Characterization and identification of PARM-1 as a new potential oncogene

The Graffi murine leukemia virus (MuLV) induces a wide spectrum of leukemias in several strains of mice, including lymphoid and non-lymphoid types [1, 2] making of this virus a good model to gain new insights on lymphoid leukemia development and progression and to identify new oncogenes. Retroviruses have been used as molecular tools to identify oncogenes or tumor suppressors directly targeted through the retroviral integration. However the microarray technology is attractive because it allows identifying, in addition to the retrovirus targeted genes, those involved in the cascade of events that leads to cell transformation, tumor progression, cancer and metastasis. We therefore used this approach to compare the transcriptome of a full panel of leukemias induced by the Graffi MuLV [2] and we focused our analyses on the lymphoid types [3]. We identified genes that were deregulated in one type of leukemia when compared to the corresponding control, therefore representing potential markers and oncogenes or tumor suppressor candidates that are specific for B, T or common to both types of leukemia. As expected, many of these genes were known to be specific to a lineage and to leukemia types (for details, see: [4]). Furthermore, we validated changes in the expression levels of 10 genes selected according to their specificity for lymphoid leukemias. These results clearly validated our approach and identified genes that now deserve more attention. Indeed, we previously reported that the Fmn2 gene harbors oncogenic potential. It was found specifically over-expressed in murine B-leukemias as well as in human pre-B-ALL especially in children bearing a t(12;21) translocation (TEL/AML1 rearrangement) [3].

In this study, we focused on genes that are associated with T-CD8⁺ leukemias. We identified Parm-1 (prostate androgen-regulated mucin-like protein 1), a gene specifically up-regulated in T-CD8⁺ leukemias induced by Graffi virus. PARM-1 is a member of the mucin family. Very little is known about the physiological and biological function of this gene and its precise role in cellular transformation has not been fully explored.

We characterized the function of PARM-1 and we investigated the oncogenic potential of mouse and human proteins. PARM-1 is a weakly secreted protein which contains a transmembrane domain (TM) and a cytoplasmic tail (CT) in addition to the extracellular (EC) domains. Both human (hPARM-1) and mouse (mPARM-1) proteins are predominantly located at the Golgi and in the early and late endosomes but transiently located at the plasma membrane. PARM-1 trafficking within the cells seems associated with the microtubule cytoskeleton. Also, PARM-1 induced both anchorage and serum-independent growth, enhanced cell proliferation and activated ERK1/2, AKT and STAT3.

Together, these results provide strong evidences for the oncogenic potential of PARM-1 and emphasize their important role in leukemogenesis.

The raw microarrays results obtained in our previous microarrays analysis were reanalyzed focusing on genes that were specifically deregulated in T-CD8⁺ leukemias when compared to T-cells control. From this analysis 50 probsets were selected (Fold-change of 4). Some of these genes were already known to be involved in T-CD8⁺ leukemias: Il2ra (expressed in primary leukemia cells from a patient with T-CD8⁺ prolymphocytic leukemia) [5]. Our microarray analysis also showed that some other genes were known to be associated with other T-leukemia sub-types or cancer as Irf4 (an oncogene locus which is frequently translocated in peripheral T-cell lymphomas) [7], Depdc6 (when over-expressed, it increases survival of hepatocellular carcinoma cells) [9] and Als2cl (a tumor-suppressor gene that contributes to the tumorigenesis of head and neck squamous cell carcinoma) [10]. These results validate our new microarray analysis. More interestingly, we also found other genes that had never been associated with leukemias nor with other types of cancer, or had no assigned function such as the Exoc3l4[23], Hectd2[24, 25] and AU014947. The complete list of these genes, which are good candidates for specific markers, oncogenes or tumour suppressors for T-CD8⁺ leukemias, is presented in Table 1.

From this list, we focused on the 9130213B05Rik that corresponds to the conserved mParm-1 gene (Additional file 1: Figure S1) and we validated the specificity of its over-expression in Graffi MuLV induced T-CD8⁺ tumors (Figure 1).

Our interest for this gene was drained by the fact that Parm-1 was poorly characterized and had never been clearly associated with cancer. Indeed, the rat Parm-1 is over-expressed in prostate epithelial cells after androgen deprivation following castration [26]. However, its human counterpart expression is increased by androgen in the LNCaP prostate cancer cell line and decreased in the CWR22 xenograft upon castration [17]. Moreover, ectopic expression of hParm-1 in human prostate cancer cell line enhances their proliferation [17]. However, the rat Parm-1 had no effect on rat cancer cell line [27]. In contrast, even if in vivo models demonstrated that over-expression of Parm-1 is not implicated in apoptosis [26], in vitro models suggested that Parm-1 is indirectly involved in the survival program [27]. Also, it was demonstrated that Parm-1 silencing in rat cardiac myocytes enhanced apoptotic response to endoplasmic reticulum stress [28]. Due to these conflicting data, we further characterized the function and determined the oncogenic potential of PARM-1.

The human mucin family can be sub-classified into secreted and membrane-associated mucin forms [14, 29, 30]. The extracellular domain of most transmembrane mucins is released from the cell surface [14]. Since PARM-1 shares similar structure with the membrane-associated mucins (Figure 2a and 2b), we determined whether the EC-domain of this highly conserved protein is also released. We showed that hPARM-1 is weakly intact secreted protein (Figure 3 and Additional file 2: Figure S2). This result, although unexpected for proteins of the mucin family, correlates with data reported for many other type I transmembrane proteins such as APP [31], N-CAM [32], insulin receptor [33], recombinant EGF precursor [34], and c-Kit receptor proto-oncogene [35].

