Vimla Band

Background: The four highly homologous human EHD proteins (EHD1-4) form a distinct subfamily of the Eps15 homology domain-containing protein family and are thought to regulate endocytic recycling. Certain members of this family have been studied in different cellular contexts; however, a lack of concurrent analyses of all four proteins has impeded an appreciation of their redundant versus distinct functions. Results: Here, we analyzed the four EHD proteins both in mammalian cells and in a cross-species complementation assay using a C. elegans mutant lacking the EHD ortholog RME-1. We show that all human EHD proteins rescue the vacuolated intestinal phenotype of C. elegans rme-1 mutant, are simultaneously expressed in a panel of mammalian cell lines and tissues tested, and variably homo- and hetero-oligomerize and colocalize with each other and Rab11, a recycling endosome marker. Small interfering RNA (siRNA) knock-down of EHD1, 2 and 4, and expression of dominant-negative EH domain deletion mutants showed that loss of EHD1 and 3 (and to a lesser extent EHD4) but not EHD2 function retarded transferrin exit from the endocytic recycling compartment. EH domain deletion mutants of EHD1 and 3 but not 2 or 4, induced a striking perinuclear clustering of co-transfected Rab11. Knock-down analyses indicated that EHD1 and 2 regulate the exit of cargo from the recycling endosome while EHD4, similar to that reported for EHD3 (Naslavsky et al. (2006) Mol. Biol. Cell 17, 163), regulates transport from the early endosome to the recycling endosome. Conclusion: Altogether, our studies suggest that concurrently expressed human EHD proteins perform shared as well as discrete functions in the endocytic recycling pathway and lay a foundation for future studies to identify and characterize the molecular pathways involved.

Our laboratory is involved in identifying genes that can be used as early diagnostic or prognostic markers in breast cancer. We previously identified a gene (NES1) that is expressed in normal but not in transformed mammary epithelial cells (MECs).NES1is located on chromosome 19q13.4 within the kallikrein locus and thus was designated as human kallikrein 10 (hK10), although we have been unable to detect any protease activity. Importantly,hK10expression is decreased in a majority of breast cancer cell lines. Transfection ofhK10intohK10-negative breast cancer cells reduces the tumorigenicity. Using methylation-specific PCR and subsequent sequencing, we demonstrate a strong correlation between hypermethylation ofhK10and loss of mRNA expression. Further analysis showed that essentially 100% of normal breast specimens hadhK10expression, whereas 46% of ductal carcinomain situ(DCIS) and the majority of infiltrating ductal carcinoma (IDC) samples lacked thehK10 mRNA. Importantly,hK10-negative DCIS diagnosed at the time of biopsy were subsequently diagnosed as IDC at the time of definitive surgery. It has been shown that hK10 protein expression is regulated by steroids. In addition to breast cancers, hK10 is downregulated in cervical cancer, prostate cancer and acute lymphocytic leukemia, whereas it is upregulated in ovarian cancers. These results point to the paradoxical role of hK10 in human cancers and underscore the importance of further studies of this kallikrein.

Human kallikrein 10 (hK10) protein is expressed in normal breast but is significantly downregulated in a majority of invasive breast cancers. Thus, understanding how hK10 expression is regulated is of substantial significance. In this study, we analyzed the promoter region of hK10 using a website software (TRANSFAC 3.0), which predicted three possible retinoic acid response elements (RAREs), RARE1 at -1041 (TGACCTCGTGATCC), RARE2 at -859 (TGACCTCCTATGA) and RARE3 at -765 (TGACCTCCTGTGA), each with a half-site of a canonical sequence (TGACCT; reverse complement AGGTCA). Using electrophoretic mobility shift assays and nucleotide competition analysis, as well as chromatin immunoprecipitation of the native hK10 promoter, we demonstrated specific binding of RXR only to RARE1. The functional importance of RARE in the hK10 promoter was demonstrated by retinoid induction of hk10 promoter-reporters; furthermore, mutation of RARE1 but not of RARE2 or RARE3 abolished the induction of the reporter. Finally, we demonstrated the induction of hK10 mRNA and protein expression upon retinoid treatment of cells. In view of the correlation of the downregulation of hK10 mRNA and protein with breast cancer progression, these findings suggest a potential approach to restore hK10 expression in cancer patients.

Normal human mammary epithelial cells (HMECs) have a finite life span and do not undergo spontaneous immortalization in culture. Critical to oncogenic transformation is the ability of cells to overcome the senescence checkpoints that define their replicative life span and to multiply indefinitely – a phenomenon referred to as immortalization. HMECs can be immortalized by exposing them to chemicals or radiation, or by causing them to overexpress certain cellular genes or viral oncogenes. However, the most efficient and reproducible model of HMEC immortalization remains expression of high-risk human papillomavirus (HPV) oncogenes E6 and E7. Cell culture models have defined the role of tumor suppressor proteins (pRb and p53), inhibitors of cyclin-dependent kinases (p16^INK4a, p21, p27 and p57), p14^ARF, telomerase, and small G proteins Rap, Rho and Ras in immortalization and transformation of HMECs. These cell culture models have also provided evidence that multiple epithelial cell subtypes with distinct patterns of susceptibility to oncogenesis exist in the normal mammary tissue. Coupled with information from distinct molecular portraits of primary breast cancers, these findings suggest that various subtypes of mammary cells may be precursors of different subtypes of breast cancers. Full oncogenic transformation of HMECs in culture requires the expression of multiple gene products, such as SV40 large T and small t, hTERT (catalytic subunit of human telomerase), Raf, phosphatidylinositol 3-kinase, and Ral-GEFs (Ral guanine nucleotide exchange factors). However, when implanted into nude mice these transformed cells typically produce poorly differentiated carcinomas and not adenocarcinomas. On the other hand, transgenic mouse models using ErbB2/neu, Ras, Myc, SV40 T or polyomavirus T develop adenocarcinomas, raising the possibility that the parental normal cell subtype may determine the pathological type of breast tumors. Availability of three-dimensional and mammosphere models has led to the identification of putative stem cells, but more studies are needed to define their biologic role and potential as precursor cells for distinct breast cancers. The combined use of transformation strategies in cell culture and mouse models together with molecular definition of human breast cancer subtypes should help to elucidate the nature of breast cancer diversity and to develop individualized therapies.

The vast majority of breast cancers are carcinomas that arise from mammary epithelial cells (MECs). One of the key early events in tumorigenic transformation is the ability of cells to overcome replicative senescence. However, the precise genetic changes that are responsible for this event in MECs is largely unknown. Here, we report that Bmi-1, originally identified as a c-Myc cooperating oncoprotein, can bypass senescence, extend the replicative life span, and immortalize MECs. Furthermore, Bmi-1 was overexpressed in immortal MECs and several breast cancer cell lines. Overexpression of Bmi-1 in MECs led to activation of human telomerase reverse transcriptase (hTERT) transcription and induction of telomerase activity. Telomerase induction by Bmi-1 was an early event in the extension of the replicative life span and immortalization. Bmi-1 was not overexpressed in hTERT-immortalized MECs, suggesting that Bmi-1 functions upstream of hTERT. Although, c-Myc has been reported to induce telomerase in MECs, Bmi-1 appeared to act independently of c-Myc binding sequences in the hTERT promoter. Deletion analysis of the Bmi-1 protein suggested that the RING finger, as well as a conserved helix-turn-helix-turn domain, were required for its ability to induce telomerase and immortalize MECs. These data suggest that Bmi-1 regulates telomerase expression in MECs and plays a role in the development of human breast cancer.