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Journal of Thoracic Oncology:
doi: 10.1097/JTO.0000000000000061
Brief Reports

HIP1–ALK, a Novel Fusion Protein Identified in Lung Adenocarcinoma

Hong, Mineui*; Kim, Ryong Nam; Song, Ji-Young; Choi, So-Jung§; Oh, Ensel; Lira, Maruja E.; Mao, Mao; Takeuchi, Kengo; Han, Joungho; Kim, Jhingook§; Choi, Yoon-La*‡#

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Departments of *Pathology, §Thoracic Surgery, Samsung Medical Center, Sungkyunkwan University College of Medicine, Seoul, Korea; Department of Pharmacy, College of Pharmacy, Seoul National University, Seoul, Korea; Laboratory of Cancer Genomics and Molecular Pathology, Samsung Biomedical Research Institute, Samsung Medical Center, Seoul, Korea; Pfizer Oncology, San Diego, California; Pathology Project for Molecular Targets of the Cancer Institute/Division of Pathology of the Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, Japan; and #Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University School of Medicine, Seoul, Korea.

Disclosure: M.E. Lira, and M. Mao are employed by and own stock in Pfizer, Inc. The other authors declare no conflict of interest.

Address for correspondence: Yoon-La Choi and Jhingook Kim, Department of Pathology and Thoracic Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Ilwon-dong, Gangnam-gu, Seoul 135–710, Korea. E-mail:;

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Introduction: The most common mechanism underlying overexpression and activation of anaplastic lymphoma kinase (ALK) in non–small-cell lung carcinoma could be attributed to the formation of a fusion protein. To date, five fusion partners of ALK have been reported, namely, echinoderm microtubule associated protein like 4, tropomyosin-related kinase-fused gene, kinesin family member 5B, kinesin light chain 1, and protein tyrosine phosphatase, nonreceptor type 3.

Methods: In this article, we report a novel fusion gene huntingtin interacting protein 1 (HIP1)–ALK, which is conjoined between the huntingtin-interacting protein 1 gene HIP1 and ALK. Reverse-transcriptase polymerase chain reaction and immunohistochemical analysis were used to detect this fusion gene’s transcript and protein expression, respectively. We had amplified the full-length cDNA sequence of this novel fusion gene by using 5'-rapid amplification of cDNA ends. The causative genomic translocation t(2;7)(p23;q11.23) for generating this novel fusion gene was verified by using genomic sequencing.

Results: The examined adenocarcinoma showed predominant acinar pattern, and ALK immunostaining was localized to the cytoplasm, with intense staining in the submembrane region. In break-apart, fluorescence in situ hybridization analysis for ALK, split of the 5' and 3' probe signals, and isolated 3' signals were observed. Reverse-transcriptase polymerase chain reaction revealed that the tumor harbored a novel fusion transcript in which exon 21 of HIP1 was fused to exon 20 of ALK in-frame.

Conclusion: The novel fusion gene and its protein HIP1–ALK harboring epsin N-terminal homology, coiled-coil, juxtamembrane, and kinase domains, which could play a role in carcinogenesis, could become diagnostic and therapeutic target of the lung adenocarcinoma and deserve a further study in the future.

Anaplastic lymphoma kinase (ALK)–positive non– small-cell lung carcinoma (NSCLC) is highly sensitive to ALK kinase inhibitors such as crizotinib.1 More than 20 variants of the EML4–ALK fusion gene have been identified along with diversity in the breakpoint and length of EML4.2 In addition, four more fusion partner genes of ALK have been identified: TRK-fused gene, kinesin family member 5B (KIF5B), kinesin light chain 1, and protein tyrosine phosphatase, nonreceptor type3.3–6 In the current study, we identified the aberrant expression of huntingtin interacting protein 1 (HIP1)-ALK and the chromosomal translocation for generating this fusion gene in NSCLCs.

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A 38-year-old Korean woman with a nonsmoking history underwent lobectomy because of an irregular mass on the lingual division of the upper-left lobe (Fig. 1A). The tumor area measured 2.5 × 2.5 cm2 and microscopic examination revealed an adenocarcinoma with predominant acinar pattern (Fig. 1C). Metastasis of one of nine lymph nodes was noticed. Immunohistochemistry showed that ALK (dilution 1:50; clone 5A4, Novocastra, Newcastle, United Kingdom) localized to the cytoplasm, with higher density in the submembrane region of tumor cells (Fig. 1D and E). In break-apart, fluorescence in situ hybridization analysis for ALK (Abbott Molecular, Abbott Park, IL), split of the 5' and 3' probe signals and isolated 3' signals were observed (Fig. 1B), confirming the chromosomal rearrangement. The tumor did not harbor either epidermal growth factor receptor (EGFR) or Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations.

