Multiple variants of the RON receptor tyrosine kinase: Biochemical properties, tumorigenic activities, and potential drug targets

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Cancer Letters 257 (2007) 157–164 www.elsevier.com/locate/canlet

Mini-review

Multiple variants of the RON receptor tyrosine kinase: Biochemical properties, tumorigenic activities, and potential drug targets Yi Lu a, Hang-Ping Yao a

b

a,*

, Ming-Hai Wang

a,b,*

Laboratory of Cancer Biology and Therapeutics, Institute of Infectious Diseases at First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310003, PR China Cancer Biology Center and Department of Pharmaceutical Sciences, Texas Tech University Health Sciences, Center School of Pharmacy, Amarillo, TX 79106, USA Received 11 April 2007; received in revised form 6 August 2007; accepted 8 August 2007

Abstract Aberrant expression of the RON (Recepteur d’Origine Nantais) receptor tyrosine kinase, accompanied by generation of multiple splicing or truncated variants, contributes to pathogenesis of epithelial cancers. Currently, six variants including ROND170, D165, D160, D155, D110, and D55 with various deletions or truncations in the extracellular or intracellular regions have been identified. The extracellular sequences contain functional structures such as sema domain, PSI motif, and IPT units. The deletion or truncation results in constitutive phosphorylation and increased kinase activities. Oncogenic ROND160, generated by exclusion of the first IPT unit, is a typical example. In contrast, the deletion adjacent to the conserved MET1254 in the kinase domain converts RON into a dominant negative agent. Among three mechanisms underlying isoform production, the switch from constitutive to alternative pre-mRNA splicing is the major event in producing RON variants in cancer cells. Most of the RON variants have the ability to activate multiple signaling cascades with a different substrate specificity and phosphorylation profile. They regulate cell migration, invasion, and proliferation, which contribute to the invasive phenotype and promote the malignant progression. Thus, determining the pathogenesis of RON variants is critical in understanding the mechanisms underlying cancer initiation and progression. Targeting oncogenic signals elicited by RON or its variants by special antibody or small interfering RNA could provide a novel strategy for the treatment of malignant epithelial cancers. Ó 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Receptor tyrosine kinase; Alternative splicing; Oncogenic signal; Invasive growth; Therapeutical target

1. Introduction The RON receptor tyrosine kinase [1], also known as stem cell-derived tyrosine kinase (STK) *

Corresponding authors. Tel.: +1 86 0571 87236586; fax: +1 806 356 4034. E-mail addresses: [email protected] (H.-P. Yao), [email protected] (M.-H. Wang).

in mouse [2], is a member of the MET proto-oncogene family [1]. The specific ligand for RON is macrophage-stimulating protein (MSP) [3–6], also known as hepatocyte growth factor-like protein [7]. RON is a 180 kDa heterodimeric protein composed of an extracellular 40 kDa a-chain and a 145 kDa transmembrane b-chain [1]. The RON gene contains 20 exons and resides in chromosome 3p21

0304-3835/$ - see front matter Ó 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2007.08.007

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region [8], which is often altered in human cancers [9]. Studies from the past several years have shown that RON is required for embryonic development [10] and is involved in the innate immune response against pathogen-induced inflammatory reactions [11]. RON is also implicated in pathogenic processes related to cancer initiation, progression, and malignant conversion [12]. The generation of various RON variants with diverse and even antagonistic functions provides an opportunity to study its oncogenic activities. Currently, the therapeutic potential of RON is under intensive investigation [13,14], which may represent a novel approach for cancer treatment. This review summarizes our current knowledge about RON variants in carcinogenesis of epithelial cancers. Our focus is on biological features of RON variants that contribute significantly to the progression of cancers. The potentials of RON variants as drug targets are also discussed. It is believed that understanding the biology of RON variants and their roles in cancer development could provide insight into mechanisms underling malignant progression of epithelial cancers. 2. RON variants and their biochemical features Production of protein isoforms is a key event for protein diversity [15]. Currently, six RON variants have been identified (Fig. 1) [12]. These variants were produced through three mechanisms: alternative pre-mRNA splicing, protein truncation, and

