of unequal segregation of the genomic material during mitosis. Finally, we demonstrate that p16(INK4a) cooperates with p21 through regulation of cyclin-dependent kinase activity to prevent centriole pair splitting. Cells with loss of p16(INK4a) activity have been found in vivo in histologically normal mammary tissue from a substantial fraction of healthy, disease-free women. Demonstration of centrosome dysfunction in cells due to loss of p16(INK4a) suggests that, under the appropriate conditions, these cells can become aneuploid. Gain or loss of genomic material (aneuploidy) may provide the necessary proproliferation and antiapoptotic mechanisms needed for the earliest stages of tumorigenesis.

An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMCs) triggers pulmonary vasoconstriction and stimulates PASMC proliferation leading to vascular wall thickening. Here, we report that STIM2 (stromal interaction molecule 2), a Ca2+ sensor in the sarcoplasmic reticulum membrane, is required for raising the resting [Ca2+]cyt in PASMCs from patients with pulmonary arterial hypertension (PAH) and activating signaling cascades that stimulate PASMC proliferation and inhibit PASMC apoptosis. Downregulation of STIM2 in PAH-PASMCs reduces the resting [Ca2+]cyt, whereas overexpression of STIM2 in normal PASMCs increases the resting [Ca2+]cyt The increased resting [Ca2+]cyt in PAH-PASMCs is associated with enhanced phosphorylation (p) of CREB (cAMP response element-binding protein), STAT3 (signal transducer and activator of transcription 3), and AKT, increased NFAT (nuclear factor of activated T-cell) nuclear translocation, and elevated level of Ki67 (a marker of cell proliferation). Furthermore, the STIM2-associated increase in the resting [Ca2+]cyt also upregulates the antiapoptotic protein Bcl-2 in PAH-PASMCs. Downregulation of STIM2 in PAH-PASMCs with siRNA (1) decreases the level of pCREB, pSTAT3, and pAKT and inhibits NFAT nuclear translocation, thereby attenuating proliferation, and (2) decreases Bcl-2, which leads to an increase of apoptosis. In summary, these data indicate that upregulated STIM2 in PAH-PASMCs, by raising the resting [Ca2+]cyt, contributes to enhancing PASMC proliferation by activating the CREB, STAT3, AKT, and NFAT signaling pathways and stimulating PASMC proliferation. The STIM2-associated increase in the resting [Ca2+]cyt is also involved in upregulating Bcl-2 that makes PAH-PASMCs resistant to apoptosis, and thus plays an important role in sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in patients with PAH. [ABSTRACT FROM AUTHOR]

Pulmonary vascular remodeling characterized by concentric wall thickening and intraluminal obliteration is a major contributor to the elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Here we report that increased hypoxia-inducible factor 2α (HIF-2α) in lung vascular endothelial cells (LVECs) under normoxic conditions is involved in the development of pulmonary hypertension (PH) by inducing endothelial-to-mesenchymal transition (EndMT), which subsequently results in vascular remodeling and occlusive lesions. We observed significant EndMT and markedly increased expression of SNAI, an inducer of EndMT, in LVECs from patients with IPAH and animals with experimental PH compared with normal controls. LVECs isolated from IPAH patients had a higher level of HIF-2α than that from normal subjects, whereas HIF-1α was upregulated in pulmonary arterial smooth muscle cells (PASMCs) from IPAH patients. The increased HIF-2α level, due to downregulated prolyl hydroxylase domain protein 2 (PHD2), a prolyl hydroxylase that promotes HIF-2α degradation, was involved in enhanced EndMT and upregulated SNAI1/2 in LVECs from patients with IPAH. Moreover, knockdown of HIF-2α (but not HIF-1α) with siRNA decreases both SNAI1 and SNAI2 expression in IPAH-LVECs. Mice with endothelial cell (EC)-specific knockout (KO) of the PHD2 gene, egln1 (egln1EC−/−), developed severe PH under normoxic conditions, whereas Snai1/2 and EndMT were increased in LVECs of egln1EC−/− mice. EC-specific KO of the HIF-2α gene, hif2a, prevented mice from developing hypoxia-induced PH, whereas EC-specific deletion of the HIF-1α gene, hif1a, or smooth muscle cell (SMC)-specific deletion of hif2a, negligibly affected the development of PH. Also, exposure to hypoxia for 48–72 h increased protein level of HIF-1α in normal human PASMCs and HIF-2α in normal human LVECs. These data indicate that increased HIF-2α in LVECs plays a pathogenic role in the development of severe PH by upregulating SNAI1/2, inducing EndMT, and causing obliterative pulmonary vascular lesions and vascular remodeling. [ABSTRACT FROM AUTHOR]