Figure 1
Figure 1
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By using total RNA extracted from tumor tissue obtained from the patient, we had prepared a first-strand cDNA library by reverse-transcriptase polymerase chain reaction (PCR) to screen fusion transcripts involving ALK as a fusion partner. Then, we amplified a double-strand cDNA fragment for the fusion region of HIP1–ALK transcript by PCR using specific primers and the first-strand cDNA library. The SMARTer 5'-rapid amplification of cDNA ends cDNA Amplification Kit (Clontech Laboratories, Inc., Mountain View, CA) was used to amplify the full-length cDNA of this novel fusion gene HIP1–ALK. Then, the PCR products were cloned into the pCR4-TOPO vector using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA) for DNA sequencing. The sequence analysis of HIP1–ALK cDNA showed that exon 21 of HIP1 was fused to exon 20 of ALK in-frame, generating a fusion mRNA harboring an intact 3762-base pair open reading frame encoding a deduced 1253-amino acid protein sequence that contains epsin N-terminal homology, coiled-coil, juxtamembrane, and kinase domains (Fig. 2A and B). The cDNA sequence of the novel fusion gene HIP1–ALK has been deposited in the National Center for Biotechnology Information database (accession number KF360988). Direct sequencing of genomic DNA also revealed that the genomic breakpoint occurred between 79th nucleotide of intron 21 of HIP1 and 1017th nucleotide upstream from the nucleotide before the beginning nucleotide of the exon 20 of ALK, thereby resulting in a chromosomal translocation t(2;7)(p23;q11.23) (Fig. 2A). Noticeably, the beginning nucleotide of this novel fusion gene’s 5'UTR was 127,263-base pair far away from the beginning nucleotide of 5'UTR of reference gene HIP1 (NM_001243198.1) in the University of California, Santa Cruz (UCSC) Genome Browser. To check whether the 5'UTR of this novel fusion gene, which had been identified by our 5'-rapid amplification of cDNA ends in this study, could be very close to another promoter which is different from the previously known one of the HIP1 reference gene (NM_001243198.1), we had searched ChIP-Seq database in the UCSC Genome Browser. Very interestingly, H3K4me3 signal, which could provide strong evidence for existence of a promoter region, appeared exactly around the beginning part of the 5'UTR of the fusion gene (Fig. 3A), confirming that HIP1–ALK could be regulated by not the conventional promoter of HIP1 but a novel promoter. The existence of this novel promoter could have been substantiated once more by our discovery in this study of a new HIP1 transcript variant (in submission process to National Center for Biotechnology Information) whose transcription starts from the same genomic nucleotide position as the first 5' nucleotide position of the HIP1–ALK and ends at the 3'UTR of the HIP1 (Fig. 3B), implying that HIP1–ALK and the new HIP1 variant gene could be regulated by the novel promoter.

Figure 2
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Figure 3
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The binding site (755–783 in HIP1–ALK amino acid sequence) for crizotinib, which is an adenosine triphosphate (ATP)-binding site in a normal condition, resides behind the breakpoint just after 691th amino acid, participating in ALK part in the fusion protein HIP1–ALK and also strongly suggesting that HIP1–ALK activity could be inhibited by crizotinib.

The patient was treated with crizotinib according to the National Comprehensive Cancer Network guideline, which recommends crizotinib as a first-line therapy for locally advanced or metastatic ALK-positive NSCLC. After 15 months of follow-up, computed tomography scans did not reveal any evidence of recurrence or metastasis in the patient.

