Differential subcellular and extracellular localisations of proteins required for insulin-like growth factor- and extracellular matrix-induced signalling events in breast cancer progression

Experimental and clinical evidence has implicated a role for the insulin-like growth factor (IGF) axis in cancer progression [1]. In fact a number of inhibitors of, and antibodies directed against, the IGF type I receptor (IGF-IR) have been reported to show anti-tumour activity in vitro and in vivo, and are currently in clinical trials [2]. These studies, combined with many others, have highlighted the complexity of the dysregulation of the IGF system in cancers. Simply targeting the IGF-IR or the IGF system in isolation may therefore not be the most efficacious strategy for treating this disease; more complex therapeutic approaches to target the IGF system and prevent tumorigenesis and, in particular, metastasis, are likely to be required.

Cancer metastasis is the main contributor to breast cancer fatalities [3]. Women with metastatic breast cancers have considerably poorer survival outcomes than women whose cancers are localised to the breast [4, 5]. Adjuvant systemic therapies for patients with breast cancer metastasis remain palliative [3]. Understanding the processes underpinning the progression of breast cancer, identifying patients likely to develop metastases and developing strategies to prevent the secondary spread of cancers are of significant clinical and financial relevance. There has also been a growing urgency to create cost-effective and appropriate prognostic methods that can accurately resolve those patients with a poor prognosis that require more intense treatment regimes. The prognostic methods currently available are unable to adequately address this issue [6, 7].

A critical component that is often overlooked during the identification and analysis of prognostic biomarkers, and one which may explain the inability to develop adequate prognostic techniques thus far, is the interplay between tumour cells, the surrounding microenvironment and the growth factors present in this milieu. Cellular attachment and interactions with the extracellular matrix (ECM) regulate biological responses vital for tumour progression. Considerable evidence indicates that interactions between proteins required for IGF-induced signalling events and those within the ECM contribute to processes leading to cancer progression. Studies by Kricker et al.[8] found that IGF-I stimulates migration of MCF-7 breast cancer cells when bound to the ECM protein vitronectin (VN) indirectly through the presence of IGF binding proteins (IGFBPs). The presence of function blocking antibodies against IGF-IR and VN-binding integrins abolished the enhanced migration of these cells [9], while, the overexpression of total-akt/protein kinase B (AKT) and phosphorylated-akt/protein kinase B (P-AKT) enhanced IGF-I: IGFBP:VN-stimulated migration [9]. Gene microarray technology has also been applied to elucidate the molecular mechanisms involved in IGF-I: IGFBP:VN-stimulated migration of breast cancer cells in in vitro cell based assays [10]. These studies have identified a number of genes, including Stratifin (SFN), enhancer-of-split and hairy-related protein 2 (Sharp-2), Tissue Factor, Claudin-1 (CLDN1), that are uniquely regulated by the IGF-I: IGFBP:VN complex. The genes are known for their roles in migration, invasion as well as cell survival. However, to date the effects of IGF and ECM protein interactions on the dissemination and progression of breast cancer in vivo are unclear. Given this, we chose to investigate the clinical relevance of proteins required for IGF-induced signalling events and those within the ECM for the development and progression of breast cancer, as well as investigate these proteins as potential prognostic biomarkers.

The capability of the ECM and IGF system proteins to regulate cell function, and consequently tumorigenesis, is highly influenced by their spatial arrangement within and around the cell. It was observed that proteins required for IGF- and ECM-induced signalling events are differentially expressed between subcellular and extracellular localisations and that the interpretation of the protein immunoreactivity data is influenced by the scoring method applied. The results described below will only refer to the results obtained for the Q score scoring method [12]. The Q score values (x), including the standard deviation (SD) and sample numbers (n) for each protein across the tissue types and cellular localisation are outlined in Table 1.

