Mark E. Sobel

We describe a gene, NM23, that is associated with the tumor metastatic process. NM23 RNA levels were highest in cells and tumors of relatively low metastatic potential in two experimental systems: (1) murine K-1735 melanoma cell lines, in which the gene was identified, and (2) N-nitroso-N-methylurea-induced rat mammary carcinomas. NM23 RNA levels did not correlate with cell sensitivity to host immunological responses and may, therefore, be associated with intrinsic aggressiveness. The predicted carboxy-terminal protein sequence encoded by the pNM23 cDNA clone is novel compared with Genebank animal, bacterial, and viral sequences.

Expression of a recently identified murine gene, nm23, has been previously proposed to be inversely correlated to tumor metastatic potential in rodent model systems. The present study was designed to investigate whether nm23 RNA was detectable in human tumor tissue, and if it was differentially expressed. nm23 RNA levels in 27 human primary infiltrating ductal breast carcinomas were determined by using Northern blots or in situ hybridization. These data were compared to traditional histopathological indicators of metastatic potential, including the number of involved (tumor bearing) lymph nodes, grade of differentiation, and hormone receptor status. A striking consistency was observed in all tumors from patients with involved lymph nodes. Using Northern blot or in situ hybridizations, all of these tumors expressed low levels of nm23 RNA. Quantitative in situ hybridization on tumors from patients with 0 involved lymph nodes identified two groups: (a) approximately 75% contained high nm23 RNA levels, and (b) 25% contained significantly (alpha = 0.05) lower nm23 RNA levels. Low nm23 RNA levels in the 0 involved lymph node tumors were accompanied by two additional histopathological indicators of high metastatic potential, low nuclear and cytoplasmic estrogen receptor content, and poorly differentiated histological grade. In contrast, none of the high nm23 RNA level tumors were both receptor negative and poorly differentiated. We conclude that nm23 RNA levels are differentially expressed in human breast tumors, and that low nm23 RNA levels are associated with histopathological indication of high metastatic potential. Short term (median follow-up of 16 months) clinical course data were consistent with nm23 RNA levels, in that 2 of 11 low nm23 RNA content patients (including one from the 0 involved lymph node group) developed metastases, while none of the high nm23 RNA patients have experienced recurrent disease.

Transformation of chick embryo fibroblasts by Rous sarcoma virus results in decreased amounts of a major cell surface protein and of collagen. To determine the mechanism accounting for the decreased production of these proteins, we have measured the relative amounts of functional mRNAs for these and other transformation-sensitive proteins. Total cellular RNAs extracted from normal cells and from cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus were translated in a cell-free system derived from wheat germ. Analysis of the in vitro translation products of RNAs from normal and transformed chick embryo fibroblasts shows a 5-fold reduction in the translatable mRNA for cell surface protein and a 10-fold reduction in translatable mRNA for two collagen precursors. In addition, increases in functional mRNA are observed for myosin and for two membrane polypeptides with molecular weights of 95,000 and 78,000; the latter two proteins increase on transformation, but the increases are in large part secondary to the depletion of glucose from the medium of transformed cells. Our data suggest that some of the major cellular changes induced by oncogenic viruses are due to changes in the activity of specific cellular genes.

Proteolytic enzymes, such as type IV collagenase, play an important role in tumor invasion and metastasis. To examine Mr 72,000 type IV collagenase expression in human colon carcinoma, blot hybridization of total RNA from 19 primary colon tumors were performed. These filters were probed with complementary DNA probes encoding the Mr 72,000 type IV collagenase metalloenzyme. The results were expressed as the ratio of the messenger RNA (mRNA) levels in the tumor tissue to that in the adjacent normal mucosa (R). The level of the 3.1-kilobase type IV collagenase mRNA was higher in the primary tumor than in the normal adjacent colonic mucosa in 13 of 18 (72%) cases with a diagnosis of adenocarcinoma. These cases were divided into high expression (R, 4.50 to 29.34) and intermediate expression (R, 2.54 to 3.31) subgroups. Both groups showed statistically significant (P less than 0.05) elevations when compared with the five cases showing the lowest levels of Mr 72,000 type IV collagenase mRNA expression (low expression subgroup; R, 0.96 to 1.48). With this demonstrated elevation of Mr 72,000 type IV collagenase mRNA in colorectal adenocarcinoma we examined concomitant expression at the protein level using immunohistochemical techniques. Immunohistochemical examination of 70 cases of colon tumors, including 30 benign adenomas, using anti-Mr 72,000 type IV collagenase antibodies demonstrated a significant correlation with Duke's classification (P less than 0.001). Our results suggest that enhanced expression of the Mr 72,000 type IV collagenase enzyme may be a marker of human colorectal tumor invasiveness.

NM23, a novel gene associated with low tumor metastatic potential, has been investigated in an experimental system in which metastasis is inhibited by the transfection of viral and cellular oncogenes. The experimental system utilizes transfection of the Adenovirus 2 Ela gene to inhibit metastasis: rat embryo fibroblasts (REF) transfected with c-Ha-ras were highly metastatic, while REF cotransfected with ras and Ela were virtually nonmetastatic. NM23 RNA levels were higher in three independently ras + Ela-cotransfected, low metastatic REF lines than in three independently ras-transfected, highly metastatic REF line. Differences in hybridizable NM23 RNA levels between the two groups of transfected cell lines ranged from 2- to 8-fold. In situ hybridization demonstrated that the relatively high NM23 RNA levels in low metastatic ras + Ela-cotransfected REF cells were not due to overexpression of the NM23 gene by a subpopulation of cells. Thus, the metastasis-inhibitory effect of the exogenously added Ela gene has been associated with increased activation of the cellular NM23 gene. This associated is particularly significant in light of the very few changes observed in translatable steady-state RNA levels between ras- and ras + Ela-transfected REF lines. The data identify NM23 as a candidate for a gene that suppresses the malignant state.