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Autophagy of damaged mitochondria, called mitophagy, is an important organelle quality control process involved in the pathogenesis of inflammation, cancer, aging, and age-associated diseases. Many of these disorders are associated with altered expression of the inner mitochondrial membrane (IMM) protein Prohibitin 1. The mechanisms whereby dysfunction occurring internally at the IMM and matrix activate events at the outer mitochondrial membrane (OMM) to induce mitophagy are not fully elucidated. Using the gastrointestinal epithelium as a model system highly susceptible to autophagy inhibition, we reveal a specific role of Prohibitin-induced mitophagy in maintaining intestinal homeostasis. We demonstrate that Prohibitin 1 induces mitophagy in response to increased mitochondrial reactive oxygen species (ROS) through binding to mitophagy receptor Nix/Bnip3L and independently of Parkin. Prohibitin 1 is required for ROS-induced Nix localization to mitochondria and maintaining homeostasis of epithelial cells highly susceptible to mitochondrial dysfunction., (© 2023. The Author(s).)

Objective: To identify risk factors associated with high-frequency ventilation (HFV) following definitive closure of the patent ductus arteriosus (PDA)., Methods: We performed a retrospective study of premature infants (<37 weeks) who were mechanically ventilated before and after surgical or transcatheter PDA closure. Primary outcome was HFV requirement within 24 h of procedure. Logistic regression was used to estimate clinical associations with post procedure HFV requirement., Results: We identified 110 infants who were mechanically ventilated before PDA closure, of which 48 (44%) escalated to HFV within 24 h after closure. In the multivariable model, surgical ligation (OR 21.5, 95% CI 1.6-284), elevated Respiratory Severity Score (RSS) 1 h post-procedure (OR 1.78, 95% CI 1.07-2.99) and 12 h post-procedure (OR 2.12, 95% CI 1.37-3.26) were independent predictors of HFV., Conclusion: Surgical ligation and elevated RSS values over the first 12 h after PDA closure are risk factors for HFV., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Objectives: Describe outcomes among preterm infants diagnosed with single-vessel primary pulmonary vein stenosis (PPVS) initially treated using conservative management (active surveillance with deferral of treatment)., Study Design: Retrospective cohort study at a single, tertiary-center (2009-2019) among infants <37 weeks' gestation with single-vessel PPVS. Infants were classified into two categories: disease progression and disease stabilization. Cardiopulmonary outcomes were examined, and a Kaplan-Meier survival analysis performed., Results: Twenty infants were included. Compared to infants in the stable group (0/10, 0%), all infants in the progressive group had development of at least severe stenosis or atresia (10/10, 100%; P < 0.01). Severe pulmonary hypertension at diagnosis was increased in the progressive (5/10, 50%) versus the stable group (0/10, 0%; P = 0.03). Survival was lower among infants in the progressive than the stable group (log-rank test, P < 0.01)., Conclusion: Among preterm infants with single-vessel PPVS, risk stratification may be possible, wherein more targeted, individualized therapies could be applied., (© 2020. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Transgenic mice expressing the Notch-4 intracellular domain (designated Int3) in the mammary gland have two phenotypes exhibited with 100% penetrance: arrest of mammary alveolar/lobular development and mammary tumorigenesis. Notch-4 signaling is mediated primarily through the interaction of Int3 with the transcription repressor/activator Rbpj. Interestingly, WAP-Int3/Rbpj knockout mice have normal mammary gland development but still developed mammary tumors with a slightly longer latency than the WAP-Int3 mice. Thus, Notch-induced mammary tumor development is Rbpj-independent. Here, we show that Int3 activates NF-κB in HC11 cells in absence of Rbpj through an association with the IKK signalosome. Int3 induced the canonical NF-κB activity and P50 phosphorylation in HC11 cells without altering the NF-κB2 pathway. The minimal domain within the Int3 protein required to activate NF-κB consists of the CDC10/Ankyrin (ANK) repeats domain. Treatment of WAP-Int3 tumor bearing mice with an IKK inhibitor resulted in tumor regression. In a soft agar assay, treatment of HC11-Int3 cells with P50-siRNA caused a significant decrease in colony formation. In addition, Wap-Int3/P50 knockout mice did not develop mammary tumors. This data indicates that the activation of NF-κB canonical signaling by Notch-4/Int3 is ANK repeats dependent, Rbpj-independent, and is mediated by IKK activation and P50 phosphorylation causing mammary tumorigenesis.

