Immortalization-associated gene signature in breast cancer

Background

Cell immortalization has been considered a hallmark of malignancy, yet the molecular signature identifying a tumor's ability to immortalize is not known. To better understand the immortalization process, we used in vitro model systems developed from clinical primary breast cancer specimens. While spontaneous immortalization of tumor cells in vitro is a rare event, immortalization can be achieved through the introduction of the hTERT component of telomerase. Because spontaneously arising cancer cell lines are already immortal and display high levels of telomerase expression, they are not amenable for study of the immortalization process in malignancy. However, finite life primary tumor cultures show low hTERT expression, providing a tool to study the effect of exogenously introduced hTERT.

Methods and results

We introduced the hTERT gene into 17 primary breast cancer cultures and analyzed global gene expression changes before and after hTERT transduction in nine cases. All transduced cultures achieved immortalization without other genetic manipulation. Using 42,000 feature cDNA microarrays, we identified 594 genes (the immortalization-associated signature [IAS]) that distinguished hTERT-transduced and non-transduced cultures. We suggest that hTERT immortalization reflects a physiologic process since expression patterns of hTERT-transduced and spontaneously immortalized primary cultures along IAS genes were correlated. To identify whether these genes relate to in vivo breast cancer, we used the IAS to supervise the analysis of 295 breast cancer patients. The signature strongly predicted distant metastasis-free (P = 0) and overall survival (P < 0.001) in ER-positive tumors but not in ER-negative tumors. Notably, hTERT gene expression level itself did not predict clinical outcome in primary tumors.

Conclusion

To our knowledge, this is the first demonstration of an immortalization signature in human breast cancer. This signature is strongly associated with specific cellular pathways in human tumors that may underlie the immortalization process and influence tumor behavior. The association between the IAP signature and outcome for ER-positive tumors but not ER-negative tumors suggests that different pathways are involved in the outcome of ER-negative tumors.