Cancer and Polio Research Fund

<div>Abstract<p><b>Purpose:</b> Cancer cells have been shown to be more susceptible to Ran knockdown than normal cells. We now investigate whether Ran is a potential therapeutic target of cancers with frequently found mutations that lead to higher Ras/MEK/ERK [mitogen-activated protein/extracellular signal-regulated kinase (ERK; MEK)] and phosphoinositide 3-kinase (PI3K)/Akt/mTORC1 activities.</p><p><b>Experimental Design:</b> Apoptosis was measured by flow cytometry [propidium iodide (PI) and Annexin V staining] and MTT assay in cancer cells grown under different conditions after knockdown of Ran. The correlations between Ran expression and patient survival were examined in breast and lung cancers.</p><p><b>Results:</b> Cancer cells with their PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways inhibited are less susceptible to Ran silencing–induced apoptosis. K-Ras–mutated, c-Met–amplified, and Pten-deleted cancer cells are also more susceptible to Ran silencing–induced apoptosis than their wild-type counterparts and this effect is reduced by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways. Overexpression of Ran in clinical specimens is significantly associated with poor patient outcome in both breast and lung cancers. This association is dramatically enhanced in cancers with increased c-Met or osteopontin expression, or with oncogenic mutations of K-Ras or PIK3CA, all of which are mutations that potentially correlate with activation of the PI3K/Akt/mTORC1 and/or Ras/MEK/ERK pathways. Silencing Ran also results in dysregulation of nucleocytoplasmic transport of transcription factors and downregulation of Mcl-1 expression, at the transcriptional level, which are reversed by inhibitors of the PI3K/Akt/mTORC1 and MEK/ERK pathways.</p><p><b>Conclusion:</b> Ran is a potential therapeutic target for treatment of cancers with mutations/changes of expression in protooncogenes that lead to activation of the PI3K/Akt/mTORC1 and Ras/MEK/ERK pathways. <i>Clin Cancer Res; 18(2); 380–91. ©2011 AACR</i>.</p></div>

Ras-related nuclear protein (Ran) is required for cancer cell survival in vitro and human cancer progression, but the molecular mechanisms are largely unknown. We investigated the effect of the v-myc myelocytomatosis viral oncogene homolog (Myc) on Ran expression by Western blot, chromatin immunoprecipitation, and luciferase reporter assays and the effects of Myc and Ran expression in cancer cells by soft-agar, cell adhesion, and invasion assays. The correlation between Myc and Ran and the association with patient survival were investigated in 14 independent patient cohorts (n = 2430) and analyzed with Spearman's rank correlation and Kaplan-Meier plots coupled with Wilcoxon-Gehan tests, respectively. All statistical tests were two-sided. Myc binds to the upstream sequence of Ran and transactivates Ran promoter activity. Overexpression of Myc upregulates Ran expression, whereas knockdown of Myc downregulates Ran expression. Myc or Ran overexpression in breast cancer cells is associated with cancer progression and metastasis. Knockdown of Ran reverses the effect induced by Myc overexpression in breast cancer cells. In clinical data, a positive association between Myc and Ran expression was revealed in 288 breast cancer and 102 lung cancer specimens. Moreover, Ran expression levels differentiate better or poorer survival in Myc overexpressing breast (χ2 = 24.1; relative risk [RR] = 9.1, 95% confidence interval [CI] = 3.3 to 24.7, P < .001) and lung (χ2 = 6.04; RR = 2.8, 95% CI = 1.2 to 6.3; P = .01) cancer cohorts. Our results suggest that Ran is required for and is a potential therapeutic target of Myc-driven cancer progression in both breast and lung cancers.

1. Unilateral changes in mammary cell number are elicited when one gland is milked more or less frequently than the contralateral gland in lactating goats. These changes were investigated using histochemical and immunocytochemical markers of mammary cell types, and the degree of mammary apoptosis was determined by end-labelling of fragmented DNA. 2. Histological analysis confirmed that unilateral cessation of milking initiated involution and cell loss preferentially in the unmilked gland. The presence of fragmented DNA and morphological characteristics consistent with apoptosis demonstrated that these changes in mammary cell number in unmilked glands were, in part, the result of programmed alveolar cell death. 3. De-differentiation of the remaining secretory cells to ductal epithelial cells occurred with an increase in staining of cytokeratin markers and decreased staining by peanut lectin and casein antisera. 4. Differential once- and thrice-daily milking of lactating goats was also associated with unilateral changes in mammary cell number and milk yield. Milk yield and alveolar size were reduced after 4 weeks of infrequent milking. The latter was due to the increased loss of secretory cells by apoptosis, as indicated by a higher degree of fragmented DNA laddering. 5. After 10 weeks of differential milking, a homogeneous secretory morphology, albeit with smaller alveoli, was maintained in thrice-daily milked glands. Once-daily milked glands possessed a heterogeneous composition of terminal structures, resulting in the simultaneous presence of secretory and involuting alveoli as well as resting ductules. 6. The differences in programmed cell death and mammary morphology between unmilked and twice-daily milked glands, and between once- and thrice-daily milked glands, suggests that mammary apoptosis is subject to modulation by intra-mammary mechanisms sensitive to the frequency of milk removal.