In this study a change in the immunoreactivity of key ECM molecules, IGF regulators and integrins was observed with breast tumour progression. These observations suggest that the ECM surrounding normal breast ductal structures is remodelled during tumour development and progression. The ECM protein FN and the cell surface β₁ integrin (the FN-binding receptor) were found to be highly expressed along the leading edge of many primary tumours in the present study. Interestingly, FN is implicated in epithelial-to-mesenchymal transition (EMT) [16] and both the β₁ integrin and FN are required in the formation of lamellipodia, filopodia and invadopodia [17, 18]; potentially supporting their role in ECM remodelling and subsequent tumour cell invasion [19]. Indeed, the β₁ integrin has been reported to be more highly expressed in primary tumours with LN metastases [20] and with poor survival outcomes [21]. This fits in well with our findings that suggest an increase in β₁ integrin immunoreactivity with increasing invasiveness. Various proteases, including matrix metalloproteinases (MMPs) have been implicated in ECM remodelling events that allow cancer cells to migrate [22]. It has been shown that the expression, activity and/or internalisation of MMPs is regulated by integrin-ECM interactions in endothelial cells [23]. Integrins, such as the α_vβ₃ integrin, cooperate with MMPs to regulate breast cancer cell migration [24]. Interestingly, IGF-I:VN: IGFBP-5-stimulated breast cell migration, which requires the IGF-IR and VN-binding integrins [9], can regulate the gene expression of proteases such as MMP13, MMP7, ADAMTS5, CPM and protease inhibitors such as SERPINE1 and TRP1 ([10] and supplementary data from [10]).

The data we report here has also provided evidence that ECM molecules and their associated membrane-bound receptors, the integrins, are internalised during breast cancer development and metastasis. Step-wise increases in cytoplasmic and concomitant decreases in stromal immunoreactivity of VN and the α_v integrin (data not shown) were evident between normal breast, primary and metastatic cancer tissues. As described previously, VN can be internalised by integrin receptor-mediated endocytosis and degraded within the lysosomes [25]. There is also evidence indicating that the VN-binding α_v integrin can be recycled to the cell membrane through intracellular signals [26]. This decrease in the stromal VN and the concomitant increase in the cytoplasmic VN with breast cancer progression suggests a potential re-shuffling or trafficking of VN from the tumour stroma to the tumour cell cytoplasm with increasing invasiveness of the tumour-type.

Given the importance of the phosphoinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling pathways in IGF-I: IGFBP:VN-stimulated migration of breast cancer cells in vitro[9], and in IGF-I-stimulated ECM re-modelling [27] and EMT events [27, 28], protein intermediates within these pathways were also investigated. Decreases in the nuclear immunoreactivity and increases in the cytoplasmic immunoreactivity in breast cancer tissues were observed in this study. We propose a number of explanations for these findings: namely, the preferential activation of substrates in the cytoplasm (of cancer cells) rather than in the nucleus; enzyme-mediated dephosphorylation; and protein internalisation. Both AKT1 and MAPK contain transportation signals which potentially enable their movement throughout a cell [29, 30]. Defects in these transportation signals during cancer tumorigenesis might explain the results of this study. Ras homolog gene family member B (RhoB), which has been shown to influence the trafficking of Total- and P-AKT in primary human endothelial cells [31], may impede the import of Total- and P-AKT into or promote the export of Total- and P-AKT from the nucleus of breast cancer cells; resulting in an accumulation of AKT in cytoplasmic compartments. Phospho-kinases, such as MAPK, are not necessarily required to enter the nucleus to regulate gene transcription. In fact, the activation of transcription factors in the cytoplasm and their movement into the nucleus for transcriptional control [32] has been reported. The duration and strength of AKT and MAPK signalling in breast epithelial cells can also be regulated in different subcellular locations through the action of various cytoplasmic and nuclear phosphatases [33]. Indeed, phosphatase and tensin homolog (PTEN), a dual lipid and protein phosphatase, can be localised to the cell nucleus [34], and if functional, may therefore de-phosphorylate nuclear AKT. As discussed by Tzivion et al.[35], the ability of a protein to interact with modifying enzymes, such as phosphatases, can be influenced by the presence of 14-3-3 proteins.

Intriguingly, 14-3-3 proteins, including SFN, have been shown to regulate the cytoplasmic sequestration and nuclear retention of cell cycle regulators, many of which are associated with and downstream of the PI3K pathway [36]. It is highly likely that 14-3-3 proteins, such as SFN, may regulate similar sequestration events for P-AKT itself. It was intriguing to find that the increases in the nuclear and cytoplasmic immunoreactivity of SFN with breast cancer development and progression as measured in this study correlated with the increase in mRNA expression of SFN reported by Kashyap et al.[10]. In contrast, other studies have reported the downregulation of SFN expression in breast cancers [37, 38]. However, Neal et al.[39] showed that overexpression of SFN reduces the overall and disease-free survival of breast cancer patients and is able to predict which patients have a high susceptibility to develop metastasis. Interestingly, it has been proposed that 14-3-3 proteins are important regulators of external environmental signals by eliciting positive and negative effects on the IGF signalling pathway. The ability of 14-3-3 proteins to bind phospho-serine enables them to bind to the IGF-IR [40]. Yang et al.[41] have shown that SFN can also bind to and inhibit the activity of AKT, preventing AKT-mediated cellular events. They also indicate that SFN expression was inversely correlated with P-AKT expression [41]; this supports our findings of decreases in nuclear P-AKT and increases in intracellular SFN with tumour development and progression.

