Exploring the Conformational and Binding Dynamics of HMGA2·DNA Complexes Using Trapped Ion Mobility Spectrometry-Mass Spectrometry.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is an intrinsically disordered DNA-binding protein expressed during embryogenesis. In the present work, the conformational and binding dynamics of HMGA2 and HMGA2 in complex with a 22-nt (DNA22) and a 50-nt (DNA50) AT-rich DNA hairpin were investigated using trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) under native starting solvent conditions (e.g., 100 mM aqueous NH4Ac) and collision-induced unfolding/dissociation (CIU/CID) as well as solution fluorescence anisotropy to assess the role of the DNA ligand when binding to the HMGA2 protein. CIU-TIMS-CID-MS/MS experiments showed a significant reduction of the conformational space and charge-state distribution accompanied by an energy stability increase of the native HMGA2 upon DNA binding. Fluorescence anisotropy experiments and CIU-TIMS-CID-MS/MS demonstrated for the first time that HMGA2 binds with high affinity to the minor groove of AT-rich DNA oligomers and with lower affinity to the major groove of AT-rich DNA oligomers (minor groove occupied by a minor groove binder Hoechst 33258). The HMGA2·DNA22 complex (18.2 kDa) 1:1 and 1:2 stoichiometry suggests that two of the AT-hook sites are accessible for DNA binding, while the other AT-hook site is probably coordinated by the C-terminal motif peptide (CTMP). The HMGA2 transition from disordered to ordered upon DNA binding is driven by the interaction of the three basic AT-hook residues with the minor and/or major grooves of AT-rich DNA oligomers.

Transcriptomic-Based Identification of the Immuno-Oncogenic Signature of Cholangiocarcinoma for HLC-018 Multi-Target Therapy Exploration.

Cholangiocarcinomas (CHOLs), hepatobiliary malignancies, are characterized by high genetic heterogeneity, a rich tumor microenvironment, therapeutic resistance, difficulty diagnosing, and poor prognoses. Current knowledge of genetic alterations and known molecular markers for CHOL is insufficient, necessitating the need for further evaluation of the genome and RNA expression data in order to identify potential therapeutic targets, clarify the roles of these targets in the tumor microenvironment, and explore novel therapeutic drugs against the identified targets. Consequently, in our attempt to explore novel genetic markers associated with the carcinogenesis of CHOL, five genes (SNX15, ATP2A1, PDCD10, BET1, and HMGA2), collectively termed CHOL-hub genes, were identified via integration of differentially expressed genes (DEGs) from relatively large numbers of samples from CHOL GEO datasets. We further explored the biological functions of the CHOL-hub genes and found significant enrichment in several biological process and pathways associated with stem cell angiogenesis, cell proliferation, and cancer development, while the interaction network revealed high genetic interactions with a number of onco-functional genes. In addition, we established associations between the CHOL-hub genes and tumor progression, metastasis, tumor immune and immunosuppressive cell infiltration, dysfunctional T-cell phenotypes, poor prognoses, and therapeutic resistance in CHOL. Thus, we proposed that targeting CHOL-hub genes could be an ideal therapeutic approach for treating CHOLs, and we explored the potential of HLC-018, a novel benzamide-linked small molecule, using molecular docking of ligand-receptor interactions. To our delight, HLC-018 was well accommodated with high binding affinities to binding pockets of CHOL-hub genes; more importantly, we found specific interactions of HLC-018 with the conserved sequence of the AT-hook DNA-binding motif of HMGA2. Altogether, our study provides insights into the immune-oncogenic phenotypes of CHOL and provides valuable information for our ongoing experimental validation.

Methylation-dependent SUMOylation of the architectural transcription factor HMGA2.

High mobility group A2 (HMGA2) is a chromatin-associated protein involved in the regulation of stem cell function, embryogenesis and cancer development. Although the protein does not contain a consensus SUMOylation site, it is shown to be SUMOylated. In this study, we demonstrate that the first lysine residue in the reported K66KAE SUMOylation motif in HMGA2 can be methylated in vitro and in vivo by the Set7/9 methyltransferase. By editing the lysine, the increased hydrophobicity of the resulting 6-N-methyl-lysine transforms the sequence into a consensus SUMO motif. This post-translational editing dramatically increases the subsequent SUMOylation of this site. Furthermore, similar putative methylation-dependent SUMO motifs are found in a number of other chromatin factors, and we confirm methylation-dependent SUMOylation of a site in one such protein, the Polyhomeotic complex 1 homolog (PHC1). Together, these results suggest that crosstalk between methylation and SUMOylation is a general mode for regulation of chromatin function.