We interviewed 328 men diagnosed with prostate cancer before the age of 75 years and 328 age-matched population controls. The principal hypotheses were that risk would increase with a high intake of total or saturated fat and would decrease with a high intake of carotene (beta-carotene equivalents) or lycopene. We also examined the associations of other nutrients and foods with risk. There was no evidence for an association between fat intake and risk, although the average fat intake was high and the range of fat intakes was narrow (medians of lower and upper thirds of percentage of energy from fat among controls were 34.3% and 42.9% respectively). Risk was lower in subjects with higher carotene intake: odds ratios 0.65 (95% CI 0.45-0.94) and 0.76 (0.53-1.10) in the middle and upper thirds of carotene intake respectively (P for trend = 0.150). Lycopene was not associated with risk. Among 13 other nutrients examined, the odds ratios in the top third of intake were below 0.8 for: potassium, 0.74 (0.51-1.09; P for trend = 0.054); zinc, 0.73 (0.49-1.08; P for trend = 0.126); iodine, 0.75 (0.51-1.11; P for trend = 0.077); vitamin B6 food only, 0.77 (0.53-1.12; P for trend = 0.077); and vitamin B6 including supplements, 0.70 (0.48-1.03; P for trend = 0.029). Among 18 foods examined, statistically significant associations were observed for: garlic as food, > or = 2/week vs never, 0.56 (0.33-0.93); garlic including supplements, > or = 2/week vs never, 0.60 (0.37-0.96); baked beans, > or = 2/week vs < 1/month, 0.57 (0.34-0.95); and garden peas, > or = 5/week vs < or = 3/month, 0.35 (0.13-0.91). This study does not support the hypothesis that fat increases risk and is equivocal in relation to carotene. The possible relationships of vitamin B6, garlic, beans and peas with risk for prostate cancer should be further investigated.

Benign rat mammary epithelial cells transfected with restriction enzyme-fragmented DNA from a human malignant metastatic cell line (Ca2-83) produces transfectants that yield metastatic tumours in syngeneic rats. The six metastasis-inducing DNAs (Met-DNAs) that have been isolated from such transfectants are subgene in size and do not code for any expressed mRNAs, but correspond to potential regulatory regions of human DNA from malignant, metastatic cells. In pilot studies the one Met-DNA tested is detectable in some human breast tumours but not in normal tissue. Transfection of all six Met-DNAs singly into the benign mammary epithelial cells causes enhanced expression of osteopontin, whilst transfection of cDNA for osteopontin also induces the metastatic state. These results show that short regulatory DNAs exist in human cancer cells that can induce metastatic spread via a common effector gene, osteopontin, in model rat mammary cell lines.

Conference Article| August 01 1996 A growth factor activity in bovine milk QING-MING LIU; QING-MING LIU 1Cancer and Polio Research Fund Laboratories, Department of Biochemistry, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK Search for other works by this author on: This Site PubMed Google Scholar MARK C WILKINSON; MARK C WILKINSON 1Cancer and Polio Research Fund Laboratories, Department of Biochemistry, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK Search for other works by this author on: This Site PubMed Google Scholar JOHN A. SMITH JOHN A. SMITH 1Cancer and Polio Research Fund Laboratories, Department of Biochemistry, University of Liverpool, PO Box 147, Liverpool L69 3BX, UK Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1996) 24 (3): 342S. https://doi.org/10.1042/bst024342s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation QING-MING LIU, MARK C WILKINSON, JOHN A. SMITH; A growth factor activity in bovine milk. Biochem Soc Trans 1 August 1996; 24 (3): 342S. doi: https://doi.org/10.1042/bst024342s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1996 Biochemical Society1996 Article PDF first page preview Close Modal You do not currently have access to this content.