alternative transcription [12]. In most cases, the changes in the RON sequence lead to the constitutive activation [16]. However, some changes such as those occurred in ROND170 results in inactivation of the RON kinase activity [17]. The similarity and difference among six RON variants are described below. ROND170 was a 170 kDa variant generated by alternative pre-mRNA splicing that eliminates exon 19 [17]. Exon 19 codes 46 amino acids belonging to the RON kinase domain [8]. The deletion also causes the reading frame-shift and creates a new stop codon at nt 3998. These changes effectively truncated 84 amino acids from the C-terminal tail including the docking site [17]. Genetic analysis indicates that a C to A nucleotide polymorphism in intron 18 of the RON gene contributes to the ROND170 mRNA transcript [18]. ROND170 is a kinase-defective variant and acts as a dominant negative agent that inhibits tumorigenic activities mediated by oncogenic variant ROND160 in colon cancer cells [17]. Thus, ROND170 has a pharmacological potential against tumorigenesis of epithelial cancers with aberrant RON expression. The ROND165 was discovered first in gastric cancer KATO III cells [19] and later in various cancer samples and cell lines [12]. The protein originates from a spliced mRNA transcript that has an inframe deletion in exon 11 [19]. Exon 11 encodes 49 amino acids and resides in the fourth IPT domain in the extracellular sequences of the RON b-chain [8]. The deletion affects the proteolytic process

-S-S-

-S-S-

-S-S-

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Alternative transcription

Deletion

Sema domain

Sema domain

truncation

- α -chain

β -chain -

-S-S-

PSI motif Deletion

Deletion

IPT units Deletion

Deletion TM P

TK -

P P

Catalytic loop

P P

RON

- Y1017 - Y1238 - Y1239 - M1254 - Y1353 - Y1360

P P P

P P P

P

P P P

P

P

P P

P P

P P

Deletion P P

RON Δ165

P P

RON Δ160

P P

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RON Δ110

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Fig 1. Schematic representation of human RON and its variants: mature RON is composed of a 40 kDa a-chain and a 150 kDa b-chain linked by a disulfide bond. The extracellular a-chain has 284 amino acids. The b-chain has 1096 amino acids and comprises an extracellular sequence, a short transmembrane (TM) segment, and a large cytoplasmic portion with intrinsic tyrosine kinase domain (TK) and a C-terminal tail. The extracellular sequences of the RON b-chain contain a sema domain, followed by a PSI motif and four IPT units. Deletion and truncation are marked with arrows. Various tyrosine residues important for RON activities were labeled.

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resulting in the accumulation of single-chain proROND165 in the cytoplasm. Because the deletion creates uneven numbers of cysteine residues, the aberrant intracellular disulfide bridges are formed, which facilitates ROND165 oligomerization leading to constitutive phosphorylation. ROND165 is an active variant that has the ability to mediate motile and invasive phenotypes in cancerous cells [19]. ROND160 is a naturally occurring oncogenic form of RON identified in human colon cancer HT-29 and Sw680 cells and primary cancer samples [16,20]. It is derived from a splicing mRNA transcript with an in-frame deletion of 109 amino acids coded by exons 5 and 6 in the first IPT domain in the RON b-chain extracellular sequences [16,20]. The deletion causes structural changes that lead to cellular transformation in vitro and tumor growth in vivo [16,20]. In colon epithelial cells, the mechanism underlying ROND160-mediated tumorigenesis is to cause abnormal accumulation of b-catenin and to enhance b-catenin target gene expression such as c-myc and cyclin D1 [21,22]. Altered expression of b-catenin is critical in carcinogenesis of colon epithelial cells [23]. ROND160 also activates signaling proteins such as disheveled [DVL] and inhibits glycogen synthase kinase-3b (GSK-3b) activities [21,22]. Both proteins are key regulators for stability of b-catenin [23]. Recently studies have demonstrated that ROND160 is highly expressed in metastatic colorectal cancer samples (our unpublished data), suggesting that ROND160 plays a role in metastatic processes. ROND155 was cloned from a splicing mRNA transcript in primary colon cancer samples [16]. The transcript has a combined deletion of exons 5, 6, and 11. The deletion of exon 11 prevents the maturation of ROND155 into the a/b two-chain form and the protein is retained in the cytoplasm [16,19]. ROND155 has the ability to transform rodent fibroblasts in vitro and cause tumor growth in athymic nude mice [16]. The underlying mechanisms of ROND155 are similar to those mediated by ROND160. ROND110 is a proteolytically truncated RON variant found in cancerous cells overexpressing RON [12]. The site of truncation is at Arg631– Lys632 in the RON b-chain extracellular sequences. Trypsin-like enzymes on the cell membrane may be responsible for ROND110 truncation. Because of truncation, ROND110 is a protein missing the entire SEMA domain, PSI domain and the part of the first IPT domain. The protein resides on the cell