Identification of HMGA2 inhibitors by AlphaScreen-based ultra-high-throughput screening assays.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a multi-functional DNA-binding protein that plays important roles in tumorigenesis and adipogenesis. Previous results showed that HMGA2 is a potential therapeutic target of anticancer and anti-obesity drugs by inhibiting its DNA-binding activities. Here we report the development of a miniaturized, automated AlphaScreen ultra-high-throughput screening assay to identify inhibitors targeting HMGA2-DNA interactions. After screening the LOPAC1280 compound library, we identified several compounds that strongly inhibit HMGA2-DNA interactions including suramin, a century-old, negatively charged antiparasitic drug. Our results show that the inhibition is likely through suramin binding to the "AT-hook" DNA-binding motifs and therefore preventing HMGA2 from binding to the minor groove of AT-rich DNA sequences. Since HMGA1 proteins also carry multiple "AT-hook" DNA-binding motifs, suramin is expected to inhibit HMGA1-DNA interactions as well. Biochemical and biophysical studies show that charge-charge interactions and hydrogen bonding between the suramin sulfonated groups and Arg/Lys residues play critical roles in the binding of suramin to the "AT-hook" DNA-binding motifs. Furthermore, our results suggest that HMGA2 may be one of suramin's cellular targets.

The Mammalian High Mobility Group Protein AT-Hook 2 (HMGA2): Biochemical and Biophysical Properties, and Its Association with Adipogenesis.

The mammalian high-mobility-group protein AT-hook 2 (HMGA2) is a small DNA-binding protein and consists of three "AT-hook" DNA-binding motifs and a negatively charged C-terminal motif. It is a multifunctional nuclear protein directly linked to obesity, human height, stem cell youth, human intelligence, and tumorigenesis. Biochemical and biophysical studies showed that HMGA2 is an intrinsically disordered protein (IDP) and could form homodimers in aqueous buffer solution. The "AT-hook" DNA-binding motifs specifically bind to the minor groove of AT-rich DNA sequences and induce DNA-bending. HMGA2 plays an important role in adipogenesis most likely through stimulating the proliferative expansion of preadipocytes and also through regulating the expression of transcriptional factor Peroxisome proliferator-activated receptor γ (PPARγ) at the clonal expansion step from preadipocytes to adipocytes. Current evidence suggests that a main function of HMGA2 is to maintain stemness and renewal capacity of stem cells by which HMGA2 binds to chromosome and lock chromosome into a specific state, to allow the human embryonic stem cells to maintain their stem cell potency. Due to the importance of HMGA2 in adipogenesis and tumorigenesis, HMGA2 is considered a potential therapeutic target for anticancer and anti-obesity drugs. Efforts are taken to identify inhibitors targeting HMGA2.

The regulation of acetylation and stability of HMGA2 via the HBXIP-activated Akt-PCAF pathway in promotion of esophageal squamous cell carcinoma growth.

High-mobility group AT-hook 2 (HMGA2) is an architectural transcription factor that plays essential roles in embryonic development and cancer progression. However, the mechanism of HMGA2 regulation remains largely uncharacterized. Here, we demonstrate that HMGA2 can be modulated by hepatitis B X-interacting protein (HBXIP), an oncogenic transcriptional coactivator, in esophageal squamous cell carcinoma (ESCC). HMGA2 expression was positively associated with HBXIP expression in clinical ESCC tissues, and their high levels were associated with advanced tumor stage and reduced overall and disease-free survival. We found that oncogenic HBXIP could posttranslationally upregulate HMGA2 protein level in ESCC cells. HBXIP induced HMGA2 acetylation at the lysine 26 (K26), resulting in HMGA2 protein accumulation. In this process, HBXIP increased the acetyltransferase p300/CBP-associated factor (PCAF) phosphorylation and activation via the Akt pathway, then PCAF directly interacted with HMGA2, leading to HMGA2 acetylation in the cells. HMGA2 K26 acetylation enhanced its DNA binding capacity and blocked its ubiquitination and then inhibited proteasome-dependent degradation. Functionally, HBXIP-stabilized HMGA2 could promote ESCC cell growth in vitro and in vivo. Strikingly, aspirin suppressed ESCC growth by inhibiting HBXIP and HMGA2. Collectively, our findings disclose a new mechanism for the posttranslational regulation of HMGA2 mediated by HBXIP in ESCC.

