Identification and transcript analysis of a novel wallaby (Macropus eugenii) basal-like breast cancer cell line

<p>Abstract</p> <p>Background</p> <p>A wide variety of animal models have been used to study human breast cancer. Murine, feline and canine mammary tumor cell lines have been studied for several decades and have been shown to have numerous aspects in common with human breast cancer. It is clear that new comparative approaches to study cancer etiology are likely to be productive.</p> <p>Results</p> <p>A continuous line of breast carcinoma cells (WalBC) was established from a primary breast cancer that spontaneously arose in a female tammar wallaby (<it>Macropus eugenii</it>). The primary tumor was 1.5 cm³and although large, did not appear to invade the stroma and lacked vimentin expression. The WalBC cell line was cultured from the primary tumor and passaged for 22 months. WalBC cells displayed an epithelial morphology when grown on plastic, were not EGF responsive, stained strongly for cyto-keratin and negatively for vimentin. WalBC cells were shown to be non-invasive within a Matrigel invasion assay and failed to produce tumors following transplantation into nude mice. Gene expression profiling of WalBC cells was performed using a cDNA microarray of nearly 10,000 mammary gland cDNA clones and compared to normal primary mammary cells and profiles of human breast cancer. Seventy-six genes were down-regulated and sixty-six genes were up-regulated in WalBC cells when compared to primary mammary cells. WalBC cells exhibited expression of known markers of basal invasive human breast cancers as well as increased KRT17, KRT 14 and KRT 19, DSP, s100A4, NDRG-1, ANXA1, TK1 and AQP3 gene expression and decreased gene expression of TIMP3, VIM and TAGLN. New targets for breast cancer treatment were identified such as ZONAB, PACSIN3, MRP8 and SUMO1 which have human homologues.</p> <p>Conclusion</p> <p>This study demonstrates how novel models of breast cancer can provide new fundamental clues regarding cancer etiology which may lead to new human treatments and therapies.</p

Acute involution in the tammar wallaby : identification of genes and putative novel milk proteins implicated in mammary gland function

AbstractMarsupials provide a suitable alternative model to studying mammary gland involution. They have evolved a different reproductive strategy from eutherians, giving birth to an altricial young and secreting milk that changes in composition during lactation. In this study, we used a marsupial-specific EST microarray to identify 47 up-regulated genes during mammary gland involution in the tammar wallaby (Macropus eugenii). These include the pro-apoptotic tumour necrosis factor receptor superfamily 21 (TNFRSF21) gene, whose expression in the mammary gland has not previously been reported. Genes encoding putative novel milk proteins which may protect the mammary gland from infection were also found to be up-regulated, such as amiloride binding protein 1 (ABP1), complement component 1QB (C1QB), complement component 4A (C4A) and colony stimulating factor 2 receptor β (CSF2Rβ). Our results show that the marsupial reproductive strategy was successfully exploited to identify genes and putative novel milk proteins implicated in mammary gland involution

Validation of Epidermal AMBRA1 and Loricrin (AMBLor) as a prognostic biomarker for non-ulcerated AJCC stage I/II cutaneous melanoma

Background: Combined expression of the autophagy-regulatory protein AMBRA1 (activating molecule in Beclin1-regulated autophagy) and the terminal differentiation marker loricrin in the peritumoral epidermis of stage I melanomas can identify tumour subsets at low risk of metastasis. Objectives: To validate the combined expression of peritumoral AMBRA1 and loricrin (AMBLor) as a prognostic biomarker able to identify both stage I and II melanomas at low risk of tumour recurrence. Methods: Automated immunohistochemistry was used to analyse peritumoral AMBRA1 and loricrin expression in geographically distinct discovery (n = 540) and validation (n = 300) cohorts of nonulcerated American Joint Committee on Cancer (AJCC) stage I and II melanomas. AMBLor status was correlated with clinical outcomes in the discovery and validation cohorts separately and combined. Results: Analysis of AMBLor in the discovery cohort revealed a recurrence-free survival (RFS) rate of 95.5% in the AMBLor low-risk group vs. 81.7% in the AMBLor at-risk group (multivariate log-rank, P < 0.001) and a negative predictive value (NPV) of 96.0%. In the validation cohort, AMBLor analysis revealed a RFS rate of 97.6% in the AMBLor low-risk group vs. 78.3% in the at-risk group (multivariate log-rank, P < 0.001) and a NPV of 97.6%. In a multivariate model considering AMBLor, Breslow thickness, age and sex, analysis of the combined discovery and validation cohorts showed that the estimated effect of AMBLor was statistically significant ,with a hazard ratio of 3.469 (95% confidence interval 1.403–8.580, P = 0.007) and an overall NPV of 96.5%. Conclusions These data provide further evidence validating AMBLor as a prognostic biomarker to identify nonulcerated AJCC stage I and II melanoma tumours at low risk of disease recurrence

