Roger Barraclough

Protein synthesis in chloroplasts IX. Assembly of newly-synthesized large subunits into ribulose bishopshate carboxylase in isolated intact pea chloroplasts

Isolated pea (Pisum sativum) chloroplasts incorporate [35S]methionine into the large subunit of the chloroplast enzyme ribulose bisphosphate carboxylase. When chloroplasts are incubated in a medium containing KCl as osmoticum, newly-synthesised large subunits are not incorporated into the holoenzyme but can be separated from pre-existing enzyme by gel electrophoresis under non-denaturating conditions. Furthermore, newly-synthesised large subunits are not precipitated by antibodies which precipitate pre-existing holoenzyme and large subunit prepared from holoenzyme. When chloroplasts are incubated in a medium containing sorbitol as osmoticum, some of the newly-synthesised large subunits comigrate with holoenzyme on both 3% and 5% polyacrylamide non-denaturing gels. Such comigrating large subunits are precipitated by antibodies raised against the holoenzyme. These results indicate assembly of large subunits into ribulose bisphosphate carboxylase in the sorbitol medium. Time course experiments indicate that there is a time-lag of several minutes between onset of synthesis of large subunits and the onset of assembly. Newly-synthesised large subunits which do not comigrate with holoenzyme on both 3% and 5% polyacrylamide non-denaturing gels are associated with a protein of subunit molecular weight 60 000. This protein may be specifically combined with newly-synthesised large subunits, and the resulting aggregate be involved in the assembly of complete molecules of ribulose bisphosphate carboxylase.

Human Homologue of Cement Gland Protein, a Novel Metastasis Inducer Associated with Breast Carcinomas

A suppression subtractive cDNA library representing mRNAs expressed at a higher level in the malignant human breast cancer cell line, MCF-7, relative to a benign breast tumor-derived cell line, Huma 123, contained a cDNA, M36, which was expressed in estrogen receptor alpha (ERalpha)-positive breast carcinoma cell lines but not in cell lines from normal/benign/ERalpha-negative malignant breast lesions. M36 cDNA had an identical coding sequence to anterior gradient 2 (AGR2), the human homologue of the cement gland-specific gene (Xenopus laevis). Screening of breast tumor specimens using reverse transcription-PCR and immunocytochemistry with affinity-purified anti-AGR2 antibodies showed that the presence of AGR2 mRNA and protein were both statistically significantly associated with ERalpha-positive carcinomas (P = 0.007, Fishers exact test) and with malignancy (P < or = 0.025). When an expression vector for AGR2 cDNA was introduced into benign nonmetastatic rat mammary tumor cells, and three separate clones and two pools of cells were transferred to the mammary glands of syngeneic hosts, there were no consistent differences in the mean latent periods of tumor formation. However, metastases occurred in the lungs of animals receiving the AGR2 transfectants in 77% to 92% of animals with primary tumors (P = 0.0001) compared with no metastases in the control groups. The AGR2 transfectants exhibited enhanced rates of adhesion to a plastic substratum and extracellular AGR2 enhanced the rate of attachment of AGR2-negative but not AGR2-positive cells. These experiments are the first to link mechanistically the developmental gene product, AGR2, with metastasis in vivo.

Calcium-binding protein S100A4 in health and disease.

The S100 proteins contain two EF-hand motifs and are of generally unknown function. One of these proteins, S100A4, is an intracellular calcium-binding protein that is present in normal rodent and human cells. In cultured rodent mammary cells, S100A4 is expressed at a higher level in some metastatic epithelial cells than in non-metastatic counterparts. Similarly, in human breast cell lines, S100A4 is present at a higher level in cultured cells from the more malignant, than in those from the more benign tumours. Gene transfer experiments have shown that rodent or human S100A4 is able to induce metastatic capability in otherwise non-metastatic breast tumour cells. Furthermore, expression of rodent S100A4 transgenes can induce metastasis of benign tumours arising in transgenic model systems. Possible mechanisms for the metastasis-inducing effect of S100A4 and the relevance of these observations to human cancer are discussed.

