Dysregulation of the C/EBPalpha differentiation pathway in human cancer

Abstract

While much is known about aberrant pathways affecting cell growth and apoptosis, our understanding of another critical step of neoplastic transformation, differentiation arrest, remains poor. The differentiation-inducing transcription factor CCAAT enhancer binding protein alpha (C/EBPalpha) is required for proper control of adipogenesis, glucose metabolism, granulocytic differentiation, and lung development. Studies investigating the function of this protein in hematopoietic malignancies as well as in lung and skin cancer have revealed numerous ways how tumor cells abrogate C/EBPalpha function. Genetic and global expression analysis of acute myeloid leukemia (AML) cases identifies C/EBPalpha-deficient AML as a separate entity yielding novel classification schemes. In patients with a dysfunctional C/EBPalpha pathway, targeted therapies may overcome the block in differentiation, and in combination with conventional chemotherapy, may lead to complete eradication of the malignant clone. Overall, a better understanding of the mechanisms of how C/EBPalpha dysregulation participates in the neoplastic process has opened new gateways for differentiation biology research.

Fig 1.

Physiological function of CCAAT enhancer binding protein alpha (C/EBPα). (A) Major sites of tissue expression of C/EBPα include the liver, the hematopoietic system, white adipose tissue, and the lung. Accordingly, mice with targeted disruption of C/EBPα (C/EBPα-knockout mice) display perinatal hypoglycemia, absence of granulocytes, lack of white adipose tissue, and hyperproliferation of interstitial lung tissue. Modified from Zhang et al, Yang et al and Flodby et al (with permission). (B) Within the hematopoietic system, C/EBPα is expressed at lower levels in hematopoietic stem cells (HSCs), and its expression increases progressively during granulocytic differentiation via common myeloid progenitor cells (CMP) and granulocyte-macrophage progenitor cells (GMP). C/EBPα is not detectable in megakaryocyte-erythrocyte progenitors (MEP), common lymphoid progenitors (CLP), or more mature lymphoid cells. Modified from Akashi et al with permission. (C) The C/EBPα mRNA is comprised of a cap sequence and 5′ untranslated region (UTR), the C/EBPα coding sequence derived from a single exon, and the 3′ UTR, which is followed by the poly A sequence (upper panel). The letters A, B1, B2, C, and D designate five ATG sites of translational start sites which are highly conserved across the six different species depicted with the exception of the A site, which is missing in the human species (middle panel). Translation from all sites except D produce a specific C/EBPα isoform that can be detected by Western blotting, and omission of each site (ie, −A) leads to the loss of the specific isoform. The major C/EBPα isoforms are produced from the B1 and C sites and are 42 kD and 30 kD in size. From Calkhoven et al with permission. NK, natural killer; Pro-T, T-cell progenitors; Pro-B, B-cell progenitors; wt, wild type.

Fig 2.

Pathogenetic mechanisms of CCAAT enhancer binding protein alpha (C/EBPα) dysfunction. C/EBPα function is disturbed at various levels in acute leukemias. Promoter methylation leads to silencing of promoter activity and decreased expression of C/EBPα. The fusion protein AML1-ETO suppresses C/EBPα transcription via disturbed autoregulation. Chromosomal translocations involving the immunoglobin H locus and the CEBPA gene (t14;19) occur in B-cell precursor acute lymphoblastic leukemia and have been described to induce aberrant C/EBPα expression in lymphoid cells. Other translocations which are associated with AML (AML1/MDS/Evi1 and CBFB/SMMHC) or chronic myelogenous leukemia (CML; BCR-ABL) have been demonstrated to increase the expression of specific RNA binding proteins which can block proper C/EBPα translation from an intact mRNA. In addition, posttranslational modifications such as Serine-21 phosphorylation by oncogenic Flt3-ITD as well as impairment of C/EBPα DNA binding by PLZF-RAR results in dysfunction of C/EBPα and a block of granulocytic differentiation. Finally, C/EBPα protein degradation is induced by Trib2 whose expression is increased by Notch1 mutations in AML. CBFB, core-binding factor β; BP, RNA-binding protein; UPD, uniparental disomy.

Fig 3.

CCAAT enhancer binding protein alpha (C/EBPα) mutations and their effect on prognosis in acute myeloid leukemia (AML). (A) and (B) Mutations of the coding sequence of C/EBPα have been described in approximately 15% of patients with AML and a normal karyotype by conventional cytogenetics. Two thirds of these patients harbor N-terminal frameshift mutations and C-terminal in-frame insertion/deletion mutations, leading to a nonfunctional 20 kD isoform and increased 30 kD isoform expressed from the N-terminally mutated allele as well as a dysfunctional 42 kD isoform expressed from the C-terminally mutated allele (ie, patient 2”). Please see Pabst et al for more details. (C) Retrospective survival analysis of patients with AML harboring C/EBPα mutations or no C/EBPα mutations showing superior overall survival of patients with C/EBPα mutations. Data from Preudhomme et al. N′ab, antibody recognizing N-terminal portion; TAD, transactivation domain; C'ab, antibody recongnizing the C-terminal domain; bZip, basic zipper region; wt, wild type.

Fig 4.

CCAAT enhancer binding protein alpha (C/EBPα) in lung cancer. (A) Generation of tissue-specific, inducible C/EBP-knockout mice. (Ba) Electron microscopy of airway epithelium in a lung-specific C/EBPα-knockout mouse demonstrates hyperproliferation of immature alveolar cells as well as intracellular accumulation of glycogen. (Bb) Histology of lung tumors in C/EBPα-knockout mice demonstrates bronchoalveolar carcinoma-like histology. (C) Immunohistochemical studies demonstrate downregulation of C/EBPα in non–small-cell lung cancer. The panels shown demonstrate strong expression of C/EBPα in normal bronchial epithelium (a) and show non–small-cell lung tumors with different degrees of expression (panels b, 0; c, 1+; d, 2+; e, 3+). (D) In vitro studies demonstrate growth arrest, (a) apoptosis, and (b) and cellular differentiation in lung cancer cell lines as a result of C/EBPα expression in H358 lung cancer cells. Reprinted with permission from Halmos et al, Costa et al, and Basseres et al. H358-MT, H358 cells transfected with a zinc-inducible metallothionein promoter-driven expression plasmid.