We were intrigued to find that SFN was differentially expressed within the stroma surrounding the tissue types examined. Although previous reports of SFN expression have been limited to the cytoplasm of malignant breast cells [38], in vitro evidence indicates that SFN can be excreted by keratinocytes into the pericellular matrix [42]. Extracellular SFN is a key regulator of MMP function and ECM degradation. Studies have shown that following the release of SFN from keratinocytes, MMP-1 mRNA [43] and MMP-1 protein synthesis [44] increases in dermal fibroblasts. Increases in mRNA encoding the β₁ integrin have also been observed in dermal fibroblasts after treatment with SFN, or in co-cultures with keratinocytes known to release SFN [45]. Under the same conditions the expression of many ECM molecules, including collagen type I, FN and α₁ integrin, decreases. This collective evidence suggests that it is highly likely that SFN may mediate similar functions regulating the degradation of ECM during epithelial tumour development and progression.

We also observed step-wise increases in cytoplasmic and decreases in nuclear immunoreactivity of SHARP-2 between the normal breast, primary and metastatic cancer tissues; this may be explained by nucleocytoplasmic shuttling events. SHARP-2 is known to possess a functional nuclear export sequence (NES) and two nuclear localisation signal (NLS) motifs [46]. A study by Ivanova et al.[46] suggests that SHARP-2 may be required in the nucleus of proliferating and differentiating cells to regulate gene transcription after stimulation by an external factor. They also propose that SHARP-2 may be sequestered in the cytoplasm following cell differentiation.

In summary, we have reported changes in the temporal and spatial distribution of IGF- and ECM-induced signalling proteins that occur during breast cancer metastasis. Specifically, our findings provide further evidence that the ECM surrounding normal breast ductal structures is remodelled during tumour development and progression, and that FN and the β₁ integrin are important for the formation of invadopodia and for the epithelial-to-mesenchymal transition events (shown by others [47]) to support dissemination. Analysis of stromal and subcellular SFN immunoreactivity suggested a causal relationship in ECM remodelling events and the localisation and activity of proteins important for IGF- and ECM-induced signalling cascades. It also appears plausible that in cells at the leading edge of tumours, SHARP-2 moves into the nucleus to repress the transcription of genes associated with the hypoxic response.

Collectively the above data highlight the possibility that there are broader biological implications of, and explanations for, the differential immunoreactivity of IGF signalling and ECM components in the stroma and/or in subcellular locations within normal breast, primary breast cancer and metastatic breast cancers. This is highly pertinent given that protein function and protein localisation are closely correlated. Studies have also shown that accounting for protein localisation can be an essential requirement to identifying correlations with other proteins when applying the IHC technique [48]. To date, very few studies have evaluated the prognostic significance of differential protein distribution within diagnostic breast cancer tissue samples. Early studies do, however, suggest that specific locations of specific proteins associated with the IGF signalling cascade and the ECM have shown potential as markers for patient prognosis and therapeutic response [49, 50]. We argue that to date the potential of many molecular species to serve as markers of patient prognosis and therapeutic response is being missed by overlooking their subcellular/extracellular distribution. In view of this, we recommend a more complete analysis of protein localisation within diagnostic pathology and improvements to reporting and inclusion of protein localisation in routine pathological examinations as our data indicates the potential role of protein localisation in the progression of disease.

There is potential that the cellular and ECM events outlined herein could be manipulated to provide clinical benefits and improve the clinical management of breast cancer. In particular, may lead to early prognostic and predictive identification of patients with poor survival outcomes. However, prior to this occurring, the prognostic significance of the cellular and ECM events reported in this study must be identified.

Helen C Plant and Abhishek S Kashyap are co-first authors.