Our results for PARM-1 subcellular localization agree with previous report [17], for hPARM-1 and extend our observations to the mPARM-1. Indeed, we show that both proteins co-localized within the Golgi and at early and late endosomes but weakly localized at the plasma membrane (Figure 4). The same localization was observed in NIH/3T3 cells transfected with ∆EC-GFP and ∆SP-GFP mutants (Figure 4g and 4h). However, EC-GFP and ∆TM-GFP mutants showed a GFP-like localization (Figure 4i and 4j) and ∆CT-GFP mutant predominantly showed plasma membrane localization (Figure 4k). These results suggest that TM probably determines the Golgi-endocytic pathway localization. Such observation had already been reported for other proteins as the type I transmembrane BACE1 protein. BACE1 is mainly located in the distal Golgi membrane but not considerably present at the plasma membrane of neuroblastoma cells. It was demonstrated that the TM-domain determines its Trans-Golgi Network (TGN) localization [36]. Our results also suggest that CT-domain inhibited plasma membrane localization (Figure 4k). This is reinforced by the fact that mutations in the CT (₂₈₇YGRL₂₉₀ to ₂₈₇AGRA₂₉₀) induced PARM-1 plasma membrane localization [17]. This YGRL motif acts as a tyrosine-based plasma membrane internalization signal [37] also present in Syntaxin-6 (STX6) protein which is localized to the TGN. Importantly, it was demonstrated that deletion of this motif prevents STX6 internalization and induces its plasma membrane accumulation [38]. Our data suggest that YGRL motif induces hPARM-1 internalization. Indeed, we showed that the internalization process of hPARM-1 was temperature-dependent, very dynamic at 37°C and dramatically inhibited at 4°C (Additional file 3: Movie S1). These results suggest a very quick internalization for hPARM-1 and may explain that the protein remains barely detectable at the plasma membrane.

It has been established that endosomes and endocytic proteins can traffic via microtubules [39, 40]. Our data indicated the important role of microtubules in PARM-1 trafficking. In fact, PARM-1 co-localized with the microtubule cytoskeleton (Figure 4l) and depolymerisation of its network with nocodazole induced a dramatic inhibition of PARM-1 trafficking accompanied by an accumulation of an important portion of PARM-1 at the cell periphery (Additional file 4: Movie S2). We also found that hPARM-1 co-localized with caveolin-1 (Figure 4m). This preliminary result suggests that PARM-1 internalization may be mediated via the caveolae. Further investigations will be needed to confirm the involvement of caveolin-1 in this process.

It is known that mucins are implicated in cancer development [29] but there were no convincing data yet on the role of Parm-1 in cellular transformation. We showed that PARM-1 enhanced the proliferative capacities (Figure 5) and confer the serum-independent growth to NIH/3T3 cells (Figure 6a) suggesting that it could induce an autocrine loop in cells thus stimulating their proliferation in absence of growth factors. Using the classical NIH/3T3 colony formation in soft agar test, we demonstrated that ectopic expression of PARM-1 conferred anchorage-independent growth to the cells and we found that both deletion mutants (∆CT-GFP and ∆EC-GFP) seem to retain part of their ability to confer this capacity to the cells (Figure 6b-d). These results let us speculate that the TM-domain should play an important role in the protein function especially in its targeting toward the appropriate cell compartment. It also suggests a complementary or collaborative role for EC- and CT-domains, respectively, with TM to induce anchorage independence. Similar results were reported for the MUC1 protein where EC- and CT-domains contribute separately to the cancer cell line invasiveness and metastasis [41].

We also analyzed the downstream signaling events leading to proliferation and provided first evidence on the role of PARM-1 in ERK1/2 and especially in AKT and STAT3 dependent signaling pathways (Figure 7). These pathways are a part of a more complex process leading to cell proliferation enhancement. In fact, the AKT is implicated in cell survival, growth and proliferation [21]. ERK1/2 is also implicated in the cell proliferation. Interestingly, these two pathways are constitutively activated in several human cancers [20]. Moreover, it is known that the STAT3 Ser-727 is phosphorylated by ERK1/2 [42, 43] and that STAT3 is also implicated in the proliferation tumor-derived cell lines [44]. In summary, activation of ERK1/2, AKT, and STAT3 shed further light on the mechanism by which PARM-1 may contribute to transformation.

Overall, our results strongly support an oncogenic role for Parm-1, member of the mucin family, especially in T-CD8⁺ leukemia and enable us to propose the following model: newly synthesized protein accumulates to the Golgi where post-transcriptional modifications occur (glycosylation and probably dimerization). A major fraction of PARM-1 protein will be retained in this compartment via its TM-domain, which seems to play a determinant role in the oncogenic potentiality of the protein. Certain amount of the protein will be packaged in vesicles for transport to the plasma membrane where a minor fraction of the entire PARM-1 will be secreted and could serve as a ligand (unknown receptor), which in turn leads to the activation of the downstream signaling pathway. In parallel, the YGRL motif will induce the rapid internalization and recycling of the intracellular protein, a prerequisite for its activity indicating that non-secreted PARM-1 could act as a new receptor or transporter. These data suggest a complex role for PARM-1. Further studies are required to better understand PARM-1 functions and could provide new tools to develop new therapeutic approaches in the treatment of human cancer.