Molecular cloning of WIF1 and HMGA2 reveals ear-preferential expression while uncovering a missense mutation associated with porcine ear size in WIF1.

Considerable diversity exists in porcine ear size, which is an important morphological feature of pig breeds. Previously, we localized four crucial candidate genes-high mobility group AT-hook 2 (HMGA2), LEM domain-containing 3 (LEMD3), methionine sulfoxide reductase B3 (MSRB3) and Wnt inhibitory factor 1 (WIF1)-on Sus Scrofa chromosome 5 affecting porcine ear size, then cloned LEMD3 and MSBR3. In this study, we performed rapid amplification of cDNA ends to obtain full-length cDNA sequences of 2338-bp WIF1 and 2998-bp HMGA2. Using quantitative real-time PCR, we revealed that WIF1 expression was highest in ear cartilage of 60-day-old pigs and that this is therefore a better candidate gene for ear size than HMGA2. We further screened coding sequence variants in both genes and identified only one missense mutation (WIF1:c.1167C>G) in a conserved epidermal growth factor-like domain from the mammalian WIF1 protein. The protein-altering mutation was significantly associated with ear size across the Large White × Minzhu hybrid and Beijing Black pig populations. When WIF1:c.1167C>G was included as fixed effect in the model to re-run a genome-wide association study in the Large White × Minzhu intercross population the P-value of the peak SNP on SSC5 from re-running the genome-wide association study dropped from 2.45E-12 to 7.33E-05. Taken together, the WIF1:c.1167C>G could be an important mutation associated with ear size. Our findings provide helpful information for further studies of the molecular mechanisms controlling porcine ear size.

HMGA2 as a functional antagonist of PARP1 inhibitors in tumor cells.

Poly(ADP-ribose) polymerase 1 inhibitors alone or in combination with DNA damaging agents are promising clinical drugs in the treatment of cancer. However, there is a need to understand the molecular mechanisms of resistance to PARP1 inhibitors. Expression of HMGA2 in cancer is associated with poor prognosis for patients. Here, we investigated the novel relationship between HMGA2 and PARP1 in DNA damage-induced PARP1 activity. We used human triple-negative breast cancer and fibrosarcoma cell lines to demonstrate that HMGA2 colocalizes and interacts with PARP1. High cellular HMGA2 levels correlated with increased DNA damage-induced PARP1 activity, which was dependent on functional DNA-binding AT-hook domains of HMGA2. HMGA2 inhibited PARP1 trapping to DNA and counteracted the cytotoxic effect of PARP inhibitors. Consequently, HMGA2 decreased caspase 3/7 induction and increased cell survival upon treatment with the alkylating methyl methanesulfonate alone or in combination with the PARP inhibitor AZD2281 (olaparib). HMGA2 increased mitochondrial oxygen consumption rate and spare respiratory capacity and increased NAMPT levels, suggesting metabolic support for enhanced PARP1 activity upon DNA damage. Our data showed that expression of HMGA2 in cancer cells reduces sensitivity to PARP inhibitors and suggests that targeting HMGA2 in combination with PARP inhibition may be a promising new therapeutic approach.

Peptide Sequence Influence on the Conformational Dynamics and DNA binding of the Intrinsically Disordered AT-Hook 3 Peptide.

The intrinsically disordered ATHP3 was studied at native conditions and in complex with DNA using single amino acid substitutions and high-resolution ion mobility spectrometry coupled to mass spectrometry (trapped IMS-MS). Results showed that ATHP3 can exist in multiple conformations at native conditions (at least 10 conformers were separated), with a variety of proline cis/trans orientations, side chain orientations and protonation sites. When in complex with AT rich DNA hairpins, the -RGRP- core is essential for stabilizing the ATHP3: DNA complex. In particular, the arginine in the sixth position plays an important role during binding to AT-rich regions of hairpin DNA, in good agreement with previous NMR and X-ray data. Mobility based correlation matrices are proposed as a way to reveal differences in structural motifs across the peptide mutants based on the conformational space and relative conformer abundance.

