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Prior to the commencement of this study in 1990, a number of reports had appeared in the literature describing the importance of insulin action during lactation in mammals (see Chapter 1). These studies investigated the changes in circulating insulin and glucagon concentrations during lactation, the relative numbers of insulin receptors in insulin-sensitive tissues, and glucose utilisation by these tissues. However, at that time, no information was available on the structure of the mammary insulin receptor. The rationale for undertaking this study was to characterise the structure of the rat mammary insulin receptor as a means of furthering our understanding of the role insulin plays during lactation.An initial requirement of this study was the development of a method for the convenient and inexpensive preparation of A14-tyrosyl[125I]iodoinsulin. A14-tyrosyl[125I]iodoinsulin displays binding characteristics which are virtually indistinguishable from the native hormone, which is a necessary requirement for tracers which are to be used in binding studies. In Chapter 2, I describe a method for the purification of A14-tyrosyl[125I]iodoinsulin from a mixture of iodinated insulin molecules which are produced following oxidation by chloramine-T in the presence of Na125iodine. In this method I employed disposable cartridges packed with a C18 support matrix to which the iodinated insulin molecules are readily adsorbed when in an aqueous solution.A 14-tyrosyl[125I]iodoinsulin absorbed most strongly to the C18 matrix and unwanted products were removed through a sequence of washes prior to the elution of the A14-tyrosyl[125I]iodoinsulin derivative using a buffer containing 50% (v/v) acetonitrile. This prodct was unambiguously shown to be A14-tyrosyl[125I]iodoinsulin by N-terminal amino acid sequencing. The quality of this radiolabel compared favourably with commercially ++ / available A14-tyrosyl[125I]iodoinsulin preparations both in terms of specific activity and stability upon storage at -20C. Furthermore, a modified method based on this protocol has been used in our and other laboratories for the isolation of other iodinated peptides with highly satisfactory results.I have established that the size of the a-subunit of the rat mammary insulin receptor is significantly diminished compared with the liver insulin receptor (125 kDa versus 130 kDa). This difference in size was present throughout all stages of lactation and was not due to proteolysis of a larger form. Furthermore, I demonstrated that both the mammary and liver insulin receptor a-subunits migrated equally on PAGE following treatment with neuraminidase, indicating that the apparent size difference may be accounted for by a variation in the extent of receptor sialation. Treatment of the mammary insulin receptor a-subunit with glycopeptidase F demonstrated that the size of the aglycoreceptor (100 kDa) was similar to that described for insulin receptors from other insulin-sensitive tissues.I characterised the distribution of mRNA encoding the two, naturally-occurring insulin receptor isoforms in mammary tissue throughout all stages of pregnancy and lactation. These insulin receptor isoforms differ due to the absence (IR-A) or presence (IR-B) of a 12 amino acid peptide, encoded by exon 11 of the insulin receptor gene, and located near the C-terminus of the insulin receptor a-subunit. Mammary tissue predominantly expressed IR-A mRNA in contrast to liver tissue, which almost exclusively expressed IRB mRNA. Furthermore, the ratio of IR-A to IR-B mRNA in mammary tissue changed significantly during the first week post-partum whilst the distribution of IR-A and IR-B mRNA in the liver remained constant throughout pregnancy and lactation. This difference in insulin receptor isoform ++ / expression between mammary and liver tissue also contributed to the estimated size difference between the insulin receptor a-subunits from these two tissues. In addition, I characterised the expression of IR-A and IR-B mRNA in several different tissues obtained from rats on day 14 of gestation through to 7 days post partum. I established that the splicing mechanism is functional at least as early as day 14 of gestation, suggesting a possible role for the preferential expression of a particular insulin receptor isoform during organogenesis. I observed that IR-A mRNA was the predominant isoform in all foetal tissue studied, and the proportion of this isoform declined as the animal matured. These changes were significant in cardiac muscle, kidney and most dramatic in the liver where the expression of IR-A mRNA changed from 53% in the 21 day old foetus (the day before parturition) to 13% in the 1 day old neonate. These results suggest that the splicing mechanism which generates the receptor isoforms is subject to acute hormonal and/or metabolic control.The current literature suggests that the carbohydrate moieties of the insulin receptor affects its affinity for insulin. Furthermore, the IR-A and IR-B isoforms have been shown to display a 2-fold difference in their insulin binding affinity when expressed in heterologous cell lines such at CHO cells or Rat-1 fibroblasts. Since both glycosylational and isoform distribution differences were evident between mammary and liver tissues, the insulin binding affinities of these receptors were compared. Estimates of the binding affinity parameters were performed at both 4 C and 37 C. At both temperatures the equilibrium binding constants for mammary and liver tissues were not significantly different suggesting that structural variations of the mammary insulin receptor had no effect on the insulin binding affinity under the ++ / conditions described in this study. Comparison of the 4 C and 37 C binding data showed that the mammary insulin receptor exhibited complex, temperature-dependent binding characteristics, similar to those previously described for the liver insulin receptor, and entirely consistent with the presence of a temperature-dependent regulatory protein that affects insulin binding.

