Extracellular matrix rigidity modulates neuroblastoma cell differentiation and N-myc expression
- Wilbur A Lam†1, 2,
- Lizhi Cao†1,
- Vaibhavi Umesh1,
- Albert J Keung3,
- Shamik Sen1 and
- Sanjay Kumar1Email author
© Lam et al; licensee BioMed Central Ltd. 2010
Received: 3 November 2009
Accepted: 10 February 2010
Published: 10 February 2010
Neuroblastoma is a pediatric malignancy characterized by tremendous clinical heterogeneity, in which some tumors are extremely aggressive while others spontaneously differentiate into benign forms. Because the degree of differentiation correlates with prognosis, and because differentiating agents such as retinoic acid (RA) have proven to decrease mortality, much effort has been devoted to identifying critical regulators of neuroblastoma differentiation in the cellular microenvironment, including cues encoded in the extracellular matrix (ECM). While signaling between tumor cells and the ECM is classically regarded to be based purely on biochemical recognition of ECM ligands by specific cellular receptors, a number of recent studies have made it increasingly clear that the biophysical properties of the ECM may also play an important role in this cross-talk. Given that RA-mediated neuroblastoma differentiation is accompanied by profound changes in cell morphology and neurite extension, both of which presumably rely upon mechanotransductive signaling systems, it occurred to us that mechanical cues from the ECM might also influence RA-mediated differentiation, which in turn might regulate clinically-relevant aspects of neuroblastoma biology. In this study, we tested this hypothesis by subjecting a series of neuroblastoma culture models to ECM microenvironments of varying mechanical stiffness and examined the regulatory role of ECM stiffness in proliferation, differentiation, and expression of tumor markers. We find that increasing ECM stiffness enhances neuritogenesis and suppresses cell proliferation. Remarkably, increasing ECM stiffness also reduces expression of N-Myc, a transcription factor involved in multiple aspects of oncogenic proliferation that is used for evaluating prognosis and clinical grading of neuroblastoma. Furthermore, the addition of RA enhances all of these effects for all ECM stiffnesses tested. Together, our data strongly support the notion that the mechanical signals from the cellular microenvironment influence neuroblastoma differentiation and do so synergistically with RA. These observations support further investigation of the role of microenvironmental mechanical signals in neuroblastoma proliferation and differentiation and suggest that pharmacological agents that modulate the underlying mechanotransductive signaling pathways may have a role in neuroblastoma therapy.
Inducing tumor cells to differentiate is an important therapeutic goal in cancer, especially in neuroblastoma, one of the few malignancies that demonstrates spontaneous differentiation and regression to a benign state . Previous research has shown that cross-talk between neuroblastoma cells and the extracellular matrix (ECM) influences differentiation , and recent studies have identified key genes that regulate those interactions [3, 4]. Neuroblastoma differentiation can also be induced pharmacologically with retinoic acid (RA) ; indeed, RA has been demonstrated to have significant clinical benefit in neuroblastoma, which has resulted in inclusion of RA in treatment regimens for high risk disease [6–8]. RA-induced signaling in neuroblastoma affects expression of N-Myc and other proto-oncogenes but also has been shown to alter the expression and activity of integrins, Rho GTPases, and the actin cytoskeleton. These molecular components act in concert to sense and process signals from the ECM [9–12] and to mediate cell shape and cytoarchitecture. In addition, altering the biochemical composition of the ECM itself induces morphological differentiation of neuroblastoma cells [13–15]. Together, these data strongly hint that signals encoded in the ECM may play a significant role in guiding NB differentiation.
While the ECM is classically regarded to instruct cell behavior primarily through biochemical recognition by cell adhesion receptors, it has become increasingly clear that another component of ECM-based signaling is fundamentally biophysical in nature. In particular, the mechanical rigidity (stiffness) of the ECM can profoundly alter cellular behavior, including morphology, motility, and proliferation [16–18]. While the mechanisms of this "rigidity sensing" remain incompletely understood, ECM rigidity appears to dictate the amount of stress or strain the cellular cytoskeleton can impose upon adhesion plaques, which is then sensed by specific mechanosensory molecules and regulates both adhesion plaque maturation and adhesion-dependent signaling [19–21]. In fact, many processes relevant to neuroblastoma, such as stem cell differentiation, neuronal maturation, neurite extension, and the malignant potential and phenotype of tumor cells have all been shown to be influenced by ECM stiffness and other biophysical properties [19, 22–30]. Interestingly, signaling pathways that underlie this biophysical cross-talk and those implicated in RA-mediated neuritogenesis partially coincide [16, 31–34], suggesting that mechanotransductive signals from the ECM may affect neuroblastoma differentiation.
