Robert A. Ross

Dr. Robert Ross, head of the Laboratory of Neurobiology in the Department of Biological Sciences, is interested in the cell and molecular biology of the childhood cancer neuroblastoma. This often incurable tumor is the most common cancer of newborns and the third most common in children, with no obvious difference in incidence between races or genders. Intriguingly, although frequently fatal, neuroblastoma tumors can also regress, with little or no therapy, in a proportion of children. Little is known about what triggers formation of the cancer or what causes unrestrained growth versus differentiation and regression. Work in Dr. Ross’s laboratory is focused on understanding the properties that make these cells highly malignant.

Neuroblastoma arises from the developing peripheral nervous system and, as such, is composed of multiple cell types differing in their biochemistry as well as their growth and differentiation potential. Early immunoblot and receptor binding analyses characterized tumor-derived cell lines with various morphologies for expression of multiple proteins which would give information about cell phenotype using immunoblot and receptor binding analyses. These analyses led to the identification by the Ross group of three different cell types typically found in tumors and their normal cell counterparts: 1) neuroblasts (N cells), which during normal embryonic development give rise to sympathetic neurons and neuroendocrine cells; 2) substrate-adherent cells (S cells), which represent precursors normally differentiating to melanocytes, Schwann cells, and others; and 3) more primitive stem cells (I cells), precursors to the other two cell types in both tumors and normal embryos (Ross et al., 2003).

The detection of stem cells in neuroblastoma is consistent with a current hypothesis that it is malignant stem cells that cause many cancers. Thus, the three cell types were assessed for their ability to grow as tumors. We have shown (Walton et al., 2004) that the three variants differ markedly in malignant potential: the stem cell phenotype is the most malignant, neuroblasts are moderately tumorigenic, and substrate-adherent cells are unable to form tumors. One current project in the laboratory is to identify genes that are either over- or under-expressed in cancer stem cells relative to the other two cell types and that might be important in malignancy.

A related project seeks to determine whether stem cells are detectable in tumor tissue as well as cell lines and, if isolated, whether they are highly malignant in vivo as well as in vitro. This work is being done by Research Associate Barbara Spengler in collaboration with pediatric oncologists at Memorial Sloan-Kettering Cancer Center in New York City. To date, immunocytochemical staining of sections of more than 50 tumors has revealed cells with staining characteristics of I-type stem cells in all tumors analyzed . However, tumors that metastasize, recur, and are ultimately fatal have greater numbers of stem cells.

A third project addresses a different attribute of aggressive neuroblastoma – amplification and overexpression of the cancer gene N-myc. This gene encodes a transcription factor found in normal neuroblasts as well. In about 30% of neuroblastoma tumors, over-production of the protein due to the presence of many additional gene copies is a bad omen for survival. We are investigating how the expression of the N-myc gene is itself regulated and what role it plays in cancer cells. Important in this process is an RNA-binding protein termed HuD, which binds and stabilizes the newly transcribed N-myc RNA, preventing its rapid degradation. The finding that cells with amplified N-myc genes have one fewer HuD gene led to the hypothesis that loss of HuD should select for N-myc amplification, an idea that is supported by both molecular and cytogenetic/FISH analyses. Conversely, replacement of the missing HuD gene would cause a loss of amplification.

We are also currently investigating additional upstream regulators and downstream targets of N-myc. Two candidates are microRNAs, a recently discovered group of small noncoding RNAs which regulate mRNA translation. Both contain E-boxes to which N-myc binds and one (miR375) regulates HuD, thus forming a negative feedback loop with N-myc.

Ross, R.A., Spengler, B.A., Domènech, C., Porubcin, M., Rettig, W.J. and Biedler, J.L. (1995). Human neuroblastoma I-type cells are malignant neural crest stem cells. Cell Growth & Diff. 6 :449-456.

Spengler, B.A., Lazarova, D.L., Ross, R.A. and Biedler, J.L. (1997). Cell lineage and differentiation state are primary determinants of MYCN gene expression and malignant potential in human neuroblatoma cells. Oncology Res. 9 :467-476.

Lazarova, D.L., Spengler, B.A., Biedler, J.L. and Ross, R.A. (1999). HuD, a neuronal-specific RNA-binding protein, is a putative regulator of N-myc pre-mRNA processing/stability in malignant human neuroblasts. Oncogene 18 :2703-2710.

Nikolopoulos, S.N., Spengler, B.A., Kisselbach, K., Evans, A.E., Biedler, J.L. and Ross, R.A. The human non-muscle a -actinin protein encoded by the ACTN4 gene suppresses tumorigenicity of human neuroblastoma cells. Oncogene 19: 380-386, 2000.

Thomas, S.K., Messam, C.A., Spengler, B.A., Biedler, J.L., and Ross, R.A. Nestin is a potential mediator of malignancy in human neuroblastoma cells. J. Biol. Chem. 279:27994-27999, 2004.

Walton, J.D., Kattan, D.R., Thomas, S.K., Spengler, B.A., Guo, H.-f., Bieldler, J.L., Cheng, N.-K.V., and Ross, R.A. Characteristics of stem cells from human neuroblastoma cell lines and tumors. Neoplasia 10:838-845, 2004.

Grandinetti, K.B., Spengler, B.A., Biedler, J.L., and Ross, R.A. Loss of one HuD allele on chromosome #1p selects for amplification of the N-myc proto-oncogene in human neuroblastoma cells. Oncogene 25: 706-712, 2006.

Ross, R.A. and Spengler, B.A. Human neuroblastoma stem cells. Semin Cancer Biol. 17:241-7, 2007.