Lawrence Berkeley National Laboratory

Abstract: Normal human somatic cells have a finite lifespan and intact tumor suppressor barriers, whereas most carcinoma cells have gained immortality and overcome multiple tumor suppressor barriers. We have developed a comprehensive human mammary epithelial cell (HMEC) culture system to examine the alterations involved in the transition from normal finite to malignant immortal; such data can suggest prevention strategies. Our studies have indicated that HMEC initially encounter a stress-associated senescence barrier (stasis) enforced by retinoblastoma (RB). Errors in the RB pathway allow stasis bypass; similar errors are seen early in progression in vivo. Replicative senescence due to telomere erosion is a second extremely stringent barrier, with critically short telomeres leading to genomic instability. Overcoming this barrier requires reactivation of endogenous telomerase, similar to what is seen in high-grade DCIS in vivo. Resulting immortal HMEC are resistant to oncogene-induced-senescence, and exposure to oncogenes that cause finite cells to senesce, can now give rise to malignancy, underscoring the critical importance of the immortalization step in progression. Also like DCIS, the molecular properties of our non/pre-malignant immortal HMEC lines are more similar to malignant immortal than normal finite cells, highlighting the abnormal, cancer-like qualities of immortalized cells. We suggest this similarity is due to a novel process that we have discovered to be involved in cancer-associated immortal transformation, that we have called conversion. HMEC that gain an error permissive for telomerase reactivation still need to undergo additional changes to assume the cancer-associated immortal phenotype: expression of sufficient telomerase activity to maintain short telomeres, mean TRF ~4 kb (which is shorter than what is present in normal finite cells). Conversion appears initiated by a mean TRF of <4 kb, and may entail changes in telomere structure. Possibly, the observed similar alterations in immortal cells ± malignancy results from changes induced by conversion. Importantly, cancer-associated immortalization is unique to cells progressing to cancer, and its inhibition may thus be a valuable therapeutic target. Surprisingly, little has been done to explore this possibility, we believe because: 1) small short-lived animals like mice do not stringently repress telomerase in adult cells; lacking a significant immortalization barrier, they cannot model this critical step in human carcinogenesis; 2) Immortally transformed cells are commonly referred to as normal or untransformed; 3) hTERT immortalization does not model cancer-associated immortalization; there is no conversion to short telomeres. Our preliminary data suggest potential approaches to inhibiting conversion. Citation Format: Martha R. Stampfer, Tara Fresques, Sun-Young Lee, Mark LaBarge, James Garbe. The immortalization process as a therapeutic target [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5818.