Kaylene J. Simpson

Generation of a functional mammary gland from a single stem cell.

The existence of mammary stem cells (MaSCs) has been postulated from evidence that the mammary gland can be regenerated by transplantation of epithelial fragments in mice. Interest in MaSCs has been further stimulated by their potential role in breast tumorigenesis. However, the identity and purification of MaSCs has proved elusive owing to the lack of defined markers. We isolated discrete populations of mouse mammary cells on the basis of cell-surface markers and identified a subpopulation (Lin-CD29hiCD24+) that is highly enriched for MaSCs by transplantation. Here we show that a single cell, marked with a LacZ transgene, can reconstitute a complete mammary gland in vivo. The transplanted cell contributed to both the luminal and myoepithelial lineages and generated functional lobuloalveolar units during pregnancy. The self-renewing capacity of these cells was demonstrated by serial transplantation of clonal outgrowths. In support of a potential role for MaSCs in breast cancer, the stem-cell-enriched subpopulation was expanded in premalignant mammary tissue from MMTV-wnt-1 mice and contained a higher number of MaSCs. Our data establish that single cells within the Lin-CD29hiCD24+ population are multipotent and self-renewing, properties that define them as MaSCs.

Identification of genes that regulate epithelial cell migration using an siRNA screening approach

To provide a systematic analysis of genes that regulate epithelial cell migration, we performed a high throughput wound healing screen with MCF-10A breast epithelial cells, using siRNAs targeting 1,081 human genes encoding phosphatases, kinases and proteins predicted to influence cell migration and adhesion. The primary screen identified three categories of hits: those that accelerate, those that inhibit and those that impair migration with associated effects on cell proliferation or metabolism. Extensive validation of all the hits yielded 66 high confidence genes that, when downregulated, either accelerated or impaired migration; 42 of these high confidence genes have not been previously associated with motility or adhesion. Time-lapse video microscopy revealed a broad spectrum of phenotypic changes involving alterations in the extent and nature of disruption of cell–cell adhesion, directionality of motility, cell polarity and shape, and protrusion dynamics. Informatics analysis highlighted three major signalling nodes, β-catenin, β1-integrin and actin, and a large proportion of the genes that accelerated migration impaired cell–cell adhesion.

Functional analysis of the contribution of RhoA and RhoC GTPases to invasive breast carcinoma

Although the RhoA and RhoC proteins comprise an important subset of the Rho GTPase family that have been implicated in invasive breast carcinomas, attributing specific functions to these individual members has been difficult. We have used a stable retroviral RNA interference approach to generate invasive breast carcinoma cells (SUM-159 cells) that lack either RhoA or RhoC expression. Analysis of these cells enabled us to deduce that RhoA impedes and RhoC stimulates invasion. Unexpectedly, this analysis also revealed a compensatory relationship between RhoA and RhoC at the level of both their expression and activation, and a reciprocal relationship between RhoA and Rac1 activation.

Reciprocal regulation of RhoA and RhoC characterizes the EMT and identifies RhoC as a prognostic marker of colon carcinoma

Understanding how RhoC expression and activation are regulated is essential for deciphering its contribution to tumorigenesis. Here, we report that RhoC expression and activation are induced by the epithelial to mesenchymal transition (EMT) of colon carcinoma. Using LIM 1863 colon cancer cells, RhoC protein expression and subsequent activation were detected coincident with the loss of E-cadherin and acquisition of mesenchymal characteristics. Several Ets-1 binding sites were identified in the RhoC promoter, and evidence was obtained using chromatin immunoprecipitation that Ets-1 can regulate RhoC expression during the EMT. Interestingly, a marked decrease in RhoA activation associated with the EMT was observed that corresponds to the increase in RhoC expression. Use of shRNA established that RhoA inhibits and RhoC promotes post-EMT cell migration, demonstrating functional significance for their coordinate regulation. To assess the importance of RhoC expression in colon cancer, immunohistochemistry was performed on 566 colorectal tumors with known clinical outcome. The level of RhoC ranged from no expression to high expression, and statistical analysis revealed that elevated RhoC expression correlates with poor outcome as well as aberrant expression and localization of E-cadherin. These data provide one mechanism for how RhoC expression is regulated in colon carcinoma and substantiate its utility as a prognostic marker.