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surface and is constitutively active [12]. The significance of ROND110 in cellular transformation and tumorigenic activities is currently unknown. ROND55, also known as short form RON, is derived from alternative transcription at Met913 coded by exon 11 in the RON gene [24]. The protein has a short extracellular domain followed by the transmembrane and entire intracellular sequences. A similar protein has been found in mouse RON homologue STK [25]. In mouse, ROND55 expression is associated with the susceptibility of animals to Friend leukemia virus-induced erythroleukemia [25]. In human, the ROND55 mRNA has been detected in colon, lung, and other cancer cell lines [24]. The transcription of ROND55 seems to be related to hypermethylation of the distal island in the RON gene promoter region [26]. Overexpression of ROND55 causes cell transformation and migration [24]. We have studied transforming activities of ROND55 in different cell models. Expression of ROND55 in NIH3T3, MDCK, and AA/C1 cells did not cause cell transformation; however, increased cell growth and migration was observed (our unpublished data). Thus, more works are needed to determine if ROND55 is a transforming agent or merely contributes to the invasive phenotypes of epithelial cancers. The diversity of RON variants suggests their importance in pathogenesis of cancer. It is expected that additional RON variants will be discovered through combined proteomic and genetic analysis. We have recently identified three novel RON variants in a panel of breast cancer cells (our unpublished data). These variants were derived by abnormal mRNA splicing. Two variants have different intron retention in the RON b-chain extracellular domain, which disrupts the first IPT domain. The other has a partial deletion in an exon that is important for RON precursor maturation. Studies are currently underway to determine the relevance of these novel RON variants to breast cancer pathogenesis. 3. Mechanisms underlying RON variant production in cancer Alternative pre-mRNA splicing is mainly responsible for generation of RON variants although proteolytic truncation and alternative transcription also play a role [12]. Genome-wide analysis indicates that more than 50% of human genes present alternative spliced forms [15]. In general, splicing can be

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either constitutive or alternative. Constitutive splicing is a process in which only one type of mRNA is generated from a given gene [15]. In contrast, alternative splicing produces various mRNAs that give rise to a variety of proteins differing in their peptide sequences and biological activities [15]. The regulation of splicing involves both cis elements (sequences within the pre-mRNA) and trans elements (cellular factors) [15]. Cis elements, known as splicing enhancers and silencers, help with the identification of the exon–intron borders and prevent pseudoexons from being induced in the mRNA. Trans-acting splicing factors are involved in the assembly of the splicesome [15] and play a major role in the alternative splicing profile of a cell. Both elements are critical in regulating the constitutive and alternative splicing processes. The generation of RON spliced variants in cancer cells has several features. First, in normal tissues, RON mRNA is mainly governed by constitutive splicing with a single sized mRNA. Northern blot analysis of multiple tissues confirmed that this is the case [1,5]. In contrast, spliced RON variants are mainly observed in primary and established cancer cell lines [16,17,19,20]. This suggests that a switch from the constitutive to alternative splicing occurs in cancer cells. In other words, the alternative splicing plays a dominant role in regulating RON diversity under tumorigenic conditions. Definitely, such a switch is not a random process. Second, no mutations at the exon–intron boundary regions have been found so far in cancer cells that produce RON variants [16,20], indicating that the production of spliced isoform is not related to genomic abnormalities. Instead, it is a deregulation in the cellular splicing machinery. Third, changes in expression and activity in trans-acting splicing factors are responsible for specific splicing events [27]. The effect of splicing factor 2 (SF2/ASF) on the formation of ROND165 transcript is a good example [27]. In addition, novel silencers and enhancers are identified in certain RON gene exons [27], which facilitate the specific splicing process. Thus, cancer-related RON mRNA splicing is a coordinated effort contributed by constitutive and alternative splicing. Finally, spliced RON variants displayed diverse and even antagonist function [12]. In ROND160, the deletion of exons 5 and 6 resulted in increased tumorigenic activities both in vitro and in vivo [16]. In contrast, the deletion of exon 19 produced ROND170 with dominant negative activities that inhibit ROND160-mediated onco-

