Our previous study demonstrated that conditional reprogramming (CR) allows the establishment of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types including breast, lung, colon and prostate. Using CR, we have established matched normal and tumor cultures, GUMC-29 and GUMC-30 respectively, from a patient's prostatectomy specimen. These CR cells proliferate indefinitely in vitro and retain stable karyotypes. Most importantly, only tumor-derived CR cells (GUMC-30) produced tumors in xenografted SCID mice, demonstrating maintenance of the critical tumor phenotype. Characterization of cells with DNA fingerprinting demonstrated identical patterns in normal and tumor CR cells as well as in xenografted tumors. By flow cytometry, both normal and tumor CR cells expressed basal, luminal, and stem cell markers, with the majority of the normal and tumor CR cells expressing prostate basal cell markers, CD44 and Trop2, as well as luminal marker, CD13, suggesting a transit-amplifying phenotype. Consistent with this phenotype, real time RT-PCR analyses demonstrated that CR cells predominantly expressed high levels of basal cell markers (KRT5, KRT14 and p63), and low levels of luminal markers. When the CR tumor cells were injected into SCID mice, the expression of luminal markers (AR, NKX3.1) increased significantly, while basal cell markers dramatically decreased. These data suggest that CR cells maintain high levels of proliferation and low levels of differentiation in the presence of feeder cells and ROCK inhibitor, but undergo differentiation once injected into SCID mice. Genomic analyses, including SNP and INDEL, identified genes mutated in tumor cells, including components of apoptosis, cell attachment, and hypoxia pathways. The use of matched patient-derived cells provides a unique in vitro model for studies of early prostate cancer.

Using conditional cell reprogramming, we generated a stable cell culture of an extremely rare and aggressive neuroendocrine cervical cancer. The cultured cells contained HPV-16, formed colonies in soft agar and rapidly produced tumors in immunodeficient mice. The HPV-16 genome was integrated adjacent to the Myc gene, both of which were amplified 40-fold. Analysis of RNA transcripts detected fusion of the HPV/Myc genes, arising from apparent microhomologous recombination. Spectral karyotyping (SKY) and fluorescent-in-situ hybridization (FISH) demonstrated coordinate localization and translocation of the amplified Myc and HPV genes on chromosomes 8 and 21. Similar to the primary tumor, tumor cell cultures expressed very high levels of the Myc protein and, in contrast to all other HPV-positive cervical cancer cell lines, they harbored a gain-of-function mutation in p53 (R273C). Unexpectedly, viral oncogene knockdown had no effect on the growth of the cells, but it did inhibit the proliferation of a conventional HPV-16 positive cervical cancer cell line. Knockdown of Myc, but not the mutant p53, significantly inhibited tumor cell proliferation. On the basis of these data, we propose that the primary driver of transformation in this aggressive cervical cancer is not HPV oncogene expression but rather the overexpression of Myc.

Fibulin-4, an extracellular glycoprotein, which plays significant roles in elastic fiber assembly, is correlated to the progression of some cancers. However, the role of fibulin-4 in endometrial cancer cell invasion and metastasis remains unexplored. In our study, fibulin-4 expression was assessed by immunohistochemistry (IHC) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in normal endometrial tissues and endometrial carcinoma tissues. Using single cell cloning, strongly, and weakly, invasive subclones were derived from KLE and Ishikawa endometrial carcinoma cell lines. RT-qPCR, western blotting, and immunocytochemistry (ICC) were used to assess mRNA and protein expressions of fibulin-4 in primary cultured endometrial cells, 4 types of endometrial cancer cell lines, and the different invasive subclones. Using lentivirus transfection, fibulin-4 shRNA and pLVX-fibulin-4 were constructed and used to infect the strongly and weakly invasive subclones. The effects of fibulin-4 on the biological characteristics of endometrial carcinoma cells were detected by cell functional assays in vitro and in vivo. Using Wnt signaling pathway inhibitor XAV-939 and activator LiCl, we detected the role of fibulin-4 in the Wnt/β-catenin pathway and the relationship with epithelial to mesenchymal transition (EMT). Fibulin-4 was decreased in endometrial carcinoma tissues, and loss of fibulin-4 expression was significantly related with poor differentiation, lymph node metastasis, and poor prognosis of endometrial carcinoma. Fibulin-4 significantly inhibited endometrial carcinoma cell proliferation, invasion, metastasis, and EMT through the Wnt/β-catenin pathway. Fibulin-4 has the ability to suppress endometrial cancer progression. These results can contribute to the development of a new potential therapeutic target for patients with endometrial carcinoma.

The Myc/Max/Mad network plays a critical role in cell proliferation, differentiation and apoptosis and c-Myc is overexpressed in many cancers, including HPV-positive cervical cancer cell lines. Despite the tolerance of cervical cancer keratinocytes to high Myc expression, we found that the solitary transduction of the Myc gene into primary cervical and foreskin keratinocytes induced rapid cell death. These findings suggested that the anti-apoptotic activity of E7 in cervical cancer cells might be responsible for negating the apoptotic activity of over-expressed Myc. Indeed, our earlier in vitro studies demonstrated that Myc and E7 synergize in the immortalization of keratinocytes. Since we previously postulated that E7 and the ROCK inhibitor, Y-27632, were members of the same functional pathway in cell immortalization, we tested whether Y-27632 would inhibit apoptosis induced by the over-expression of Myc. Our findings indicate that Y-27632 rapidly inhibited Myc-induced membrane blebbing and cellular apoptosis and, more generally, functioned as an inhibitor of extrinsic and intrinsic pathways of cell death. Most important, Y-27632 cooperated with Myc to immortalize keratinocytes efficiently, indicating that apoptosis is a major barrier to Myc-induced immortalization of keratinocytes. The anti-apoptotic activity of Y-27632 correlated with a reduction in p53 serine 15 phosphorylation and the consequent reduction in the expression of downstream target genes p21 and DAPK1, two genes involved in the induction of cell death.

EFEMP1, a kind of extracellular matrix (ECM) protein, has been suggested to correlate with the development of different types of carcinoma. However, its functions in ovarian cancer remain unclear. In our study, we performed cDNA microarray analysis and identified EFEMP1 dramatically elevated in the highly invasive subclone, compared with the low invasive subclone. Lentivirus transfection experiments were constructed afterwards. The results demonstrated that knockdown of EFEMP1 significantly inhibited ovarian cancer cell proliferation and induced cell cycle arrest at the G1/G0 phase. We also found that decreased the activity of phospho-AKT could suppress cell invasion and metastasis. Meanwhile, the increased phospho-AKT activity induced by the overexpression of EFEMP1 had significantly enhanced the abilities of ovarian cancer cells to invade and migrate. In addition, the vivo nude mice model confirmed that EFEMP1 was tightly correlated with the development of tumor. The results of RT2 Profiler EMT PCR array further indicated that decreased EFEMP1 suppressed epithelial-to-mesenchymal transition (EMT). Collectively, by activating AKT signaling pathway, EFEMP1 contributed to ovarian cancer invasion and metastasis as a positive regulator. Overall, EFEMP1 had showed the potential use in the development of new therapeutic strategies for ovarian cancer.