A complex transcriptional circuitry is essential in maintaining the self-renewal capacity and the undifferentiated state of embryonic stem cells (ESCs). Key factors in this pluripotency network include Sox2, Nanog, and Oct3/4, which form multiple positive and negative feedback loops to regulate an array of downstream genes. Although it was previously thought that pluripotency factors are downregulated upon differentiation, the phenotypic similarities between ESCs and cancer cells have led to speculation that common underlying regulatory mechanisms may exist. For example, the cancer phenotype includes a block in differentiation, limitless replicative potential and increased proliferation, characteristics that are also observed in pluripotent embryonic stem cells. Taken together, these observations regarding similarities in tumor cells and embryonic stem cells suggest regulatory mechanisms that may be shared in development and cancer. In the studies presented here, I investigated the expression, localization, and function of Nanog, Sox2, Oct4 and particularly the Oct3 splice variant protein in cancer.

I performed a variety of biochemical, genetic and pharmacological experiments to elucidate potential roles of these pluripotency factors in maintaining the cancer phenotype, using a panel of breast cancer cells as a model system. Expression of Sox2 and the Oct3 isoform was detected in all breast cancer cell lines investigated, whereas Nanog and Oct4 expression were not detected. Moreover, Sox2 was localized to both the cytoplasm and nucleus, but Oct3 expression was detected exclusively the cytoplasm of breast cancer cells. This study also shows that Oct3 and Sox2 are upregulated in a number of breast cancer cell lines when compared to expression levels in mammary epithelial cells. I also determined that Src is an upstream regulator of Oct3 and Sox2 expression.

Given the known function of Sox2 as a regulator of the cell cycle, I focused subsequent studies on the characterization and function of Oct3. Results showed that the Oct3 N-terminal domain contains an auto-inhibitory sequence that blocks its nuclear translocation despite the presence of a functional nuclear localization signal. Additional experiments to investigate Oct3 function suggested that Oct3 may be involved in regulating the cell cycle and that this regulation may be in concert with Sox2 and β-catenin to modulate cyclin D1 expression. However, studies of Oct3 function in cancer remain inconclusive due to technical difficulties in Oct3 RNAi that precluded achieving consistent Oct3 knockdown. Despite these difficulties, the data presented here suggest that Oct3 may play a role in maintaining the cancer phenotype, warranting further functional studies.

The leading cause of death in breast cancer patients is metastasis. Cancer metastasis involves a complex series of events, including cell detachment from the primary tumor, invasion into the surrounding tissue, intravasation into the circulatory system, extravasation, and growth in a new organ. Src Family Kinases (SFKs) comprise a group of non-receptor tyrosine kinases that regulate a variety of pathways that promote cell survival, growth, proliferation, motility, and invasion. Previous studies have found a correlation between the activity of SFKs and the progression of breast cancer into metastasis. Since the pathways regulated by SFKs are often activated in metastasis, I investigated whether SFKs play a role in the metastasis of breast cancer cells.

To determine whether SFKs regulate breast cancer metastasis, I analyzed the role of SFKs in the migration and invasion of a metastatic breast cancer cell line, MDA-MB-231 cells. SFKs were inhibited with expression of dominant negative Src or by treatment with the pharmacological inhibitors PP2 and SU6656. The inhibition of SFKs in MDA-MB-231 cells led to decreased cell migration and invasion through transwell migration and invasion chambers. To explore the mechanism by which SFKs regulate cellular migration and invasion, I investigated whether SFKs regulate the Akt1 and Akt2 isoforms in MDA-MB-231 breast cancer cells. Akt isoforms are serine/threonine kinases that are often activated in cancer cells. The activity of Akt isoforms were decreased in SFK-inhibited MDA-MB-231 cells as determined by immunoblot detection of the phosphorylation of Threonine 308 and Serine 473 residues, in vitro kinase assays, and immunoblot detection of the phosphorylation of an Akt substrate. To determine whether the Akt isoforms play a role in the migration and/or invasion of breast cancer cells, small interfering RNAs were used to knockdown the expression of each Akt isoform. Akt2 knockdown specifically led to decreased MDA-MB-231 cell migration and invasion by mechanisms that did not involve the attachment of cells onto extracellular matrix or the regulation of Pak1. Co-immunoprecipitation and mass spectrometry assays were used in an effort to identify an Akt2-specific binding partner that could mediate its role in breast cancer cell migration and invasion. The Arf6-GAP ACAP2 was identified as a protein that co-immunoprecipitated with Akt2-Flag, but additional analysis found that ACAP2 was immunoprecipitated non-specifically by the mouse anti-Flag antibody and does not interact with Akt2.

These results point to a role of SFKs in breast cancer metastasis, specifically in promoting cell migration and invasion. Although SFKs regulate the activation of both Akt1 and Akt2 isoforms in MDA-MB-231 cells, only Akt2 is required for the migration and invasion of MDA-MB-231 cells.