Transgenic mice expressing the Notch 4 intracellular domain (ICD) (Int3) in the mammary gland have two phenotypes: arrest of mammary alveolar/lobular development and mammary tumorigenesis. Notch4 signaling is mediated primarily through the interaction of Int3 with the transcription repressor/activator Rbpj. We have conditionally ablated the Rbpj gene in the mammary glands of mice expressing whey acidic protein (Wap)-Int3. Interestingly, Rbpj knockout mice (Wap-Cre(+)/Rbpj(-/-)/Wap-Int3) have normal mammary gland development, suggesting that the effect of endogenous Notch signaling on mammary gland development is complete by day 15 of pregnancy. RBP-J heterozygous (Wap-Cre(+)/Rbpj(-/+)/Wap-Int3) and Rbpj control (Rbpj(flox/flox)/Wap-Int3) mice are phenotypically the same as Wap-Int3 mice with respect to mammary gland development and tumorigenesis. In addition, the Wap-Cre(+)/Rbpj(-/-)/Wap-Int3-knockout mice also developed mammary tumors at a frequency similar to Rbpj heterozygous and Wap-Int3 control mice but with a slightly longer latency. Thus, the effect on mammary gland development is dependent on the interaction of the Notch ICD with the transcription repressor/activator Rbpj, and Notch-induced mammary tumor development is independent of this interaction.

Transgenic mice overexpressing Notch4 intracellular domain (Int3) under the control of the whey acidic protein (WAP) or mouse mammary tumor virus-long terminal repeat promoters, develop mammary tumors. Microarray analysis of these tumors revealed high levels of c-Kit expression. Gleevec is a tyrosine kinase inhibitor that targets c-Kit, platelet-derived growth factor receptors (PDGFRs) and c-Abl. This led us to speculate that tyrosine kinase receptor activity might be a driving force in the development of Int3 mammary tumors. WAP-Int3 tumor-bearing mice were treated with continuous release of Gleevec using subcutaneously implanted Alzet pumps. Phosphorylation of c-Kit, PDGFRs and c-Abl is inhibited in Int3 transgenic mammary tumors by Gleevec. Inhibition of these enzymes is associated with a decrease in cell proliferation and angiogenesis, and an induction of apoptosis. To examine the signaling mechanisms underlying Notch4/Int3 tumorigenesis, we employed small interfering RNA (siRNA) to knock down c-Kit, PDGFRs and c-Abl alone or in combination and observed the effects on soft agar growth of HC11 cells overexpressing Int3. Only siRNA constructs for c-Kit and/or PDGFR-alpha were able to inhibit HC11-Int3 colony formation in soft agar. Our data demonstrate an inhibitory effect of Gleevec on Int3-induced transformation of HC11 cells and mammary tumors and indicate an oncogenic role for c-Kit and PDGFR-alpha tyrosine kinases in the context of Int3 signaling.

We present evidence that Notch4ICD attenuates TGF-beta signaling. Cells expressing the activated form of the Notch4 receptor (ICD4) were resistant to the growth-inhibitory effects of TGF-beta. Notch4ICD was found to bind to Smad2, Smad3 and Smad4 but with higher affinity to Smad3. Deletion analysis showed that binding of Smad3 to ICD4 was mediated by its MH2 domain and was not dependent on the presence of the RAM23 region in ICD4. Using two TGF-beta/Activin reporter luciferase assays, RT-PCR and Western blot analysis, we demonstrate that ICD4 and ICD4 deltaRAM23 inhibit Smad-binding element and 3TP luciferase reporter activity and PAI-1 gene expression. MCF-7 human breast cancer cells express Notch4ICD (ICD4) and are resistant to the growth-inhibitory effects of TGF-beta. Blockage of Notch4 processing to ICD4 by gamma-secretase inhibitor renders MCF-7 cells sensitive to growth inhibition by TGF-beta. The interplay between these two signaling pathways may be a significant determinant during mammary tumorigenesis.