Capsaicin is an active component of chili pepper and a pain relief drug. Capsaicin can activate transient receptor potential vanilloid 1 (TRPV1) channels to increase cytosolic Ca2+ concentration ([Ca2+]cyt). A rise in [Ca2+]cyt in pulmonary artery smooth muscle cells (PASMCs) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. In this study, we observed that a capsaicin-induced increase in [Ca2+]cyt was significantly enhanced in PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH) compared with normal PASMCs from healthy donors. In addition, the protein expression level of TRPV1 in IPAH PASMCs was greater than in normal PASMCs. Increasing the temperature from 23 to 43°C, or decreasing the extracellular pH value from 7.4 to 5.9 enhanced capsaicin-induced increases in [Ca2+]cyt; the acidity (pH 5.9)- and heat (43°C)-mediated enhancement of capsaicin-induced [Ca2+]cyt increases were greater in IPAH PASMCs than in normal PASMCs. Decreasing the extracellular osmotic pressure from 310 to 200 mOsmol/l also increased [Ca2+]cyt, and the hypo-osmolarity-induced rise in [Ca2+]cyt was greater in IPAH PASMCs than in healthy PASMCs. Inhibition of TRPV1 (with 5′-IRTX or capsazepine) or knockdown of TRPV1 (with short hairpin RNA) attenuated capsaicin-, acidity-, and osmotic stretch-mediated [Ca2+]cyt increases in IPAH PASMCs. Capsaicin induced phosphorylation of CREB by raising [Ca2+]cyt, and capsaicin-induced CREB phosphorylation were significantly enhanced in IPAH PASMCs compared with normal PASMCs. Pharmacological inhibition and knockdown of TRPV1 attenuated IPAH PASMC proliferation. Taken together, the capsaicin-mediated [Ca2+]cyt increase due to upregulated TRPV1 may be a critical pathogenic mechanism that contributes to augmented Ca2+ influx and excessive PASMC proliferation in patients with IPAH. [ABSTRACT FROM AUTHOR]

Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca2+ signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca2+] ([Ca2+]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca2+ eliminated the plateau phase increase of [Ca2+]cyt in lung cancer cells, indicating that the plateau phase of [Ca2+]cyt increase is due to Ca2+ influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca2+ or chelating intracellular Ca2+ with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/ Bax ratio, indicating that a rise in [Ca2+]cyt through Ca2+ influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression. [ABSTRACT FROM AUTHOR]

Aneuploidy, frequently observed in premalignant lesions, disrupts gene dosage and contributes to neoplastic progression. Theodor Boveri hypothesized nearly 100 years ago that aneuploidy was due to an increase in centrosome number (multipolar mitoses) and the resultant abnormal segregation of chromosomes. We performed immunocytochemistry, quantitative immunofluorescence, karyotypic analysis, and time-lapse microscopy on primary human diploid epithelial cells and fibroblasts to better understand the mechanism involved in the production of supernumerary centrosomes (more than two microtubule nucleating bodies) to directly demonstrate that the presence of supernumerary centrosomes in genomically intact cells generates aneuploid daughter cells. We show that loss of p16INK4a generates supernumerary centrosomes through centriole pair splitting. Generation of supernumerary centrosomes in human diploid epithelial cells was shown to nucleate multipolar spindles and directly drive production of aneuploid daughter cells as a result of unequal segregation of the genomic material during mitosis. Finally, we demonstrate that p16INK4a cooperates with p21 through regulation of cyclin-dependent kinase activity to prevent centriole pair splitting. Cells with loss of p16INK4a activity have been found in vivo in histologically normal mammary tissue from a substantial fraction of healthy, disease-free women. Demonstration of centrosome dysfunction in cells due to loss of p16INK4a suggests that, under the appropriate conditions, these cells can become aneuploid. Gain or loss of genomic material (aneuploidy) may provide the necessary proproliferation and antiapoptotic mechanisms needed for the earliest stages of tumorigenesis. [ABSTRACT FROM AUTHOR]

Pulmonary vascular remodeling characterized by concentric wall thickening and intraluminal obliteration is a major contributor to the elevated pulmonary vascular resistance in patients with idiopathic pulmonary arterial hypertension (IPAH). Here we report that increased hypoxia-inducible factor 2α (HIF-2α) in lung vascular endothelial cells (LVECs) under normoxic conditions is involved in the development of pulmonary hypertension (PH) by inducing endothelial-to-mesenchymal transition (EndMT), which subsequently results in vascular remodeling and occlusive lesions. We observed significant EndMT and markedly increased expression of SNAI, an inducer of EndMT, in LVECs from patients with IPAH and animals with experimental PH compared with normal controls. LVECs isolated from IPAH patients had a higher level of HIF-2α than that from normal subjects, whereas HIF-1α was upregulated in pulmonary arterial smooth muscle cells (PASMCs) from IPAH patients. The increased HIF-2α level, due to downregulated prolyl hydroxylase domain protein 2 (PHD2), a prolyl hydroxylase that promotes HIF-2α degradation, was involved in enhanced EndMT and upregulated SNAI1/2 in LVECs from patients with IPAH. Moreover, knockdown of HIF-2α (but not HIF-1α) with siRNA decreases both SNAI1 and SNAI2 expression in IPAH-LVECs. Mice with endothelial cell (EC)-specific knockout (KO) of the PHD2 gene, egln1 (egln1 EC-/- ), developed severe PH under normoxic conditions, whereas Snai1/2 and EndMT were increased in LVECs of egln1 EC-/- mice. EC-specific KO of the HIF-2α gene, hif2a, prevented mice from developing hypoxia-induced PH, whereas EC-specific deletion of the HIF-1α gene, hif1a, or smooth muscle cell (SMC)-specific deletion of hif2a, negligibly affected the development of PH. Also, exposure to hypoxia for 48-72 h increased protein level of HIF-1α in normal human PASMCs and HIF-2α in normal human LVECs. These data indicate that increased HIF-2α in LVECs plays a pathogenic role in the development of severe PH by upregulating SNAI1/2, inducing EndMT, and causing obliterative pulmonary vascular lesions and vascular remodeling.