genic activities [17]. Clearly, the outcome of different splicing has different consequences. Currently, the detailed mechanisms underlying the generation of individual RON splicing variants are largely unknown. However, a recent study has provided insight about how ROND165 was generated [27], which might have relevance to other RON variants. The central finding from the study is that exon 11 splicing is controlled by a silencer and an enhancer located in exon 12. Specifically, the identified exonic splicing enhancer (ESE) contains a purine-rich sequence that matches the consensus-binding motif for SF2/ASF [28]. SF2/ASF belongs to a group of highly conserved serine/arginine rich [SR] proteins and is essential for constitutive splicing as well as regulation of alternative splicing [28]. In cancer cells expressing ROND165, the levels of SF2/ASF parallel the activities of ESE and the amounts of ROND165 transcripts [27]. Molecular analysis further confirmed that SF2/ASF directly binds the ESE element but not the mutated sequence [27]. In addition, the binding is specific only to SF2/ASF but not to other SR family members [27]. Overexpression of SF2/ASF in cells naturally expressing RON regulated alternative splicing and promoted the production of the ROND165 mRNA transcript [27]. Moreover, SF2/ASF regulated ROND165 production, which leads to epithelial to mesenchymal transition [29], a phenotype often observed in tumor cells expressing RON variants including ROND165 [30]. 4. Tumorigenic signals mediated by RON variants Oncogenic RON variants such as ROND160 and ROND155 transduce multiple signals that direct a unique program known as invasive tumorigenesis [12,16]. This is featured by oncogenic transformation followed by loss of epithelial properties and gain of mesenchymal phenotypes with increased cell migration, invasion, and replication. Activation of Ras [31], Erk1/2 [32], p38 MAP kinase [32], PI-3 kinase [33], AKT [32], JNK [34], b-Catenin [21], DVL [21], GSK-3b [21], Smad [30], and NF-jB [35], dependent on cellular context, has been implicated in the RON variant-mediated sequential tumorigenic progression. The distinct features of RON variants in tumorigenic activities that differ from RON are the increased catalytic efficiency of the kinase activities and the changes in substrate profiles [21,34]. The changes in the critical domains