Nanoscale Assembly of High-Mobility Group AT-Hook 2 Protein with DNA Replication Fork.

High mobility group AT-hook 2 (HMGA2) protein is composed of three AT-hook domains. HMGA2 expresses at high levels in both embryonic stem cells and cancer cells, where it interacts with and stabilizes replication forks (RFs), resulting in elevated cell proliferation rates. In this study, we demonstrated that HMGA2 knockdown reduces cell proliferation. To understand the features required for interaction between HMGA2 and RFs, we studied the solution structure of HMGA2, free and in complex with RFs, using an integrated host of biophysical techniques. Circular dichroism and NMR experiments confirmed the disordered state of unbound HMGA2. Dynamic light scattering and sedimentation velocity experiments demonstrated that HMGA2 and RF are monodisperse in solution, and form an equimolar complex. Small-angle x-ray scattering studies revealed that HMGA2 binds in a side-by-side orientation to RF where 3 AT-hooks act as a clamp to wrap around a distorted RF. Thus, our data provide insights into how HMGA2 interacts with stalled RFs and the function of the process.

The Fragment HMGA2-sh-3p20 from HMGA2 mRNA 3'UTR Promotes the Growth of Hepatoma Cells by Upregulating HMGA2.

High mobility group A2 (HMGA2) plays a crucial role in the development of cancer. However, the mechanism by which HMGA2 promotes the growth of hepatocellular carcinoma (HCC) remains unclear. Here, we explore the hypothesis that HMGA2 may enhance the growth of hepatoma cells through a fragment based on the secondary structure of HMGA2 mRNA 3'-untranslated region (3'UTR). Bioinformatics analysis showed that HMGA2 mRNA displayed a hairpin structure within its 3'UTR, termed HMGA2-sh. Mechanistically, RNA immunoprecipitation assays showed that the microprocessor Drosha or DGCR8 interacted with HMGA2 mRNA in hepatoma cells. Then, Dicer contributes to the generation of the fragment HMGA2-sh-3p20 from the HMGA2-sh. HMGA2-sh-3p20 was screened by PCR analysis. Interestingly, HMGA2-sh-3p20 increased the expression of HMGA2 through antagonizing the tristetraprolin (TTP)-mediated degradation of HMGA2. HMGA2-sh-3p20 inhibited the expression of PTEN by targeting the 3'UTR of PTEN mRNA. In addition, the overexpression of PTEN could downregulate HMGA2 expression. Significantly, we documented the ability of HMGA2-sh-3p20 to promote the growth of hepatoma cells in vitro and in vivo. Thus, we conclude that the fragment HMGA2-sh-3p20 from HMGA2 mRNA 3'UTR promotes the growth of hepatoma cells by upregulating HMGA2. Our finding provides new insights into the mechanism by which HMGA2 enhances hepatocarcinogenesis.

Low-molecular-mass secretome profiling identifies HMGA2 and MIF as prognostic biomarkers for oral cavity squamous cell carcinoma.

The profiling of cancer cell secretomes is considered to be a good strategy for identifying cancer-related biomarkers, but few studies have focused on identifying low-molecular-mass (LMr) proteins (<15 kDa) in cancer cell secretomes. Here, we used tricine-SDS-gel-assisted fractionation and LC-MS/MS to systemically identify LMr proteins in the secretomes of five oral cavity squamous cell carcinoma (OSCC) cell lines. Cross-matching of these results with nine OSCC tissue transcriptome datasets allowed us to identify 33 LMr genes/proteins that were highly upregulated in OSCC tissues and secreted/released from OSCC cells. Immunohistochemistry and quantitative real-time PCR were used to verify the overexpression of two candidates, HMGA2 and MIF, in OSCC tissues. The overexpressions of both proteins were associated with cervical metastasis, perineural invasion, deeper tumor invasion, higher overall stage, and a poorer prognosis for post-treatment survival. Functional assays further revealed that both proteins promoted the migration and invasion of OSCC cell lines in vitro. Collectively, our data indicate that the tricine-SDS-gel/LC-MS/MS approach can be used to efficiently identify LMr proteins from OSCC cell secretomes, and suggest that HMGA2 and MIF could be potential tissue biomarkers for OSCC.