Induction of Metastasis by S100P in a Rat Mammary Model and Its Association with Poor Survival of Breast Cancer Patients

S100P, an EF-hand calcium-binding protein, has been reported to be associated with the progression of many types of cancers. Transfection of an expression vector for S100P into a benign, nonmetastatic rat mammary cell line causes a 4- to 6-fold increase in its level in all four transformant cell clones. When the resultant transformant cell lines are introduced in turn into the mammary fat pads of syngeneic Furth-Wistar rats, there is a significant 3-fold increase in local muscle invasion and a significant induction of metastasis in 64% to 75% of tumor-bearing animals. In a group of 303 breast cancer patients followed for up to 20 years, antibodies to S100P immunocytochemically stain 161 primary tumors. Survival of patients with S100P-positive carcinomas is significantly worse by about 7-fold than for those with negatively stained carcinomas. There is also a significant association between the class level of immunocytochemical staining of the carcinoma cells and decreased patient survival. Positive staining for S100P is significantly associated with that for two other metastasis-inducing proteins, S100A4 and osteopontin. Patients with tumors that stained positively for both S100P and S100A4 have a significantly reduced survival of 1.1% over patients with either S100 protein alone. Multivariate regression analysis identifies S100P, S100A4, and osteopontin as the most significant independent indicators of death in this group of patients. These results suggest that stratification of patients into groups according to expression of multiple metastasis-inducing proteins may lead to a more accurate prediction of patient survival.

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 ERα‐negative cases). In ERα‐positive cases, levels of XBP‐1U mRNA correlated with ERα 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: ERα‐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 ERα‐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.

Expression of S100A4 protein is associated with metastasis and reduced survival in human bladder cancer

The calcium‐binding protein S100A4 induces the metastatic phenotype in rodent models of breast cancer and its expression correlates strongly with reduced survival in human breast cancer. The expression of S100A4 in normal bladders and 101 bladder tumours has been studied using immunocytochemistry. Moderate or strong expression of S100A4 was found in 28% of the tumours, whilst the remaining tumours and normal urothelium either failed to stain or showed weak staining. S100A4 staining was more frequently observed in invasive bladder tumours than in non‐invasive tumours (p<0.05). In invasive tumours, S100A4 staining was usually strongest in invasive regions and single infiltrating cells. Statistically significant associations were found between S100A4 expression and metastasis (p=0.0003) and reduced survival (p<0.0001). It is concluded that S100A4 expression may play an important role in bladder cancer and may identify a subgroup of patients at increased risk of metastasis who should be considered for adjuvant systemic therapy. Copyright

Human S100A4 (p9Ka) induces the metastatic phenotype upon benign tumour cells.

The rodent S100-related calcium-binding protein, S100A4 induces metastasis in non-metastatic rat and mouse benign mammary cells and co-operates with benign-tumour-inducing changes in two transgenic mouse models, to yield metastatic mammary tumours. Co-transfection of the human gene for S100A4 with pSV2neo into the benign rat mammary cell line, Rama 37, yielded cells which expressed a low level of the endogenous S100A4 mRNA, and either high or undetectable levels of human S100A4 mRNA. The cells which expressed a high level of human S100A4 mRNA induced metastasis in the benign rat mammary cell line Rama 37 in an in vivo assay, whereas the cells which expressed an undetectable level of human S100A4 did not induce any detectable metastases. The primary tumours arising from the S100A4-expressing cells contained high levels of immunocytochemically-detected S100A4 and this high level of S100A4 and the metastatic potential were maintained when cells from a metastasis were re-injected into syngeneic rats. The results show that the human S100A4 possesses metastasis-inducing capabilities.