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in the RON sequences are the molecular basis for these abnormalities. The extracellular sequence of RON contains several unique structures such as sema domain, PSI motif, and IPT units [8], which are often subjected for deletion and truncation under pathological conditions. For example, the IPT units are required to maintain the integrity of RON and its activity. By eliminating the first IPT unit, ROND160 acquires cancer-causing activities [16,20]. Moreover, IPT is involved in proper maturation of the RON receptor. Deletion of the fourth IPT unit, as shown in ROND165, resulted in accumulation of single-chain pro-ROND165 in the cytoplasm [19]. The significance of the RON extracellular domains is also illustrated in ROND55. By alternative transcription that eliminates the majority of the extracellular domains, ROND55 acquired tumorigenic activities that inhibit E-cadherin expression and promote invasive phenotype [24]. The juxtamembrane sequences play a role in regulating RON stability [36]. This is achieved by phosphorylation of Y1017 that recruits c-Cbl, an ubiquitin–protein ligase [37], to RON through the c-Cbl tyrosine kinase-binding domain [36] and promotes ubiquitination and degradation of RON [36]. Oncogenic conversion escapes the c-Cbl-mediated ubiquitination [38]. We have found that ROND160 is highly resistant to c-Cbl-mediated down-regulation (our unpublished data), suggesting that oncogenic RON variants display increased protein stability against degradation, which could facilitate cancer progression. The core region of the RON kinase domain comprises several sub-domains including N-helix, catalytic loop, and activation loop [8]. The role of N-helix in RON is unclear, although it has an auto-inhibitory effect on MET kinase activities [39]. Phosphorylation of Y1238 and Y1239 in the catalytic loop is essential for the kinase activities. In oncogenic RON variants, persistent and prolonged phosphorylation of Y1238 and Y1239 exists [16,20,21], which sustains the signal transduction leading to increased tumorigenic activities. The C-terminal tail plays two roles in regulating RON activities. One is mediated by a Y1353VQLXXX-Y1360MNL sequence known as the multiple docking site [40]. The site served as an anchor to recruit downstream signaling molecules such as PI-3K, Smad, Grb2, and others [12]. However, results from tumorigenic studies using RONM1254T containing the double mutations in the Y1353 and

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Y1360 have shown that the docking site is not required for oncogenic activities [41]. The other role is to regulate RON kinase activities. In vitro biochemical analysis has showed that the entire C-terminal tail has an auto-inhibitory effect on the RON kinase activities [42], probably by interaction with the kinase catalytic domain. To validate these data, we have generated two RON variants, RON-cf and ROND160-cf, both lacking the entire C-terminal tail. Stable NIH3T3 cells expressing high levels of RON-cf or RON160-cf were established. Results from phosphorylation assays indicated that both RON-cf and ROND160-cf did not show any spontaneous or MSP-induced tyrosine phosphorylation. They also failed to activate downstream signaling events. In addition, RON-cf or RON160-cf had no effects on cell scattering, matrix invasion, and cellular transformation (our unpublished data). These in vivo results, contradicted to those reported by in vitro analysis [42], suggest that the entire C-terminal tail is required for RON or ROND160-mediated tumorigenic activities. 5. RON variants as potential therapeutical targets Aberrant expression of receptor tyrosine kinases is a valid target for cancer therapy. Application of small chemical inhibitors and specific antibodies in treatment of metastatic colon, lung, and breast cancers has achieved clinical significance [43]. Evidence accumulated from the last several years has indicated that altered RON expression is a therapeutical target. First, RON is overexpressed in various epithelial cancers [13,14,16,44,45]. Immunohistochemical analysis of tumor tissue microarray [13,45] has indicated that RON is highly accumulated in breast, colon, lung [adenocarcinoma only], thyroid, skin, bladder, and pancreas cancer samples [45]. Generation of RON variants in cancer cells is another pathogenic feature. In contrast, expression of RON and its variants is minimal in corresponding normal epithelial cells [45]. These observations provide the rationale for selecting RON and its variants as potential drug targets. Second, RON variant expression facilitates tumor progression towards malignancy [16,46]. Studies from in vitro analysis have shown that RON and its variants cause epithelial cell transformation, promote invasive phenotypes, and mediate tumor growth in vivo [12]. In transgenic models, increased RON expression in lung or breast led to

