Metastasis-promoting anterior gradient 2 protein has a dimeric thioredoxin fold structure and a role in cell adhesion.

Anterior gradient 2 (AGR2) is a normal endoplasmic reticulum protein that has two important abnormal functions, amphibian limb regeneration and human cancer metastasis promotion. These normal intracellular and abnormal extracellular roles can be attributed to the multidomain structure of AGR2. The NMR structure shows that AGR2 consists of an unstructured N-terminal region followed by a thioredoxin fold. The protein exists in monomer-dimer equilibrium with a K(d) of 8.83µM, and intermolecular salt bridges involving E60 and K64 within the folded domain serve to stabilize the dimer. The unstructured region is primarily responsible for the ability of AGR2 to promote cell adhesion, while dimerization is less important for this activity. The structural data of AGR2 show a separation between potential catalytic redox activity and adhesion function within the context of metastasis and development.

Microarray analysis of suppression subtracted hybridisation libraries identifies genes associated with breast cancer progression.

BACKGROUND: A major challenge of cancer research is to identify key molecules which are responsible for the development of the malignant metastatic phenotype, the major cause of cancer death. METHODS: Four subtracted cDNA libraries were constructed representing mRNAs differentially expressed between benign and malignant human breast tumour cells and between micro-dissected breast carcinoma in situ and invasive carcinoma. Hundreds of differentially expressed cDNAs from the libraries were micro-arrayed and screened with mRNAs from human breast tumor cell lines and clinical specimens. Gene products were further examined by RT-PCR and correlated with clinical data. RESULTS: The combination of subtractive hybridisation and microarray analysis has identified a panel of 15 cDNAs which shows strong correlations with estrogen receptor status, malignancy or relapse. This panel included S100P, which was associated with aneuploidy in cell lines and relapse/death in patients, and AGR2 which was associated with estrogen receptor and with patient relapse. X-box binding protein-1 is also an estrogen-dependent gene and is associated with better survival for breast cancer patients. CONCLUSIONS: The combination of subtracted cDNA libraries and microarray analysis has thus identified potential diagnostic/prognostic biomarkers and targets for cancer therapy, which have not been identified from common prognostic gene signatures.

Molecular analysis of a collection of clinical specimens stored at 4 degrees C as an alternative to snap-freezing.

It is critical for both basic and clinical translational cancer research to use high quality DNA, RNA and proteins from specimens with clinical outcome in order to validate novel diagnostic biomarkers and to monitor successful treatments for patients. However, using current standard procedures, the collection of specimens is often limited by the availability of liquid nitrogen in some hospitals and liquid nitrogen can be hazardous to transport. These problems would be eased if the tissue could be stored unfixed at 4 degrees C, conditions that are readily available in hospitals. Thus the effect of storing tissue specimens at 4 degrees C on the quality of DNA, RNA and protein has been examined. Clinical tissue samples were halved and kept either at 4 degrees C for up to 24 h or snap-frozen in liquid nitrogen within 30 min of removal from the patient. The results show that the quality of RNA, DNA and protein isolated from the specimens stored at 4 degrees C up to overnight is equal to that obtained from snap-frozen material. In conclusion, simplifying the collection procedure may allow for greater flexibility of conducting studies in units where liquid nitrogen is not readily available.

Expression and splicing of the unfolded protein response gene XBP-1 are significantly associated with clinical outcome of endocrine-treated breast cancer.

X-box binding protein 1 (XBP-1) is stimulated by endoplasmic reticulum stress as part of the unfolded protein response (UPR), which can promote apoptosis or cell survival. Non-conventional splicing, stimulated during the UPR, converts mRNA for "unspliced" XBP-1U to "spliced" XBP-1S mRNA. XBP-1 mRNA is oestrogen-responsive, but XBP-1S confers oestrogen independence and anti-oestrogen resistance to breast cancer cell lines. We therefore evaluated XBP-1 mRNA splicing as a factor in response of breast cancer patients to endocrine treatment. XBP-1 isoforms were measured by quantitative RT-PCR in 100 primary breast cancer patients treated with adjuvant tamoxifen (including 30 ER alpha-negative cases). In ER alpha-positive cases, levels of XBP-1U mRNA correlated with ER alpha mRNA levels and were lower in grade 3 tumors. Higher levels of XBP-1U mRNA were significantly associated with breast cancer survival (Log-rank p = 0.002; Cox hazard ratio (HR) 0.2, p = 0.005), independent of grade, size, nodal status and progesterone receptor status. However, in the full cohort, higher ratios of XBP-1S/XBP-1U mRNA (indicating enhanced splicing) were associated with poor survival (Log-rank p = 0.03; Cox HR 2.3, p = 0.03) and related factors: ER alpha-negative status, progesterone receptor negative status, grade 3 tumors and greater proliferation. Significant associations with poor outcome were also seen for XBP-1 splicing in ER alpha-positive cases. Our findings, that XBP-1 isoforms are differently associated with outcome of endocrine therapy for patients, can be explained by higher levels of dominant-negative XBP-1U favouring apoptosis of tumor cells and higher levels of XBP-1S increasing tumor survival.