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 ERa-negative cases). In ERa-positive cases, levels of XBP-1U mRNA correlated with ERa 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 5 0.002; Cox hazard ratio (HR) 0.2, p 5 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 5 0.03; Cox HR 2.3, p 5 0.03) and related factors: ERa-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 ERa-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.

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.

The secreted metastasis-inducing protein, human anterior gradient 2 (AGR2), has been independently reported to be associated with either a reduced or an increased survival of different groups of patients with breast cancer. We now aim to analyze the expression of AGR2 in a third completely independent group of patients using a specific AGR2 monoclonal antibody (mAb). Primary tumors from a group of 315 patients suffering from operable (stage I and II) breast cancer with 20-years follow-up were immunocytochemically stained with a specific mAb to AGR2 and associations with prognostic factors and patient survival were analyzed. The mAb specifically recognized AGR2 in Western blots, and positive staining for AGR2 was significantly associated with involved lymph nodes and staining for estrogen receptor ␣, progesterone receptor, and the metastasis-inducing proteins osteopontin, S100P, and S100A4. After 20 years of follow-up, only 26% of patients with AGR2-positive carcinomas survived compared with 96% of those with AGR2 negative carcinomas, with the highly significant difference in median survival times of 68 and >216 months, respectively (P < 0.0001). Cox's multivariate regression analysis showed that staining for AGR2 was one of the most significant independent prognostic indicators, with a corrected relative risk of 9. Anterior gradient 2 (AGR2) protein is a secreted protein first described in Xenopus laevis embryos, where it induces the formation of the forebrain and the mucussecreting cement gland.1 Human AGR2 is also found co-expressed with estrogen receptor ␣ (ER␣) in breast cancer cell lines 2 and its presence significantly correlates with ER␣ in breast carcinoma specimens.3 Subsequent studies have found elevated expression of AGR2 in adenocarcinomas of the esophagus, pancreas, prostate, and non-small cell lung cancer, showing that it is a widely overexpressed protein in human carcinomas. 4 -10 We have shown that human AGR2 is expressed at higher levels in malignant, rather than in benign breast tumors, 11 and that, when introduced in an expression vector into the benign, nonmetastatic rat mammary cell line, Rama 37, 12 it causes metastasis in syngeneic rats. 11 These results suggest that AGR2's metastasis-inducing properties may contribute toward the malignant progression of some breast cancers. Certain molecules shown to induce metastasis in experimental breast cancer in rodents, for example, S100A4, S100P, and osteopontin (OPN), [13][14][15][16] provide a potential source for markers that may be useful as prognostic factors in predicting patient outcome in human breast cancer.

Osteoporosis is the most common age-related bone disease worldwide and is usually clinically asymptomatic until the first fracture happens. MicroRNAs are critical molecular regulators in bone remodelling processes and are stabilised in the blood. The aim of this project was to identify circulatory microRNAs associated with osteoporosis using advanced PCR arrays initially and the identified differentially-expressed microRNAs were validated in clinical samples using RT-qPCR. A total of 161 participants were recruited and 139 participants were included in this study with local ethical approvals prior to recruitment. RNAs were extracted, purified, quantified and analysed from all serum and plasma samples. Differentially-expressed miRNAs were identified using miRNA PCR arrays initially and validated in 139 serum and 134 plasma clinical samples using RT-qPCR. Following validation of identified miRNAs in individual clinical samples using RT-qPCR, circulating miRNAs, hsa-miR-122-5p and hsa-miR-4516 were statistically significantly differentially-expressed between non-osteoporotic controls, osteopaenia and osteoporosis patients. Further analysis showed that the levels of these microRNAs were associated with fragility fracture and correlated with the low bone mineral density in osteoporosis patients. The results show that circulating hsa-miR-122-5p and hsa-miR-4516 could be potential diagnostic biomarkers for osteoporosis in the future.

FANCD2, a pivotal protein in the Fanconi anemia and BRCA pathway/network, is monoubiquitylated in the nucleus in response to DNA damage. This study examines the subcellular location and relationship with prognostic factors and patient survival of FANCD2 in breast cancer. Antibodies to FANCD2 were used to immunocytochemically stain 16 benign and 20 malignant breast specimens as well as 314 primary breast carcinomas to assess its association with subcellular compartment and prognostic factors using Fisher's Exact test or with patient survival over 20 years using Wilcoxon-Gehan statistics. Immunoreactive FANCD2 was found in the nucleus and cytoplasm of all 16 benign tissues, but nuclear staining was lost from a significant 19/20 malignant carcinomas (P < 0.0001). Antibodies to FANCD2 stained the cytoplasm of 196 primary carcinomas, leaving 118 as negatively stained. Negative cytoplasmic staining was significantly associated with positive staining for the metastasis-inducing proteins S100A4, S100P, osteopontin, and AGR2 (P < 0.002). Survival of patients with FANCD2-negative carcinomas was significantly worse (P < 0.0001) than those with positively stained carcinomas, and only 4% were alive at the census date. Multivariate regression analysis identified negative staining for cytoplasmic FANCD2 as the most significant indicator of patient death (P ‫؍‬ 0.001). Thus FANCD2's cytoplasmic loss in the primary carcinomas may allow the selection of cells overexpressing proteins that can induce metastases before surgery. Previous reports have shown that the expression of four proteins that can induce metastasis in experimental rats are highly significantly correlated with each other and separately with early patient death in human breast cancer. These four proteins are S100A4, osteopontin (OPN), AGR2, and S100P.1-4 If their expression is coordinated, then markers of the underlying mechanism should be even more highly correlated with patient demise. One possible coordination mechanism is the generation of an unstable genome by failure of a DNA repair pathway or some other protective mechanism. The majority of familial breast cancer is associated with individuals heterozygous for the BRCA1 or BRCA2 genes that encode proteins important for the repair of DNA double strand breaks and interstrand crosslinks by homologous recombination. 5,6 Biallelic inactivation of BRCA2 results in one form of the cancer-prone syndrome Fanconi anemia (complementation group FA-D1) and the BRCA2/FANCD1 protein operates with 12 other Fanconi anemia (FA) proteins and BRCA1 in a multifaceted response to DNA damage known as the FA/ BRCA tumor suppressor pathway/network. 7-10 Eight of the 13 proteins, together with two FA-associated-proteins, participate in a nuclear core-complex that is required for the monoubiquitylation of FANCD2 and FANCI.

We propose that these MIPs enhance endometrial cell invasiveness and contribute to the establishment of ectopic endometriotic deposits after retrograde menstruation.

Ovarian cancer is often asymptomatic and is diagnosed at an advanced stage with poor survival rates, thus there is an urgent need to develop biomarkers for earlier detection of ovarian cancer. In the present study, we demonstrate for the first time that the previously reported metastasis-inducing protein AGR2 (anterior gradient protein 2) can be detected in the blood of ovarian cancer patients. Using a newly developed ELISA, we show significantly increased concentrations of AGR2 protein in plasma from cancer patients relative to normal controls. Plasma AGR2 concentrations were highest in stages II and III ovarian cancer patients and were similarly elevated in patients with both serous and non-serous tumours. The identification of elevated plasma concentrations of AGR2 may provide a useful biomarker to aid in the discrimination of normal and ovarian cancer patients particularly when used in combination with CA125.

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