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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

The ubiquitin ligase CUL4A has been implicated in tumorigenesis, but its contributions to progression and metastasis have not been evaluated. Here, we show that CUL4A is elevated in breast cancer as well as in ovarian, gastric, and colorectal tumors in which its expression level correlates positively with distant metastasis. CUL4A overexpression in normal or malignant human mammary epithelial cells increased their neoplastic properties in vitro and in vivo, markedly increasing epithelial-mesenchymal transition (EMT) and the metastatic capacity of malignant cells. In contrast, silencing CUL4A in aggressive breast cancer cells inhibited these processes. Mechanistically, we found that CUL4A modulated histone H3K4me3 at the promoter of the EMT regulatory gene ZEB1 in a manner associated with its transcription. ZEB1 silencing blocked CUL4A-driven proliferation, EMT, tumorigenesis, and metastasis. Furthermore, in human breast cancers, ZEB1 expression correlated positively with CUL4A expression and distant metastasis. Taken together, our findings reveal a pivotal role of CUL4A in regulating the metastatic behavior of breast cancer cells.

The ubiquitin ligase CUL4A has been implicated in tumorigenesis, but its contributions to progression and metastasis have not been evaluated. Here, we show that CUL4A is elevated in breast cancer as well as in ovarian, gastric, and colorectal tumors in which its expression level correlates positively with distant metastasis. CUL4A overexpression in normal or malignant human mammary epithelial cells increased their neoplastic properties in vitro and in vivo, markedly increasing epithelial--mesenchymal transition (EMT) and the metastatic capacity of malignant cells. In contrast, silencing CUL4A in aggressive breast cancer cells inhibited these processes. Mechanistically, we found that CUL4A modulated histone H3K4me3 at the promoter of the EMT regulatory gene ZEB1 in a manner associated with its transcription. ZEB1 silencing blocked CUL4A-driven proliferation, EMT, tumorigenesis, and metastasis. Furthermore, in human breast cancers, ZEB1 expression correlated positively with CUL4A expression and distant metastasis. Taken together, our findings reveal a pivotal role of CUL4A in regulating the metastatic behavior of breast cancer cells. [ABSTRACT FROM AUTHOR]