Recently, we have identified a novel 1.8 kb human Notch4/Int3 RNA species (designated h-Int3sh). The h-Int3sh RNA encodes a protein that is missing the CBF1-binding region (RAM23) of the Notch 4/Int3 intracellular domain (ICD). Expression of h-Int3sh in the MCF10A 'normal' human mammary epithelial cell line has been previously shown to induce changes characteristic of oncogenic transformation, including anchorage-independent growth in soft agar. To study the consequences of h-Int3sh expression in vivo on mammary gland development and tumorigenesis, three transgenic mouse lines were established, in which the transgene is the Whey acidic protein (WAP) promoter linked to h-Int3sh. Expression of WAP-Int3sh was detectable in the mammary gland at day 15 of pregnancy in each transgenic line. Mammary gland development in all founder lines is normal and the females can lactate. WAP-h-Int3sh females from each of the founder lines develop mammary tumors, but with a long latency (average age of 18 months). Tumor development was associated with activation of Notch pathway, as evidenced by upregulation of Hes-1. The long latency of mammary tumors in WAP-h-Int3sh mice could be due in part to the subcellular localization of h-Int3sh. Immunofluorescence analysis of transfected COS-1 cells showed that h-Int3sh is localized in the cytoplasm and nucleus, while Int3-ICD is detected only in the nucleus. We speculate that the Notch4/Int3 ICD-induced block to mammary gland development and tumorigenesis are consequences of an increasing gradient of CBF1-dependent Notch4/Int3 signaling.

Int6/eIF3-p48 was first identified as a common integration site for MMTV in mouse mammary tumors. In all cases, the MMTV integration event resulted in an interruption of the normal Int6 transcript from one allele leaving the second allele intact and operative. We hypothesize that insertion of MMTV into Int6 results in a mutated allele that encodes a shortened Int6 mRNA and protein (Int6sh), which either modifies normal Int6 function or possesses a new independent function. To confirm the transforming potential of the mutation and its dominant function, we transfected two mammary epithelial cell lines, MCF10A (human), and HC11 (mouse), with Int6sh under the control of the elongation factor-1alpha (eEF1A) promoter. Expression of Int6sh in MCF10A and HC11 mammary epithelial cells leads to anchorage-independent growth in soft agar indicative of a transformed phenotype. Colonies selected from agar exhibited high levels of mutated Int6sh and wild type Int6 RNA transcripts by RT-PCR and Northern blot analysis. In addition, Int6sh transformed MCF10A and HC11 cells formed nodular growths, in vivo, in immune compromised hosts. NIH3T3 cells, mouse embryo fibroblasts, were also transformed to anchorage-independent growth in vitro by Int6sh expression. These observations provide direct evidence that the Int6 mutations observed in MMTV-induced tumors and hyperplasia contribute to the malignant transformation of the mammary epithelial cells.

The study of the mouse mammary tumor virus (MMTV) has provided important insights into the mechanisms of gene transcription regulation by steroid hormones, the mode of action of heritable super antigens and the progressive nature of neoplastic transformation in the mammary gland. Here we describe the current situation with respect to the latter aspect of MMTV biology and the prospects for further advance in our understanding of breast cancer in humans that may be expected from a continued study of MMTV-induced mammary neoplasia. MMTV is a heritable somatic mutagen whose target range is limited. Commonly, the tumorigenic capacity of MMTV is restricted to mammary gland, whereas infection is found in a variety of cell types. In order to replicate, proviral DNA must be inserted into the cell DNA and cell division is required to fix the mutation. Yet only in the mammary epithelium does this lead to neoplastic transformation. This suggests a unique relationship between MMTV and mammary epithelium. In evaluating this relationship, we and others have discovered genes and potential gene pathways that are pertinent in mammary differentiation and neoplasia. In addition, the clonal nature of these progressive events from normal to malignant phenotype has become increasingly clear. The weight of these observations compel us to conclude that mammary neoplasms arise from multipotent mammary epithelial cells through a process of acquired mutations that are reflected in the increasingly malignant nature of the population of progeny produced by these damaged stem cells.

Ectopic expression of the intracellular domain of NOTCH-4/INT-3 leads to tumorigenesis in the mouse mammary gland. This results from a gain-of-function mutation. To evaluate gain-of-function NOTCH-4/INT-3 activity in human cancers we have surveyed human breast, lung, and colon carcinoma tissue culture cell lines for evidence of increased NOTCH-4/INT-3 RNA expression. High levels of a 1.8 Kb NOTCH-4/INT-3 RNA species are detected in normal human testis but not in other tissues where a 6.5 Kb species is prevalent. Transformed human cancer cell lines express the 1.8 Kb NOTCH-4/INT-3 RNA species. We show that this RNA species encodes a truncated form of the NOTCH-4/INT-3 intracellular domain (ICD). This novel NOTCH-4/INT-3 protein includes the CDC10 repeats and amino acid residues C-terminal to them, but is missing the CBF-1 binding region of the NOTCH-4/INT-3 ICD. This suggests that it has a different mode of action. Furthermore, we show that a transgene which expresses the 1.8 Kb NOTCH-4/INT-3 RNA species in the 'normal' human mammary epithelial cell line MCF-10A enables these cells to grow in soft agar.