The tammar wallaby : A model to study putative autocrine-induced changes in milk composition

The marsupial newborn is immature and the mother has the capacity to alter milk composition significantly during lactation, presumably to meet the nutritional requirements of the developing young. Furthermore, macropodid marsupials may practice asynchronous concurrent lactation (ACL)7whereby the mother provides milk which differs in all the major components from adjacent mammary glands for two young of different ages. This phenomenon suggests that local regulation of mammary function, in addition to endocrine stimuli, is likely to be important for controlling milk composition. This paper explores the possibility that changes in sucking patterns of the young represent the first step in a mechanism to signal the mammary gland for putative autocrine-induced changes in milk composition.

Maternal Regulation of Milk Composition, Milk Production, and Pouch Young Development During Lactation in the Tammar Wallaby (Macropus eugenii )

Abstract Specific changes in milk composition during lactation in the tammar wallaby (Macropus eugenii) were correlated with the ages of the developing pouch young (PY). The present experiment was designed to test the hypothesis that the sucking pattern of the PY determines the course of mammary development in the tammar wallaby. To test this hypothesis, groups of 60-day-old PY were fostered repeatedly onto one group of host mothers so that a constant sucking stimulus on the mammary gland was maintained for 56 days to allow the lactational stage to progress 42 days ahead of the age of the young. Analysis of the milk in fostered and control groups showed the timing of changes in the concentration of protein and carbohydrate were essentially unaffected by altering the sucking regime. The only change in milk protein secretion was a small delay in the timing of down-regulation of the secretion of whey acidic protein and early lactation protein in the host tammars. In addition, the rates of growth and development of the foster PY were significantly increased relative to those of the control PY because of ingesting more milk with a higher energy content and different composition than normal for their age. The present study demonstrates that the lactating tammar wallaby regulates both milk composition and the rate of milk production and that these determine the rates of PY growth and development, irrespective of the age of the PY.

FOXO3a promotes tumor cell invasion through the induction of matrix metalloproteinases.

ABSTRACT The role of the Forkhead transcription factor FOXO3a in processes that promote tumor metastasis is poorly defined. Here, we show that depletion of FOXO3a from cancer cells leads to decreased tumor size specifically due to attenuated invasive migration. During tumor progression, an increase in tumor mass is concomitant with serum deprivation prior to tumor angiogenesis. We show that nuclear retention of FOXO3a due to serum starvation results in greatly increased cancer cell invasion. Exploration of the mechanism by which FOXO3a promotes invasive migration revealed that it induces the expression of matrix metalloproteinase 9 (MMP-9) and MMP-13, both of which have been causally linked to the invasion and progression of numerous human solid tumors. Our results link Forkhead transcription factors to a previously unexplored function in cancer progression by promoting extracellular matrix degradation, allowing tumors to invade neighboring tissues and ultimately metastasize to distant organs.

The Gene for a Novel Member of the Whey Acidic Protein Family Encodes Three Four-disulfide Core Domains and Is Asynchronously Expressed during Lactation

Secretion of whey acidic protein (WAP) in milk throughout lactation has previously been reported for a limited number of species, including the mouse, rat, rabbit, camel, and pig. We report here the isolation of WAP from the milk of a marsupial, the tammar wallaby (Macropus eugenii). Tammar WAP (tWAP) was isolated by reverse-phase HPLC and migrates in SDS-polyacrylamide gel electrophoresis at 29.9 kDa. tWAP is the major whey protein, but in contrast to eutherians, secretion is asynchronous and occurs only from approximately days 130 through 240 of lactation. The full-length cDNA codes for a mature protein of 191 amino acids, which is comprised of three four-disulfide core domains, contrasting with the two four-disulfide core domain arrangement in all other known WAPs. A three-dimensional model for tWAP has been constructed and suggests that the three domains have little interaction and could function independently. Analysis of the amino acid sequence suggests the protein belongs to a family of protease inhibitors; however, the predicted active site of these domains is dissimilar to the confirmed active site for known protease inhibitors. This suggests that any putative protease ligand may be unique to either the mammary gland, milk, or gut of the pouch young. Examination of the endocrine regulation of thetWAP gene showed consistently that the gene is prolactin-responsive but that the endocrine requirements for induction and maintenance of tWAP gene expression are different during lactation.