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tumor growth and distant metastasis [47,48]. These results demonstrated that RON overexpression is a causative or pathogenic factor contributing to epithelial carcinogenesis. Third, various approaches aimed at inhibition of RON activities have showed pharmacological effects on tumorigenic phenotypes mediated by RON or its variants. For example, a five amino acid peptide in the CD44 v6 containing isoform has the ability to block MSP-induced RON activation and subsequent cell migration [49]. A soluble RON-sema domain has been shown to inhibit ligand-dependent RON activation and subsequent biological responses in vitro [50]. The effect of a kinase-dead RON has been found to modulate RON-mediated tumorigenic activities [26]. ROND170, a variant with a defective kinase domain, also showed the dominant negative effect that inhibits the growth of colon cancer HT-29 cells and reverses their invasive phenotypes [17]. Silencing RON gene expression by siRNA techniques is another example that significantly inhibits cancer cell proliferation in vitro and tumorigenic growth in vivo [22]. Currently, small molecule inhibitors specific to RON or its variants are not available. However, two inhibitors, known as PHA-665752 [51] and PF-2341066 [52], highly specific to the MET receptor, have been shown to inhibit RON phosphorylation in tumor cell lines with IC50 values of 0.9 and 2.0 lM, respectively [51,52]. Using NIH3T3 cells expressing RON or RON160, we found that PHA-665752 hardly inhibits ligand-dependent or independent tyrosine phosphorylation even high concentrations up to 20 lM were used. PHA665752 also failed to inhibit RON160-mediated cell migration, invasion, and tumorigenic transformation (our unpublished data). The detailed analysis of PF-2341066 on RON activities is currently not available. Nevertheless, available data suggested that structural differences in the kinase domain exist between RON and MET. Thus, it is desirable to design RON specific small chemical inhibitors based on its unique kinase domain to block pathogenic activities of RON. Finally, studies from a tumor xenograft model have showed that a specific RON antibody inhibits tumor growth mediated by cancer cells derived from colon, lung, and pancreas [13]. The antibody also inhibits RON-mediated signaling events and biological activities such as cell migration [13]. By selecting a panel of mAb, we have recently found that a mouse mAb 2F2 has the inhibitory effect in vivo

on ROND160-mediated tumorigenesis. 2F2 is the IgG mAb specific to a distinct epitope on the RON extracellular sequences. The binding caused the internalization of RON with diminished signaling activities. In Balb/c mice injected subcutaneously with 3T3-RON160 cells, 2F2 treatment (15 mg mAb/kg, twice a week for 3 weeks) significantly prevented tumor formation and subsequent growth. More than 65% inhibition in tumor volume was achieved in comparison with mice treated with control IgG. Similar results were also obtained when athymic nude mice were used in which human colon or breast cancer cells were used (our unpublished data). These results, together with those described above [13], provide compelling evidence that targeting RON expression has cancer therapeutical significance. Thus, development of antibody or small chemical inhibitor based therapeutical strategies could represent a novel approach for the treatment of malignant tumors such as colon and breast cancer in the future. Acknowledgements This work was supported by grants from National Natural Science Foundation of China #30430700 (to M.H.W.), Zhejiang Provincial Foundation for Medical and Health Sciences #2006A034 (to H.P.Y.), and US National Institute of Health R01 CA91980 (to M.H.W.). References [1] C. Ronsin, F. Muscatelli, M.G. Mattei, et al., A novel putative receptor protein tyrosine kinase of the met family, Oncogene 8 (1993) 1195–1202. [2] A. Iwama, K. Okano, T. Sudo, et al., Molecular cloning of a novel receptor tyrosine kinase gene, STK, derived from enriched hematopoietic stem cells, Blood 83 (1994) 3160– 3169. [3] A. Skeel, T. Yoshimura, S.D. Showalter, S. Tanaka, E. Appella, E.J. Leonard, Macrophage-stimulating protein: purification, partial amino acid sequence, and cellular activity, J. Exp. Med. 173 (1991) 1227–1234. [4] T. Yoshimua, N. Yuhki, M.H. Wang, et al., Cloning sequencing and expression of human macrophage stimulating protein [MSP] confirms MSP as a member of the family of kringle proteins and locates the MSP gene on chromosome 3, J. Biol. Chem. 268 (1993) 15462–15468. [5] G. Gaudino, A. Follenzi, L. Naldini, et al., RON is a heterodimeric tyrosine kinase receptor activated by the HGF homologue MSP, EMBO J. 13 (1994) 3524–3532. [6] M.H. Wang, C. Ronsin, M.C. Gesnel, et al., Identification of the ron gene product as the receptor for the human macrophage stimulating protein, Science 266 (1994) 117–119.

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