Identification of differential and functionally active miRNAs in both anaplastic lymphoma kinase (ALK)+ and ALK- anaplastic large-cell lymphoma

Abstract

Aberrant anaplastic lymphoma kinase (ALK) expression is a defining feature of many human cancers and was identified first in anaplastic large-cell lymphoma (ALCL), an aggressive non-Hodgkin T-cell lymphoma. Since that time, many studies have set out to identify the mechanisms used by aberrant ALK toward tumorigenesis. We have identified a distinct profile of micro-RNAs (miRNAs) that characterize ALCL; furthermore, this profile distinguishes ALK(+) from ALK(-) subtypes, and thus points toward potential mechanisms of tumorigenesis induced by aberrant ALK. Using a nucleophosmin-ALK transgenic mouse model as well as human primary ALCL tumor tissues and human ALCL-derived cell lines, we reveal a set of overlapping deregulated miRNAs that might be implicated in the development and progression of ALCL. Importantly, ALK(+) and ALK(-) ALCL could be distinguished by a distinct profile of "oncomirs": Five members of the miR-17-92 cluster were expressed more highly in ALK(+) ALCL, whereas miR-155 was expressed more than 10-fold higher in ALK(-) ALCL. Moreover, miR-101 was down-regulated in all ALCL model systems, but its forced expression attenuated cell proliferation only in ALK(+) and not in ALK(-) cell lines, perhaps suggesting different modes of ALK-dependent regulation of its target proteins. Furthermore, inhibition of mTOR, which is targeted by miR-101, led to reduced tumor growth in engrafted ALCL mouse models. In addition to future therapeutical and diagnostic applications, it will be of interest to study the physiological implications and prognostic value of the identified miRNA profiles.

Fig. 1.

Common deregulated miRNA in ALK⁺ ALCL cell lines (A) and ALK⁺ and ALK⁻ cell lines and a murine ALCL model (B). (A) Analysis of differentially expressed miRNAs identified in three human ALK⁺ ALCL cell lines (SR-786, Karpas-299, and SUDHL-1) reveals a common profile of 41 deregulated miRNAs. (B) ALK⁻ human ALCL cell lines Mac2a and FE-PD share a common set of 18 deregulated miRNAs shown here in comparison to the common ALK⁺ miRNA profile as well as the murine ALCL model.

Fig. 2.

Role of ALK status in miRNA expression of ALCL. (A) Venn diagram of differentially regulated miRNAs in two human ALK⁻ ALCL cell lines (Mac2a and FE-PD). (B) In a k-means–based clustering using the miRNA array data, we identified six miRNAs that were differentially expressed in ALCL cell lines depending on their ALK status. (C) Sequence alignment of the five miRNAs that are members of the miR17–92 cluster. (D) Using qPCR, we tested the expression (exp.) of miR-886-3p, miR-20b, miR-106a, miR-17, miR-20a, and miR-155 in RNA extracted from primary tissue sections of 14 patients with ALCL (9 ALK⁺ and 5 ALK⁻). As reference tissue, we used RNA extracted from paraffin tissue specimens of nonpathological lymph nodes. Three replicate qPCR assays were performed for each tested tumor specimen (mean value ± SD).

Fig. 3.

Expression of miR-101 in primary ALK⁺ ALCL tissue. Using qPCR, we tested the expression of miR-101 in tumors isolated from nine different patients. As reference tissue, we used paraffin tissue specimens of nonpathological lymph node tissue and purified T cells from healthy donors. Compared with the lymph node, control miR-101 was down-regulated in primary ALCL tissue from 2- to 20-fold. Three replicate qPCR assays were performed for each tested tumor specimen (mean value ± SD).

Fig. 4.

miR-101 reintroduction and rapamycin analogue CCI-779 reduce proliferation in ALCL cell lines. Two ALK⁺ (SR-786 and K299) and two ALK⁻ (Mac2a and FE-PD) cell lines were transfected with miRNA mimics of miR-101 and a nontargeting control oligonucleotide (A) or supplemented with 1, 5, and 10 nM CCI-779 (B) (●, SR-786; ▲, Karpas-299; ○, Mac2a; △, FE-PD). Cells were counted after the times indicated. Three independent experiments were performed.

Fig. 5.

Treatment of murine NPM-ALK tumor xenografts with the rapamycin analogue CCI-779 leads to reduced tumor growth. The murine tumor cell lines NPMALK_4 and NPMALK_417 were established as tumors in 16 and 14 immunodeficient mice, respectively. Half of the mice were treated with CCI-779 (1.5 mg·kg·d) or PBS as a control, and tumor size was monitored over 10 d. Data represent the tumor size at the end of the treatment period. vol., volume.