Capsaicin is an active component of chili pepper and a pain relief drug. Capsaicin can activate transient receptor potential vanilloid 1 (TRPV1) channels to increase cytosolic Ca 2+ concentration ([Ca 2+ ] cyt ). A rise in [Ca 2+ ] cyt in pulmonary artery smooth muscle cells (PASMCs) is an important stimulus for pulmonary vasoconstriction and vascular remodeling. In this study, we observed that a capsaicin-induced increase in [Ca 2+ ] cyt was significantly enhanced in PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH) compared with normal PASMCs from healthy donors. In addition, the protein expression level of TRPV1 in IPAH PASMCs was greater than in normal PASMCs. Increasing the temperature from 23 to 43°C, or decreasing the extracellular pH value from 7.4 to 5.9 enhanced capsaicin-induced increases in [Ca 2+ ] cyt ; the acidity (pH 5.9)- and heat (43°C)-mediated enhancement of capsaicin-induced [Ca 2+ ] cyt increases were greater in IPAH PASMCs than in normal PASMCs. Decreasing the extracellular osmotic pressure from 310 to 200 mOsmol/l also increased [Ca 2+ ] cyt , and the hypo-osmolarity-induced rise in [Ca 2+ ] cyt was greater in IPAH PASMCs than in healthy PASMCs. Inhibition of TRPV1 (with 5'-IRTX or capsazepine) or knockdown of TRPV1 (with short hairpin RNA) attenuated capsaicin-, acidity-, and osmotic stretch-mediated [Ca 2+ ] cyt increases in IPAH PASMCs. Capsaicin induced phosphorylation of CREB by raising [Ca 2+ ] cyt , and capsaicin-induced CREB phosphorylation were significantly enhanced in IPAH PASMCs compared with normal PASMCs. Pharmacological inhibition and knockdown of TRPV1 attenuated IPAH PASMC proliferation. Taken together, the capsaicin-mediated [Ca 2+ ] cyt increase due to upregulated TRPV1 may be a critical pathogenic mechanism that contributes to augmented Ca 2+ influx and excessive PASMC proliferation in patients with IPAH., (Copyright © 2017 the American Physiological Society.)

Adenosine triphosphate (ATP) is a ubiquitous extracellular messenger elevated in the tumor microenvironment. ATP regulates cell functions by acting on purinergic receptors (P2X and P2Y) and activating a series of intracellular signaling pathways. We examined ATP-induced Ca(2+) signaling and its effects on antiapoptotic (Bcl-2) and proapoptotic (Bax) proteins in normal human airway epithelial cells and lung cancer cells. Lung cancer cells exhibited two phases (transient and plateau phases) of increase in cytosolic [Ca(2+)] ([Ca(2+)]cyt) caused by ATP, while only the transient phase was observed in normal cells. Removal of extracellular Ca(2+) eliminated the plateau phase increase of [Ca(2+)]cyt in lung cancer cells, indicating that the plateau phase of [Ca(2+)]cyt increase is due to Ca(2+) influx. The distribution of P2X (P2X1-7) and P2Y (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11) receptors was different between lung cancer cells and normal cells. Proapoptotic P2X7 was nearly undetectable in lung cancer cells, which may explain why lung cancer cells showed decreased cytotoxicity when treated with high concentration of ATP. The Bcl-2/Bax ratio was increased in lung cancer cells following treatment with ATP; however, the antiapoptotic protein Bcl-2 demonstrated more sensitivity to ATP than proapoptotic protein Bax. Decreasing extracellular Ca(2+) or chelating intracellular Ca(2+) with BAPTA-AM significantly inhibited ATP-induced increase in Bcl-2/Bax ratio, indicating that a rise in [Ca(2+)]cyt through Ca(2+) influx is the critical mediator for ATP-mediated increase in Bcl-2/Bax ratio. Therefore, despite high ATP levels in the tumor microenvironment, which would induce cell apoptosis in normal cells, the decreased P2X7 and elevated Bcl-2/Bax ratio in lung cancer cells may enable tumor cells to survive. Increasing the Bcl-2/Bax ratio by exposure to high extracellular ATP may, therefore, be an important selective pressure promoting transformation and cancer progression., (Copyright © 2016 the American Physiological Society.)