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This is the first report of a novel HIP1–ALK fusion gene in NSCLC. Kalchman et al.7 reported that HIP1 is essential for the membrane–cytoskeletal integrity in the brain. HIP1 plays an important role in clathrin trafficking and cell survival. The epsin N-terminal homology domain–containing protein binds to polyphosphoinositide-signaling lipids and has been found in cofactors of clathrin-mediated trafficking.8 HIP1 is overexpressed in various human cancer cell lines, which indicates that HIP1 could provide a selective growth advantage to cancer cells. Among them, mRNA level of HIP1 increased in prostate and colon cancer cell lines compared with that in normal epithelial cell lines of prostate and colon.9 The first description of chromosomal translocation of HIP1 in the pathogenesis had come out from chronic myelomonocytic leukemia with platelet-derived growth factor β receptor. HIP1– platelet-derived growth factor β receptor fusion with t(5;7)(q33;q11.2) translocation may contribute to leukemogenesis and lead to transformation of hematopoietic cells.10 The HIP1–ALK fusion protein comprises the coiled-coil domain of HIP1 and the juxtamembrane intracellular region of ALK, which plays an important role in maintaining ALK kinase activity (Fig. 2B). Together with dimerization through the coiled-coil domain, the ALK tyrosine kinase activity of the fusion protein may be activated aberrantly, thus facilitating oncogenesis in the lung.11,12 Although further studies are needed to ascertain the transforming properties of this HIP1–ALK fusion protein in cellular and mouse models, it is likely that this novel fusion protein also may harbor transforming activities from its chimeric protein structure. Takeuchi et al.4 found a novel ALK fusion protein KIF5B–ALK in lung cancer using an immunohistochemistry-based diagnostic system. They suggested that the subcellular localization of ALK fusion proteins probably depends on the fusion partner. Whereas nucleophosmin–ALK exhibits both nuclear and cytoplasmic staining, EML4–ALK stains in the cytoplasm, but not in the nucleus. The perinuclear halo pattern observed in KIF5B–ALK may indicate the accumulation of fusion protein at the periphery of the cytoplasm. In this case, the HIP1–ALK staining pattern is similar to that of KIF5B–ALK, which resembles submembrane granular staining.

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To the best of our knowledge, HIP1–ALK is the first novel fusion transcript reported in ALK-positive lung cancer. Given that the HIP1 protein contains coiled-coil domains, the fusion protein possibly dimerizes constitutively and, thereby, could possess a strong transforming potential.12 Patients with HIP1–ALK translocation may therefore respond to treatment with ALK inhibitors. This case report could provide novel diagnostic and therapeutic candidate target for patients with NSCLC.

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This work was supported by the National Research Foundation of Korea grant funded by the Korea government (MSIP) (NRF-2013R1A2A2A01068922).

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1. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature. 2007;448:561–566

2. Sasaki T, Rodig SJ, Chirieac LR, Jänne PA. The biology and treatment of EML4-ALK non-small cell lung cancer. Eur J Cancer. 2010;46:1773–1780

3. Rikova K, Guo A, Zeng Q, et al. Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell. 2007;131:1190–1203

4. Takeuchi K, Choi YL, Togashi Y, et al. KIF5B-ALK, a novel fusion oncokinase identified by an immunohistochemistry-based diagnostic system for ALK-positive lung cancer. Clin Cancer Res. 2009;15:3143–3149

5. Togashi Y, Soda M, Sakata S, et al. KLC1-ALK: a novel fusion in lung cancer identified using a formalin-fixed paraffin-embedded tissue only. PLoS One. 2012;7:e31323

6. Jung Y, Kim P, Jung Y, et al. Discovery of ALK-PTPN3 gene fusion from human non-small cell lung carcinoma cell line using next generation RNA sequencing. Genes Chromosomes Cancer. 2012;51:590–597

7. Kalchman MA, Koide HB, McCutcheon K, et al. HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain. Nat Genet. 1997;16:44–53

8. Itoh T, Koshiba S, Kigawa T, Kikuchi A, Yokoyama S, Takenawa T. Role of the ENTH domain in phosphatidylinositol-4,5-bisphosphate binding and endocytosis. Science. 2001;291:1047–1051

9. Rao DS, Hyun TS, Kumar PD, et al. Huntingtin-interacting protein 1 is overexpressed in prostate and colon cancer and is critical for cellular survival. J Clin Invest. 2002;110:351–360

10. Ross TS, Bernard OA, Berger R, Gilliland DG. Fusion of Huntingtin interacting protein 1 to platelet-derived growth factor beta receptor (PDGFbetaR) in chronic myelomonocytic leukemia with t(5;7)(q33;q11.2). Blood. 1998;91:4419–4426

11. Li R, Morris SW. Development of anaplastic lymphoma kinase (ALK) small-molecule inhibitors for cancer therapy. Med Res Rev. 2008;28:372–412

12. Mano H. Non-solid oncogenes in solid tumors: EML4-ALK fusion genes in lung cancer. Cancer Sci. 2008;99:2349–2355

Lung cancer; Anaplastic lymphoma kinase; HIP1; Translocation

Copyright © 2014 by the European Lung Cancer Conference and the International Association for the Study of Lung Cancer.


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