Species-specific cell-matrix interactions are essential for differentiation of alveoli like structures and milk gene expression in primary mammary cells of the Cape fur seal (Arctocephalus pusillus pusillus)

Few models are in place for analysis of extreme lactation patterns such as that of the fur seals which are capable of extended down regulation of milk production in the absence of involution. During a 10&ndash;12 month lactation period, female fur seals suckle pups on shore for 2&ndash;3 days, and then undertake long foraging trips at sea for up to 28 days, resulting in the longest intersuckling bouts recorded. During this time the mammary gland down regulates milk production. We have induced Cape fur seal (Arctocephalus pusillus pusillus) mammary cells in vitro to form mammospheres up to 900 &mu;m in diameter, larger than any of their mammalian counterparts. Mammosphere lumens were shown to form via apoptosis and cells comprising the cellular boundary stained vimentin positive. The Cape fur seal GAPDH gene was cloned and used in RT-PCR as a normalization tool to examine comparative expression of milk protein genes (&alpha;S2-casein, &beta;-lactoglobulin and lysozyme C) which were prolactin responsive. Cape fur seal mammary cells were found to be unique; they did not require Matrigel for rapid mammosphere formation and instead deposited their own matrix within 2 days of culture. When grown on Matrigel, cells exhibited branching/stellate morphogenesis highlighting the species-specific nature of cell&ndash;matrix interactions during morphological differentiation. Matrix produced in vitro by cells did not support formation of human breast cancer cell line, PMC42 mammospheres. This novel model system will help define the molecular pathways controlling the regulation of milk protein expression and species specific requirements of the extracellular matrix in the cape fur seal.<br /

Molecular analysis of tammar (Macropus eugenii) mammary epithelial cells stimulated with lipopolysaccharide and lipoteichoic acid

The immunological function of the metatherian mammary gland plays a crucial part in neonatal survival of the marsupial young. Marsupial pouch young do not develop adult like immune responses until just prior to leaving the pouch. The immune components of the maternal milk secretions are important during this vulnerable early post-partum period. In addition, infection of the mammary gland has not been recognized in metatherians, despite the ready availability of pathogens in the pouch. Regardless of which, little is known about the immunobiology of the mammary gland and the immune responses of mammary epithelial cells in metatherians. In this study, a molecular approach was utilized to examine the response of tammar (Macropus eugenii) mammary epithelial cells to Escherichia coli derived lipopolysaccharide (LPS) and Staphylococcus aureus derived lipoteichoic acid (LTA). Using custom-made cDNA microarrays, candidate genes were identified in the transciptome, which were involved in antigen presentation, inflammation, cell growth and proliferation, cellular damage and apoptosis. Quantification of mRNA expression of several of these candidate genes, along with seven other genes (TLR4, CD14, TNF-α, cathelicidin, PRDX1, IL-5 and ABCG2) associated with innate immunity in LPS and LTA challenged mammary epithelial cells and leukocytes, was assessed for up to 24 h. Differences in genes associated with cellular damage and pro-inflammatory cytokine production were seen between stimulated mammary epithelial cells and leukocytes. LTA challenge tended to result in lower level induction of pro-inflammatory cytokines, increased PRDX1 mRNA levels, suggesting increased oxidative stress, and increased CD14 expression, but in a non-TLR4-dependent manner. The use of functional genomic tools in the tammar identified differences in the response of tammar mammary epithelial cells (MEC) and leukocytes to challenge with LPS and LTA, and validates the utility of the approach. The results of this study are consistent with a model in which tammar mammary epithelial cells have the capacity to elicit a complex and robust immune response to pathogens.13 page(s