People

Our laboratory studies cancer and cellular differentiation. Basic questions which we are addressing include: Why do hematopoietic stem cells remain quiescent for long periods of time? We are characterizing and dissecting molecular controls of this phenomenon. These studies will shed light on the regulation of cell numbers in the blood both in cancer and during normal development.  Another question is what coordinates the exit from the cell cycle as blood cells undergo terminal differentiation. We are using structure-function mutations and tandem affinity purification techniques to examine how cdk-inhibitors and regulatory proteins such as C/EBP isoforms choreograph these transitions. This work should help to clarify how normal differentiation is blocked in leukemias. We are using direct protein transduction techniques to modulate pathways involving cdk-inhibitors both in leukemias and other cancers.  Another question which we are studying is why cancer cells grow past confluence?  Our approach to this question examines the response of STAT signaling pathways to cell confluence.

Goals outside the laboratory include optimizing knowledge and understanding of cancer in the community.  Projects have included development of a trading card game teaching about cancer and research examining ways to make health websites more accessible to low-literacy individuals.  A collaboration with Hampton University, a highly-respected minority-training institution, seeks to develop a cancer curriculum at Hampton.

Research Details:

Modulation of p27 in cancer cells

p27 was initially identified as a protein which inhibited cellular replication in the setting of nutritional deprivation or crowding.  It has since been identified as one of the most significant indicators of prognosis in cancer. Cancers in which p27 is low or is relocated to the nucleus to the cytoplasm are more aggressive and respond to treatment poorly. Patients bearing cancers with low p27 levels have shorter time to relapse and lower survival.

Our laboratory was the first to identify shifting of p27 to the cytoplasm in normal differentiation and to uncover the presence of p27 within signaling centers in the cell membrane. Molecular mediators of p27 translocation to the cytoplasm have now been characterized, with the proteins jab1 and AKT playing a role in this pathway.

Prevention of p27 loss is therefore a rational strategy for a therapeutic approach to cancer. Although there is growing recognition of the importance of nuclear p27 expression to a favorable outcome in cancer, no therapeutics have been developed specifically to prevent p27 degradation in cancers. We hypothesized that the by blocking proteins involved in the breakdown or translocation of p27, we can specifically kill or normalize the behavior of cancer cells. Our strategy utilizes cell-permeable peptides coding for regions of p27 which will competitively bind and inhibit proteins in the p27 degradation and translocation pathway.

Our goal in this project is to study the oncogenicity of p27 domains when they are expressed in the cytoplasm, to generate p27 peptides which bind to p27-binding partners cyclinD/E, jab1, AKT and grb2, and to determine whether these peptides can disrupt p27-binding partner interactions and thereby increase nuclear p27 in cancer cells

Methods include direct protein transduction, gateway cloning, confocal microscopy and live cell imaging.

Modulation of stem cell quiescence and differentiation

Stem cell biology may uncover new ways to treat diseases through tissue engineering. In order to recognize this potential, it is important to understand how stem cell replication and differentiation is regulated.  We have been approaching this question by studying the expression and function of cell cycle regulators which play dual roles in controlling cellular replication and differentiation.  A group of proteins known as cyclin-dependent-kinase inhibitors (or cdki’s) are key candidates for controlling the rate of entry of stem cells into the cell cycle.  We have characterized the levels of a cdki called p21 in primitive human blood cells. We found that a stem cell compartment (one ten-millionth of umbilical cord white blood cells) remained quiescent for months and expressed very high levels of p21.  Functional studies by other laboratories confirmed that p21 controlled the replication of blood stem cells.

Goals of this project are to dissect mechanisms through which p21 and a related protein, p27, influence replication and differentiation of blood cells. Of particular interest is the need better to understand the role of p21 in differentiating blood cells which are actively replicating despite increasing p21 expression. Questions which we are asking include what is the effect of artificially downmodulating p21 at different points along the differentiation pathway on cellular quiescence and acquisition of the differentiated phenotype? We hypothesize that cytoplasmic p21 directly affects differentiated cell shape and motility and survival and that p21 either inhibits or promotes replication depending on the relative level of specific binding partners. We further hypothesize that interactions between p21 and a transcriptional regulator known as C/EBP alpha regulate passage of blood cells along the granulocyte pathway; that high p21 levels promote early stages of differentiation and inhibit late differentiation as a result of this interaction.

Methods include transduction of inducible genes and gene mutants, interfering RNA technology, tandem affinity purification technology, single cell manipulation and confocal microscopy.

Findings include demonstration of partial hematopoietic differentiation induced by p21, the finding of stage-specific expression and cross-regulation of cdk-inhibitors, and the demonstration that p21 could inhibit apoptotic cell death of myeloid cells by stabilizing an apoptosis inhibitor protein known as c-IAP1.

Ongoing studies seek to dissect out the nuclear and cytoplasmic binding partners of p21 and p27 at discrete stages of differentiation using tandem affinity purification techniques. This will enable directed targeting and/or artificial recapitulation of these protein-protein interactions in order to assess their functional effects and importance.

Future studies will extend this work to adult human stem cells and determine whether and how p21 and p27 modulate the development of other lineages.

Control of Stat signaling pathway

Cells respond to their environment by transducing signals from receptors on their surface into their nucleus to change gene expression. Many hormones and cytokines alter cellular behavior by activating a protein called Stat3 (Signal transducer and activator of transcription-3). High levels of Stat3 activation occur in many tumors--in fact, cells manipulated to express an artificial active Stat3 are highly oncogenic. On the other hand, Stat3 is required for normal cell signaling, movement and for normal development of blood, breast and the immune system.

We noted that Stat3 was activated in cell lines at high density and hypothesized that cell-cell contact was another mechanism for activating the Stat3 pathway. We found that in fact Stat3 was activated both by cell-cell contact and by a feedback mechanism related to the growth state of the cells. Inhibition of a proliferation pathway (cyclin-dependent kinase 2) either artificially or by high cell density led to activation of Stat3. Our findings support the hypothesis that Stat3 is not oncogenic by increasing cell growth. Rather, it is likely that it’s oncogenicity derives from its ability to inhibit the death of cancer cells, particularly at high cell density.

Future work will dissect out how cdk2 inhibition leads to activation of Stat3 and will test the hypothesis that Stat3 inhibits apoptosis of cancer cells at high density. Methods will use inducible expression of Stat3 and Stat3 inhibitors, cdk2 and cdk2 inhibitors, and interfering RNA technology.