Telomerase activity is associated with cell cycle deregulation in human breast cancer.

Deregulation of the cell cycle by abnormal expression of one or several cell cycle regulatory proteins is a common finding in malignant tumors and might be a prerequisite for cancer development. Telomerase activity is an immortalization marker that is found in most cancers and for which an association with an active cell cycle has been implicated. In the tissue of 106 human breast carcinomas, we analyzed the relationship between telomerase activity levels and defects in the cell cycle machinery with a focus on the retinoblastoma protein (pRB) pathway(s). The fraction of telomerase-positive tumors was 85%, and large differences in telomerase activity were found. Overexpression of cyclin D1 and/or cyclin E, in combination with a normal pRB, was a typical feature of tumors with high telomerase activity levels. Down-regulation of p16INK4 was not related per se to telomerase activity, but tumors with low p16INK4 in combination with cyclin D1 or E overexpression demonstrated high activity. Tumor cell proliferation, determined by Ki-67 expression, correlated significantly to telomerase activity levels. There was, however, not a strict association between proliferation rate and telomerase activity, because tumors with inactivated pRB had the highest Ki-67 fractions but intermediate telomerase activity. Also, cyclin D1 overexpression was associated with high telomerase levels without an increase in tumor cell proliferation. The present study indicates that telomerase activation occurs preferentially in breast cancers with certain cell cycle regulatory defects and that telomerase activity levels may depend on the specific defect(s).

Deregulation of cyclin E and D1 in breast cancer is associated with inactivation of the retinoblastoma protein.

Inactivation of the retinoblastoma protein (pRB) by mutations or abnormal phosphorylation is a mechanism by which tumour cells can subdue normal growth control. Among molecules involved in control of pRB phosphorylation, cyclin D1 and E have been found to be deregulated and overexpressed in various types of cancers. In order to study the cell cycle regulatory mechanisms in breast cancer, we have analysed the protein expression of cyclin D1 and E in 114 tumour specimens from patients with primary breast cancer using Western blotting. Twenty-five out of 34 tumours with overexpression of cyclin E showed uniform low cyclin D1 expression, and by immunohistochemical analysis of pRB we present evidence for the existence of pRB defects in approximately 40% of these tumours in contrast to no pRB defects in the other group of tumours. This result was supported by a high protein expression of the cyclin-dependent kinase inhibitor p16 in 44% of the tumours with high cyclin E and low D1 expression, and all immunohistochemical pRB defect tumours showed a high p16 protein level. Additionally, an abnormal low pRB phosphorylation in relation to a high proliferative activity and loss of heterozygosity of the retinoblastoma susceptibility gene locus were found in all but one tumour with immunohistochemical defect pRB. Interestingly, tumours with high cyclin E and low D1 expression were generally oestrogen receptor negative suggesting a role for cell cycle regulators in the mechanisms leading to oestrogen independent tumour growth. Furthermore, the prognosis differed markedly for the patients in the various groups of tumours, indicating that the heterogeneous nature of breast cancer pathogenesis and the clinical course in part could be explained by different and distinctive sets of cell cycle defects.

G1-S transition defects occur in most breast cancers and predict outcome.

Cell cycle deregulation is frequently observed in tumors and has moreover been proposed to be a requirement for tumor development. By analyzing the expression of p27 by immunohistochemistry in 100 primary breast tumors and combining the analyses with our earlier characterization of cyclin E, D1, p16, and the retinoblastoma protein (pRB), we have been able to cover the majority of potential G1-S transition defects and observed that 90% of the tumors had alterations in one or several cell cycle regulatory proteins. Considerable variations in protein levels were found among tumors, with low p16 expression as the most common alteration followed by cyclin E or cyclin D1 overexpression, low p27 expression or pRB inactivation in decreasing prevalence. Tumors were grouped according to observed combinations of defects and the proliferative capacity was determined for each group by analyzing Ki-67 labeling index. Low proliferation was observed in tumors with: low p16; high cyclin D1 with normal or high p16 expression; and in tumors without cell cycle defects. Tumors with high cyclin E/low p27 or pRB defects showed higher proliferation. The survival differed noticeably for patients with various combinations of cell cycle defects, and four distinctive clusters were identified showing significantly different breast cancer specific survival (p<0.0001) for both node-positive (p = 0.0006) and node-negative patients (p<0.0001). In summary, we have shown that G1-S transition defects are nearly obligatory in breast tumors and that the specific type of cell cycle defect influences the clinical behavior of the tumor.