In most cells, transferrin receptor (TfR1)-mediated endocytosis is a major pathway for cellular iron uptake. We recently cloned the human transferrin receptor 2 (TfR2) gene, which encodes a second receptor for transferrin (Kawabata, H., Yang, R., Hirama, T., Vuong, P. T., Kawano, S., Gombart, A.

Transferrin receptor (TfR) plays a major role in cellular iron uptake through binding and internalizing a carrier protein transferrin (Tf). We have cloned, sequenced, and mapped a human gene homologous to TfR, termed TfR2. Two transcripts were expressed from this gene: ␣ (ϳ2.9 kilobase pairs), and ␤ (ϳ2.5 kilobase pairs). The predicted amino acid sequence revealed that the TfR2-␣ protein was a type II membrane protein and shared a 45% identity and 66% similarity in its extracellular domain with TfR. The TfR2-␤ protein lacked the aminoterminal portion of the TfR2-␣ protein including the putative transmembrane domain. Northern blot analysis showed that the ␣ transcript was predominantly expressed in the liver. In addition, high expression occurred in K562, an erythromegakaryocytic cell line. To analyze the function of TfR2, Chinese hamster ovary TfR-deficient cells (CHO-TRVb cells) were stably transfected with FLAG-tagged TfR2-␣. These cells showed an increase in biotinylated Tf binding to the cell surface, which was competed by nonlabeled Tf, but not by lactoferrin. Also, these cells had a marked increase in Tfbound 55 Fe uptake. Taken together, TfR2-␣ may be a second transferrin receptor that can mediate cellular iron transport.

The CCAAT/enhancer binding protein ε (C/EBPε) is a nuclear transcription factor expressed predominantly in myeloid cells and implicated as a potential regulator of myeloid differentiation. We show that it was rapidly induced in the acute promyelocytic leukemia (APL) cell line NB4 during granulocytic differentiation after exposure to retinoic acid (RA). Our data suggest that induction of C/EBPε expression was through the retinoic acid receptor α (RARα) pathway. Reporter gene studies showed that C/EBPε promoter/enhancer activity increased in a retinoid-dependent fashion via the retinoic acid response element (RARE) present in the promoter region of C/EBPε. The RA-induced expression of C/EBPε markedly increased in U937 myelomonoblasts that were induced to express promyelocytic leukemia/RARα (PML/RARα), but not in those induced to express promyelocytic leukemia zinc finger/RARα (PLZF/RARα). In retinoid-resistant APL cell lines, C/EBPε either is not induced or is induced only at very high concentrations of RA (≥10 -6 M). In addition, forced expression of C/EBPε in the U937 myelomonoblastic leukemia cells mimicked terminal granulocytic differentiation, including morphologic changes, increased CD11b/CD66b expression, and induction of secondary granule protein expression. Our data strongly suggest that C/EBPε is a downstream target gene responsible for RA-induced granulocytic differentiation of APL cells.

Human tumors show a high level of genetic heterogeneity, but the processes that influence the timing and route of metastatic dissemination of the subclones are unknown. Here we have used whole-exome sequencing of 103 matched benign, malignant and metastatic skin tumors from genetically heterogeneous mice to demonstrate that most metastases disseminate synchronously from the primary tumor, supporting parallel rather than linear evolution as the predominant model of metastasis. Shared mutations between primary carcinomas and their matched metastases have the distinct A-to-T signature of the initiating carcinogen dimethylbenzanthracene, but non-shared mutations are primarily G-to-T, a signature associated with oxidative stress. The existence of carcinomas that either did or did not metastasize in the same host animal suggests that there are tumor-intrinsic factors that influence metastatic seeding. We also demonstrate the importance of germline polymorphisms in determining allele-specific mutations, and we identify somatic genetic alterations that are specifically related to initiation of carcinogenesis by Hras or Kras mutations. Mouse tumors that mimic the genetic heterogeneity of human cancers can aid our understanding of the clonal evolution of metastasis and provide a realistic model for the testing of novel therapies.