c-Myb/MYB is an established and key player in hematopoietic malignancies but more recently a strong case for c-Myb as an oncogene in breast cancer has emerged. c-Myb and its transcriptional target genes have direct bearing on tumour initiation and progression and thus this has opened new opportunities to the development of therapeutic approaches in a range of cancer types with the aim of treating cancer at its various stages. In this study, the requirement of c-Myb during mammary gland tumourigenesis is being examined. In addition a direct therapeutic approach to targeting c-Myb-driven gene grp78/GRP78 in the context of primary and metastatic breast cancer was assessed. / The first aim of this study is to examine the expression of c-Myb during normal mammary gland development. The expression of c-Myb is extensively characterised in a temporal and spatial fashion. Nuclear staining of c-Myb by immunohistochemisty was found to be most elaborately expressed in the ductal epithelium during early mammary gland development. Mouse mammary gland lacking c-myb showed disorganised ductal structure in virgin mice, but did not affect subsequent pregnancy and lactation. / To extend the view that c-Myb is involved in mammary tumourigenesis c-myb-transduced immortalised mammary epithelial cells and two mammary tumour prone transgenic mouse models were examined. NMuMG cells transduced with c-myb showed enhanced proliferation and reduced Annexin V staining consistent with the protection from apoptosis. This reduced apoptosis is consistent with, and perhaps contingent upon, the elevated expression observed for bcl-2 and grp78. The data assembled by expression studies raised the possibility that c-Myb is essential for the establishment of mammary gland tumor in both MMTV-Neu and MMTV-PyMT spontaneous mammary gland tumor models. Loss of c-Myb expression in these models significantly delayed and in most instances completely abolished the onset of mammary gland tumours in both models. Preliminary evidence also indicated that Stat3 phosphorylation may underpin the elevated c-Myb expression in mouse mammary tumour cells. / The focus of my thesis then shifted to examining ways to exploit elevated c-Myb target gene GRP78 expression on the cell surface of mammary tumour cells. To do this I employed a GRP78 binding pro-apoptotic chimera peptide that specifically binds to GRP78 where I examined its efficacy against primary and metastatic breast cancer models. My results demonstrated the anti-tumour activity of the GRP78-chimera peptide both in vitro and in vivo. More importantly, the peptide is also effective at prolonging disease-free survival in mice with established metastatic disease. / Evidence obtained within these studies suggests that c-Myb plays an important role in mammary gland development and tumourgenesis. Although it may be difficult to directly target c-Myb in malignant disease, alternative anti-tumoural therapy may be developed against c-Myb-regulated target genes that are also implicated in mammary tumours. Collectively my thesis studies have advanced our understanding of c-Myb in mammary cancer initiation, progression and as a direct or indirect therapeutic target.

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The mammary gland consists of an organized network of epithelial ducts and lobules. This histoarchitecture can be recapitulated in vitro by culturing mammary epithelial cells as 3D spheroids embedded in a reconstituted basement membrane. I first used this assay to characterize the role of cell-cell and cell-ECM adhesion in the formation and polarization of the apical junction complexes in normal mammary epithelial cells. Cell-cell adhesion alone was sufficient to initiate polarized junction assembly. However, the addition of exogenous ECM generated a spatial polarity signal dependent on laminin-1 and α6 and β1 integrins. This caused clusters of mammary epithelial cells to re-localize the junctional complexes to the center of the spheroid prior to lumen formation. In ductal breast carcinoma, a critical hallmark is the loss of normal polarized tissue architecture without the induction of an epithelial-to-mesenchymal transformation (EMT). Thus, misregulation of molecules that function as polarity determinants may contribute to ductal tumor progression. Podocalyxin is an anti-adhesive glycoprotein that may be involved, as it is important in epithelial morphogenesis, and its overexpression in clinical breast tumors is associated with poor outcome. Despite this, overexpression of podocalyxin in normal mammary epithelial cells did not disrupt 3D morphogenesis or apicobasal polarity. However, its overexpression in non-metastatic breast tumor cells did perturb the architecture and growth of tumor spheroids in vitro and it facilitated subcutaneous tumor growth in vivo without causing an EMT. Mechanistically, podocalyxin localized to and expanded non-adhesive membrane domains and induced microvillus formation that was dependent on its extracellular domain and Rho GTPase-regulated actin polymerization. Podocalyxin also recruited its intracellular binding partners NHERF-1 and ezrin via its cytoplasmic tail. Strikingly, the formation of this protein complex was not required for microvillus formation. Additionally, podocalyxin delayed cell-cell aggregation and decreased the initial adhesion, spreading and strength of attachment of tumor cells to fibronectin where it restricted β1 integrin localization to the basal/attached domain. These alterations in adhesion possibly contributed to podocalyxin's ability to increase growth factor-dependent tumor cell migration. Altogether, these data indicate that podocalyxin overexpression may facilitate a ductal tumor-like progression that involves EMT-independent alterations in tissue architecture.