The Dimerization State of the Mammalian High Mobility Group Protein AT-Hook 2 (HMGA2).

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a chromosomal architectural transcription factor involved in cell transformation and oncogenesis. It consists of three positively charged "AT-hooks" and a negatively charged C-terminus. Sequence analyses, circular dichroism experiments, and gel-filtration studies showed that HMGA2, in the native state, does not have a defined secondary or tertiary structure. Surprisingly, using combined approaches of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) chemical cross-linking, analytical ultracentrifugation, fluorescence resonance energy transfer (FRET), and mass spectrometry, we discovered that HMGA2 is capable of self-associating into homodimers in aqueous buffer solution. Our results showed that electrostatic interactions between the positively charged "AT-hooks" and the negatively charged C-terminus greatly contribute to the homodimer formation.

Isomerization kinetics of AT hook decapeptide solution structures.

The mammalian high mobility group protein HMGA2 contains three DNA binding motifs associated with many physiological functions including oncogenesis, obesity, stem cell youth, human height, and human intelligence. In the present paper, trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) has been utilized to study the conformational dynamics of the third DNA binding motif using the "AT hook" decapeptide unit (Lys(1)-Arg(2)-Prol(3)-Arg(4)-Gly(5)-Arg(6)-Prol(7)-Arg(8)-Lys(9)-Trp(10), ATHP) as a function of the solvent state. Solvent state distributions were preserved during electrospray ion formation, and multiple IMS bands were identified for the [M + 2H](2+) and for the [M + 3H](3+) charge states. Conformational isomer interconversion rates were measured as a function of the trapping time for the [M + 2H](2+) and [M + 3H](3+) charge states. Candidate structures were proposed for all IMS bands observed. Protonation site, proline residue conformation, and side chain orientations were identified as the main motifs governing the conformational interconversion processes. Conformational dynamics from the solvent state distribution to the gas-phase "de-solvated" state distribution demonstrated that ATHP is "structured", and relative abundances are associated with the relative stability between the proposed conformers. The most stable ATHP [M + 2H](2+) conformation at the "de-solvated" state corresponds to the AT hook motif observed in AT-rich DNA regions.

HMGA2 exhibits dRP/AP site cleavage activity and protects cancer cells from DNA-damage-induced cytotoxicity during chemotherapy.

HMGA proteins are not translated in normal human somatic cells, but are present in high copy numbers in pluripotent embryonic stem cells and most neoplasias. Correlations between the degree of malignancy, patient prognostic index and HMGA levels have been firmly established. Intriguingly, HMGA2 is also found in rare tumor-inducing cells which are resistant to chemotherapy. Here, we demonstrate that HMGA1a/b and HMGA2 possess intrinsic dRP and AP site cleavage activities, and that lysines and arginines in the AT-hook DNA-binding domains function as nucleophiles. We also show that HMGA2 can be covalently trapped at genomic abasic sites in cancer cells. By employing a variety of cell-based assays, we provide evidence that the associated lyase activities promote cellular resistance against DNA damage that is targeted by base excision repair (BER) pathways, and that this protection directly correlates with the level of HMGA2 expression. In addition, we demonstrate an interaction between human AP endonuclease 1 and HMGA2 in cancer cells, which supports our conclusion that HMGA2 can be incorporated into the cellular BER machinery. Our study thus identifies an unexpected role for HMGA2 in DNA repair in cancer cells which has important clinical implications for disease diagnosis and therapy.

Macroscopic differences in HMGA oncoproteins post-translational modifications: C-terminal phosphorylation of HMGA2 affects its DNA binding properties.