Maximal stimulation of DNA synthesis in quiescent rat mammary (Rama) 27 fibroblasts is elicited by epidermal growth factor (EGF) or basic fibroblast growth factor (bFGF) 18 h after the initial addition of the growth factors-the 'lag' period. At maximally-stimulating concentrations, EGF and bFGF are interchangeable 9 h after their initial addition. When the initial concentration of growth factor is below that required to elicit a maximal response, it is possible to increase the level of DNA synthesis by increasing the concentration of growth factor 9 h after its initial addition. When the initial concentration of growth factor is high, substitution by a lower concentration of growth factor after 9 h allows a greater proportion of cells to synthesize DNA than would be expected from a continuous low dose of growth factor. Similar results are obtained when both the growth factor and its concentration are changed 9 h after the initial addition of growth factor. However, when EGF at a low concentration is substituted for a high concentration of EGF or bFGF the resulting increase in the levels of DNA synthesis is greater when EGF rather than bFGF is added for a second time. The half-life of the growth-stimulatory signals delivered by EGF and by bFGF 9 h after their initial addition is 1-2 h. These results suggest that to stimulate DNA synthesis: (i) EGF or bFGF must deliver a signal(s) continuously; (ii) the initial signals produced by EGF and bFGF are equivalent; (iii) the signals produced between 9-18 h by EGF may be different to those produced by bFGF.

To examine the potential role of fibroblast growth factors (FGF) in tumorigenesis and metastasis, plasmid constructs containing the human basic FGF (bFGF) gene, with or without fusion to a secretory signal peptide (IgbFGF), were transfected into the diploid rat mammary epithelial cell line Rama 37. All transfectants possessed multiple copies of the transfected cDNA, which was expressed as the corresponding mRNA and the protein. The amount of bFGF protein was usually greater than the bFGF growth-stimulatory activity that could be recovered from the transfected cells. Nevertheless, the amount of bFGF growth-stimulatory activity secreted by the IgbFGF transfectants (0.08–0.8 ng/ml/24 hr) was sufficient to induce growth in responsive cells. However, the transfectants themselves were refractory to stimulation by exogenously added bFGF, despite possessing a small number of high-affinity receptors for bFGF. When the bFGF or the IgbFGF transfectants were inoculated into the mammary fat pads of syngeneic rats, the tumour incidence was low (0–50%). However, when cells cultured from these tumours were inoculated into the fat pad of syngeneic rats, the tumour incidence was 100%. Tumours were in all cases benign and no metastases were observed. Our results suggest that the role of bFGF in metastasis is not simply one of autocrine/paracrine stimulation of cell growth and that other events may also be required. © 1996 Wiley-Liss, Inc.

To examine the potential role of fibroblast growth factors (FGF) in tumorigenesis and metastasis, plasmid constructs containing the human basic FGF (bFGF) gene, with or without fusion to a secretory signal peptide (IgbFGF), were transfected into the diploid rat mammary epithelial cell line Rama 37. All transfectants possessed multiple copies of the transfected cDNA, which was expressed as the corresponding mRNA and the protein. The amount of bFGF protein was usually greater than the bFGF growth-stimulatory activity that could be recovered from the transfected cells. Nevertheless, the amount of bFGF growth-stimulatory activity secreted by the IgbFGF transfectants (0.08-0.8 ng/ml/24 hr) was sufficient to induce growth in responsive cells. However, the transfectants themselves were refractory to stimulation by exogenously added bFGF, despite possessing a small number of high-affinity receptors for bFGF. When the bFGF or the IgbFGF transfectants were inoculated into the mammary fat pads of syngeneic rats, the tumour incidence was low (0-50%). However, when cells cultured from these tumours were inoculated into the fat pad of syngeneic rats, the tumour incidence was 100%. Tumours were in all cases benign and no metastases were observed. Our results suggest that the role of bFGF in metastasis is not simply one of autocrine/paracrine stimulation of cell growth and that other events may also be required.

mRNA for basic Fibroblast Growth Factor (bFGF) was expressed in a series of SV40-transformed human mammary cell lines as molecules of 7.1, 3.6, 2.0 and 1.2 kb. This expression was much weaker in those lines of epithelial morphology than in myoepithelial-like cell lines derived from them. It was confirmed, using northern hybridization to single-stranded RNA probes, that the multiple mRNAs were transcribed from the coding strand for bFGF. bFGF activity was detected in extracts of the cells and the relative amounts of activity corresponded in general to the amounts of mRNA found. Similar results were obtained from spontaneously transformed cell lines derived from a human benign breast lesion. The presence of bFGF protein in the extracts was confirmed by western blotting, which showed a band of 18-19 kDa, migrating in the same position as authentic bFGF; in addition, the myoepithelial-like cells showed prominent bands of bFGF at 24 and 26 kDa. No FGF receptor was detectable by the binding of 125I-bFGF to the SV40-transformed cell lines or to the epithelial cell lines from the benign breast lesion, but both high- and low-affinity receptors were found on myoepithelial-like cells derived from the latter. The results indicate that differentiation to the human myoepithelial-like phenotype in culture is associated with the enhanced expression of bFGF, and it is suggested that bFGF, immunocytochemically detected in the basement membrane of the human breast, may arise, at least in part, from the myoepithelial cells of the mammary parenchyma.