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The microRNAs in the miR-34 family, consisting of miR-34a, miR-34b and miR-34c, are tumour suppressors. The annotated human miR-34b-5p has one additional base at the 5' end of the common miR-34 family seed sequence, compared to miR-34a-5p and miR-34c-5p. This extra base results in a shift of the seed sequence, which would affect the target gene repertoire and have functional consequences. During our studies of miR-34 functions, we investigated the precise sequence of mature miR-34b-5p in human cells by deep sequencing. We found that a miR-34b-5p without the extra base was the predominant form in both non-malignant and malignant cells derived from several human tissues, indicating that the miR-34b annotation is misleading. We evaluated the functional implications of the seed shift, by comparing the effect of mimics representing the alternative miR-34b-5p sequences in MDA-MB-231 cells. In contrast to the annotated miR-34b, the endogenously expressed miR-34b displayed tumour suppressive characteristics in vitro similarly to miR-34c. These data demonstrate the importance of determining the precise sequence of a mature microRNA before exploring miRNA functions.

PRUNE, the human homologue of the Drosophila gene, is located in 1q21.3, a region highly amplified in human sarcomas, malignant tumours of mesenchymal origin. Prune protein interacts with the metastasis suppressor nm23-H1, but shows impaired affinity towards the nm23-H1 S120G mutant associated with advanced neuroblastoma. Based on these observations, we previously suggested that prune may act as a negative regulator of nm23-H1 activity. We found amplification of PRUNE in aggressive sarcoma subtypes, such as leiomyosarcomas and malignant fibrous histiocytomas (MFH) as well as in the less malignant liposarcomas. PRUNE amplification was generally accompanied by high mRNA and moderate to high protein levels. The sarcoma samples expressed nm23-H1 mostly at low or moderate levels, whereas mRNA and protein levels were moderate to high in breast carcinomas. For the more aggressive sarcoma subtypes, 9/13 patients with PRUNE amplification developed metastases. A similar situation was observed in all breast carcinomas with amplification of PRUNE. Infection of NIH3T3 cells with a PRUNE recombinant retrovirus increased cell proliferation. Possibly, amplification and overexpression of PRUNE has the same effect in the tumours. We suggest that amplification and overexpression of PRUNE could be a mechanism for inhibition of nm23-H1 activity that affect the development or progression of these tumours.

Clinical and experimental evidence suggests that tumor cells shed into the circulation from solid cancers are ineffective in forming distant metastasis unless the cells are able to respond to growth conditions offered by the secondary organs. To identify the phenotypic properties that are specific for such growth response, we injected carcinoma cells, which had been recovered from bone marrow micrometastases in a breast cancer patient who was clinically devoid of overt metastatic disease and established in culture, into the systemic circulation of immunodeficient rats. The animals developed metastases in the central nervous system, and metastatic tumor cells were isolated with immunomagnetic beads coated with an antibody that was reactive with human cells. The segregated cell population was compared with the injected cells by means of differential display analysis, and two candidate fragments were identified as up-regulated in the fully metastatic cells. The first was an intracellular effector molecule involved in tyrosine kinase signaling, known to mediate nerve growth factor-dependent promotion of cell survival. The second was a novel gene product (termed candidate of metastasis-1), presumably encoding a DNA-binding protein of helix-turn-helix type. Constitutive expression of candidate of metastasis-1 seemed to distinguish breast cancer cells with metastatic potential from cells without metastatic potential. Hence, our experimental approach identified factors that may mediate the growth response of tumor cells upon establishment in a secondary organ and, thereby, contribute to the metastatic phenotype.