Synthetic lethality between CCNE1 amplification and loss of BRCA1

Significance Women with high-grade serous ovarian cancer (HGSC) harboring Cyclin E1 (CCNE1) gene amplification generally face a poor clinical outcome. These tumors comprise a significant group of ∼20% of HGSCs that are not associated with BRCA1/2 mutation and are unlikely to respond to standard cytotoxic or poly-ADP-ribose polymerase inhibitors. We identified a specific dependency on BRCA1 and members of the ubiquitin pathway in CCNE1-amplified tumors. The requirement for BRCA1 seems to account for the mutual exclusivity of mutations observed in primary tumors. We propose a unique therapeutic strategy involving inhibition of the proteasome and homologous recombination function with bortezomib. Our findings are likely to have relevance to the treatment of other tumor types with CCNE1 amplification, including triple negative breast cancer. High-grade serous ovarian cancers (HGSCs) are characterized by a high frequency of TP53 mutations, BRCA1/2 inactivation, homologous recombination dysfunction, and widespread copy number changes. Cyclin E1 (CCNE1) gene amplification has been reported to occur independently of BRCA1/2 mutation, and it is associated with primary treatment failure and reduced patient survival. Insensitivity of CCNE1-amplified tumors to platinum cross-linking agents may be partly because of an intact BRCA1/2 pathway. Both BRCA1/2 dysfunction and CCNE1 amplification are known to promote genomic instability and tumor progression. These events may be mutually exclusive, because either change provides a path to tumor development, with no selective advantage to having both mutations. Using data from a genome-wide shRNA synthetic lethal screen, we show that BRCA1 and members of the ubiquitin pathway are selectively required in cancers that harbor CCNE1 amplification. Furthermore, we show specific sensitivity of CCNE1-amplified tumor cells to the proteasome inhibitor bortezomib. These findings provide an explanation for the observed mutual exclusivity of CCNE1 amplification and BRCA1/2 loss in HGSC and suggest a unique therapeutic approach for treatment-resistant CCNE1-amplified tumors.

Transformation of a grain legume (Lupinus angustifolius L.) via Agrobacterium tumefaciens-mediated gene transfer to shoot apices

Transgenic plants of Lupinus angustifolius L. (cvs. Unicrop and Merrit) were routinely generated using Agrobacterium-mediated gene transfer to shoot apices. The bar gene for resistance to phosphinothricin (PPT, the active ingredient of the herbicide Basta) was used as the selectable marker. After co-cultivation, the shoot apex explants were transferred onto a PPT-free regeneration medium and their tops were thoroughly wetted with PPT solution (2 mg/ml). The multiple axillary shoots developing from the shoot apices were excised onto a medium containing 20 mg/l PPT. The surviving shoots were transferred every second week onto fresh medium containing 20 mg/l PPT. At each transfer, the number of surviving shoots decreased, until it stabilized. Indeed, some of these chimeric shoots surviving the PPT selection, eventually produced new green healthier axillary shoots which could be transferred to soil. This whole process took from 5 to 9 months after co-cultivation. Average transformation frequencies of 2.8% for cv. Unicrop and of 0.4% for the commercial cultivar Merrit were achieved. Molecular analysis of T0, T1, and T2 generations demonstrated stable integration of the foreign gene into the plant genome and expression of the integrated gene. Transformed plants of the T1 and T2 generations were resistant in glasshouse trials where the herbicide Basta (0.1 mg/ml) was sprayed onto whole plants. These results demonstrate that Agrobacterium-mediated gene transfer to preorganised meristematic tissue combined with axillary regeneration can form the basis of a routine transformation system for legume crop species which are difficult to regenerate from other explants.