S100A4 regulates cell motility and invasion in an in vitro model for breast cancer metastasis

Elevated levels of the calcium-binding protein S100A4 are associated with poor patient survival in breast cancer patients and induce metastasis in rodent models. To investigate the effects of S100A4 on different components of the metastatic process, epithelial cells lines have been isolated from nonmalignant tumours in neu transgenic mice and from malignant tumours in neu/S100A4 double transgenic mice. Additional cell lines expressing both Neu and S100A4 have also been derived by transfection of rat S100A4 cDNA into tumour cell lines cloned from neu single transgenic mice. Using these cells in transfilter migration assays, it has been shown that increases in either motility or invasive properties correlate with each other and with the level of S100A4 protein. Injection of three of the cell lines separately into the mammary fat pads of nude mice showed that elevated levels of S100A4 correlated with the degree of metastasis to the lungs. In contrast, changes in cell proliferation and cell–substrate adhesion did not correlate with S100A4 levels. Neither motility nor invasiveness correlated with proteolytic degradation of gelatin as measured by zymography. Thus, the results suggest that the main effect of increases in S100A4 levels in metastasis is to generate increased cell motility and invasion and that this latter change is not dependent upon an increased ability to degrade the intercellular matrix.

Comparison of the metastasis-inducing protein S100A4 (p9ka) with other prognostic markers in human breast cancer.

Our aim was to compare the occurrence and prognostic significance over 14–20 years of immunocytochemically detected S100A4 and other tumour variables in primary tumours from 349 patients with operable breast cancer. For a cut‐off of 1% staining of the malignant cells, the antibody to S100A4 stains positively 56% of the carcinomas. There was a significant association of staining for S100A4 with tumours fixed to the chest wall, staining for c‐erbB‐2, c‐erbB‐3, pS2, cathepsin D and, inversely, at borderline levels with staining for estrogen receptor. Using Wilcoxon statistics in univariate analyses, staining for S100A4, nodal status, tumour class, histological grade and staining for c‐erbB‐2, p53 were associated negatively and staining for estrogen receptor, progesterone receptor were associated positively with patient survival times. The survival times of patients with S100A4‐negative carcinomas with or without one of the other tumour variables showed no significant differences, whilst those of patients with S100A4‐positive carcinomas showed significant differences in a negative or a positive way. Multivariate regression analysis for 137 patients showed that staining for S100A4 is most highly correlated with patient deaths, but involved lymph nodes, fixed tumours, high histological grade and staining for progesterone receptor were also significant independent prognostic variables. Our results suggest that in this set of patients, the tumour variable most tightly correlated with patient death is S100A4. Int. J. Cancer (Pred. Oncol.) 89:198–208, 2000.

Association of S100A4 and osteopontin with specific prognostic factors and survival of patients with minimally invasive breast cancer.

PURPOSE: S100A4 and the estrogen-inducible osteopontin are alone capable of inducing angiogenesis and metastasis in rodent models for breast cancer. The present study assesses the relationship of S100A4 and osteopontin with vessel density and estrogen receptor alpha (ERalpha) in primary tumors and with survival of patients to ascertain their involvement in metastatic breast cancer. EXPERIMENTAL DESIGN: Primary tumors from 312 patients treated for minimally invasive human breast cancer were immunocytochemically stained and then assessed for the significance of their association with each other using Fishers exact test or with patient survival over 18 years of follow-up using Kaplan-Meier plots and Wilcoxon-Gehan statistics. RESULTS: Antibodies to S100A4 significantly stained endothelial cells of vessels adjacent to S100A4-staining groups of carcinoma cells, and antibodies to osteopontin significantly stained groups of carcinoma cells staining for ERalpha (P < 0.0001). There was a significant association of tumors staining for S100A4 with those with high vessel density (P = 0.021) and of tumors staining for osteopontin with those staining for ERalpha (P = 0.034). The association of staining for S100A4, osteopontin, or vessel density with patient death was significant (P < 0.0001, P = 0.005, and P = 0.014, respectively). The difference in cumulative proportion surviving between S100A4-positive patients with higher or lower vessel density increased up to about 12 years, but thereafter decreased to virtually zero after 18 years of follow-up. Patients with both S100A4-positive and osteopontin-positive primary tumors showed a statistically significant reduction in survival time over those with either one alone (P < 0.019), although in multivariate regression analysis, only staining for S100A4 was significant (P < 0.001). CONCLUSIONS: It is suggested that in human breast cancer, S100A4 exerts some of its effects through angiogenesis, and that osteopontin is dependent on ERalpha for its expression.