HMGA is a family of nuclear proteins involved in a huge number of functions at the chromatin level. It consists of three members, HMGA1a, HMGA1b, and HMGA2, having high sequence homology and sharing the same structural organization (three highly conserved DNA-binding domains, an acidic C-terminal tail, and a protein-protein interaction domain). They are considered important nodes in the chromatin context, establishing a complex network of interactions with both promoter/enhancer sequences and nuclear factors. They are involved in a plethora of biological processes and their activities are finely tuned by several different post-translational modifications. We have performed an LC/MS screening on several different cell lines to investigate HMGA proteins expression and their post-translational modifications in order to detect distinctive modification patterns for each. Our analyses evidenced relevant macroscopic differences in the phosphorylation and methylation patterns of these proteins. These differences occur both within the HMGA family members and in the different cell types. Focusing on HMGA2, we have mapped its in vivo phosphorylation sites demonstrating that, similarly to the HMGA1 proteins, it is highly phosphorylated on the acidic C-terminal tail and that these modifications affect its DNA binding properties.

Physcomitrella HMGA-type proteins display structural differences compared to their higher plant counterparts.

High mobility group (HMG) proteins of the HMGA family are chromatin-associated proteins that act as architectural factors in nucleoprotein structures involved in gene transcription. To date, HMGA-type proteins have been studied in various higher plant species, but not in lower plants. We have identified two HMGA-type proteins, HMGA1 and HMGA2, encoded in the genome of the moss model Physcomitrella patens. Compared to higher plant HMGA proteins, the two Physcomitrella proteins display some structural differences. Thus, the moss HMGA proteins have six (rather than four) AT-hook DNA-binding motifs and their N-terminal domain lacks similarity to linker histone H1. HMGA2 is expressed in moss protonema and it localises to the cell nucleus. Typical of HMGA proteins, HMGA2 interacts preferentially with A/T-rich DNA, when compared with G/C-rich DNA. In cotransformation assays in Physcomitrella protoplasts, HMGA2 stimulated reporter gene expression. In summary, our data show that functional HMGA-type proteins occur in Physcomitrella.

Specific recognition of AT-rich DNA sequences by the mammalian high mobility group protein AT-hook 2: a SELEX study.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a transcriptional factor involved in cell differentiation and transformation. Disruption of its normal expression pattern is directly linked to oncogenesis and obesity. HMGA2 contains three "AT-hook" DNA binding domains, which specifically bind to the minor groove of AT-rich sequences. Using a PCR-based systematic evolution of ligands by exponential enrichment (SELEX) procedure, we have identified two consensus sequences for HMGA2, 5'-ATATTCGCGAWWATT-3' and 5'-ATATTGCGCAWWATT-3', where W represents A or T. These two consensus sequences have a unique and interesting feature: the first five base pairs are AT-rich, the middle four base pairs are GC-rich, and the last six base pairs are AT-rich. Our results showed that all three of these segments are critical for high-affinity binding of HMGA2 to DNA. For example, if one of the AT-rich sequences is mutated to a non-AT-rich sequence, the DNA binding affinity of HMGA2 is reduced at least 100-fold. Intriguingly, if the GC-segment is replaced by an AT-rich segment, the binding affinity of HMGA2 is reduced approximately 5-fold. Identification of the consensus sequences for HMGA2 represents an important step toward finding its binding sites within the genome.

The second AT-hook of the architectural transcription factor HMGA2 is determinant for nuclear localization and function.

High Mobility Group A (HMGA) is a family of architectural nuclear factors which play an important role in neoplastic transformation. HMGA proteins are multifunctional factors that associate both with DNA and nuclear proteins that have been involved in several nuclear processes including transcription. HMGA localization is exclusively nuclear but, to date, the mechanism of nuclear import for these proteins remains unknown. Here, we report the identification and characterization of a nuclear localization signal (NLS) for HMGA2, a member of the HMGA family. The NLS overlaps with the second of the three AT-hooks, the DNA-binding domains characteristic for this group of proteins. The functionality of this NLS was demonstrated by its ability to target a heterologous beta-galactosidase/green fluorescent protein fusion protein to the nucleus. Mutations to alanine of basic residues within the second AT-hook resulted in inhibition of HMGA2 nuclear localization and impairment of its function in activating the cyclin A promoter. In addition, HMGA2 was shown to directly interact with the nuclear import receptor importin-alpha2 via the second AT-hook. HMGA proteins are overexpressed and rearranged in a variety of tumors; our findings can thus help elucidating their role in neoplastic transformation.