The INT3 gene is frequently rearranged in mouse mammary tumor virus (MMTV)-induced mammary tumors of the CzechII mouse strain. We have completed the nucleotide sequence of the normal 6.5 Kb INT3 RNA and defined the intron/exon boundaries of the gene. The open reading frame of INT3 RNA should encode a 200 kd protein which shares 60% homology with the mouse homologue of Drosophila NOTCH. INT3 is unique among other members of the NOTCH family by containing 29 instead of 36 EGF-like repeats in the extracellular domain of the gene product. Five novel EGF-like repeats have been created as consequence of apparent small deletions which have occurred within the coding region for the extracellular domain during evolution. Nucleotide sequence analysis of host-viral junction fragments from nine independent MMTV-induced mammary tumors containing a rearranged INT3 gene reveals that all of the integration events occur within a 174 bp region 3' of the sequences encoding the LIN12 repeats in the INT3 extracellular domain and 5' of the sequences encoding the transmembrane domain. Therefore, the only tumorigenic INT3 mutations resulting from MMTV proviral insertions are those which results in the expression of the intracellular domain. This strongly suggests that MMTV-induced activation of INT3 is manifest in the absence of the regulatory action of the extracellular domain, including the LIN12 repeat sequences, leaving the expressed intracellular domain constitutively free to function in its role in mammary tumorigenesis.

p53 mutations are frequent in human breast cancer. In order to understand the role of p53 in the context of the accumulation of mutations in breast cancer, a model of non transformed mammary cells was sought. The HC11 cells are immortalized, non transformed rodent mammary epithelial cells which synthesize milk proteins following stimulation with lactogenic hormones. p53 protein was readily detected in HC11 protein extracts with the PAb421 antibody. Two mutations were identified in the p53 cDNA from HC11 cells: a missense mutation at codon 138, substituting Trp for Cys, and a microdeletion, codon 123 to 130, of exon 5. The latter results from an intronic mutation of the splice acceptor site at the intron 4/exon 5 junction. The mutations affect separate p53 alleles, and no wt allele was found. Wt p53 was introduced into HC11 cells by means of a retroviral vector, under the control of a Cd(++)-inducible promoter. In the presence of CdSO4 a dramatic growth inhibition was observed. A temperature-sensitive mutant p53 gene was also transfected into HC11 cells. This resulted in a marked inhibition of cells growth at 32 degrees C, when the p53 is in the wt conformation, while no effect was observed at 37 degrees C, when the mutant conformation is predominant. wt p53-mediated inhibition of monolayer growth does not involve induction of programmed cell death and does not activate de novo synthesis of differentiation-specific milk proteins. We conclude that mutations in the p53 gene likely played a role in their immortalization. The HC11 cells provide a model for assessing the cooperative action of other mutations in mammary tumorigenesis.

Gene amplification is a relatively frequent event in human malignant tumors and is believed to have an important function in neoplastic transformation and tumor progression. Our attention has been focused on the amplification and the expression of the int-2 gene for several reasons: (1) In the mouse mammary tumorigenesis int-2 is frequently activated by MMTV proviral integration. (2) The human homolog of int-2, located on chromosome 11q13, is frequently amplified in human primary tumors and is comprised in an amplification unit encompassing the hst gene, which is often coamplified; the amplification at the 11q13 locus in breast carcinomas correlates with a poor outcome of the disease. (3) int-2 and hst belong to the basic FGF gene family. All these observations raise the possibility that the human int-2 gene plays an active role in the neoplastic process, but this will prove to be true only if int-2 is expressed in human tumors. In the present study we used RNA:RNA in situ hybridization and Northern blot analysis to show that int-2 gene is expressed in a number of human carcinomas amplified at the same locus.

The int-2 gene, a member of the fibroblast growth factor (FGF) super-gene family, has previously been shown to be amplified in 16% of the 110 human breast tumors examined. In order to characterize the amplification unit containing the int-2 gene (chromosome 11q13), the same panel of breast tumors was screened for possible amplifications of other markers mapping between 11q11 and 11q24. Out of the eight additional genes analysed, simultaneous amplification of bcl-1 (11q13, a locus involved in hematopoietic malignancies) and hst (11q13, another member of the FGF family) was observed in 17/18 tumors with increased copy number of the int-2 gene. A single breast tumor showed amplification of int-2 oncogene only. Neither the bcl-1 nor the hst locus was individually amplified in any of the tumor DNAs examined.