In this study, we tested the hypothesis that mechanical cues from the ECM might influence spontaneous and RA-induced neuroblastoma differentiation and the expression of clinically-relevant markers of cell proliferation and differentiation. We cultured neuroblastoma cells on ECM substrates of various stiffnesses, exposed the cells to defined doses of RA, and measured neurite extension and Ki67 and N-Myc expression. To control ECM stiffness, we fabricated polyacylamide (PA) hydrogels whose stiffness could be controlled by setting the monomer (acrylamide): crosslinker (bis-acrylamide) ratio and which could be rendered suitable for cell adhesion by covalently conjugating collagen I. We and others have previously validated this platform [35, 36] and demonstrated that ECM rigidity can be varied independently of biochemical composition and ligand density [29, 37, 38].
Signals from the ECM canonically direct cell behavior through biochemical recognition by receptors at the cell surface, which in turn triggers specific signaling networks that alter cell behavior. In this study, we show that mechanical signals encoded in the ECM (i.e., ECM rigidity) can modulate neuroblastoma cell behaviors relevant to tumorigenesis. Specifically, we have shown ECM rigidity potentiates neuroblastoma cell differentiation and decreases cell proliferation and N-Myc expression. Furthermore, ECM rigidity appears to act synergistically with RA-induced cell differentiation as evidenced by morphometric analysis and measurements of cell proliferation and N-Myc expression. Further studies are required to elucidate the underlying mechanisms of this phenomenon, but the decreased cell proliferation observed with increasing ECM rigidity may be due in part to the attenuation of N-Myc expression. It is also highly likely that components of the cellular mechanotransductive machinery, such as integrins, adhesion proteins, Rho family GTPases, and cytoskeletal proteins are key players, and further study will be needed to narrow this list. In addition, while our results show that mechanical inputs can control N-Myc expression, it remains to be seen whether this relationship is bidirectional; i.e., whether manipulation of N-Myc expression can control mechanosensitivity. In addition, future experiments involving primary human neuroblastoma cells should help determine to what extent our results with culture models are directly translatable to the clinical disease. While many biochemical and cellular factors have been previously shown to influence neuroblastoma cell differentiation and sensitivity to chemotherapeutic agents, to our knowledge this is the first evidence that a purely biophysical parameter can directly influence biological indicators of neuroblastoma differentiation that correlate with prognosis and sensitivity to a pharmacologic agent.
This study raises novel questions about both neuroblastoma biology and therapy that warrant further investigation. For example, RA is insufficient as a single agent to achieve sustained clinical remission, and our results raise the intriguing prospect that this may be due in part to the absence of additional microenvironmental cues needed to achieve optimal differentiation. In particular, neuroblastoma metastasizes to a wide variety of tissues and it is possible that the diversity of mechanical microenvironments encountered in these tissues might modulate either malignant phenotype or RA chemosensitivity. Interestingly, additional studies revealed that ECM rigidity did not affect neuroblastoma chemosensitivity to doxorubicin (Additional File 2), suggesting that the observed synergy between ECM rigidity and pharmacological agents may not occur with standard chemotherapy but only with differentiation agents such as RA. In addition, our results beg the question of whether pharmacologic modulation of mechanotrandsductive signaling pathways may have a role in neuroblastoma therapy. Finally, the observation that a biophysical signal from the ECM can modulate tumor cell differentiation has implications beyond neuroblastoma, as N-Myc overexpression can contribute to the pathophysiology of other cancers, such as retinoblastoma, small cell lung carcinoma, and glioblastoma .
Materials and methods
Cell lines, stains, pharmacological agents
SK-N-DZ, SH-SY5Y, and SK-N-SH cell lines (ATCC) were cultured on polyacrylamide gels of varying rigidities at 5% CO2, 37°C in DMEM/10% FBS with or without 13-cis-RA (Sigma). Neurite length measurements were made via direct visualization with phase-contrast microscopy. Ki67, DAPI, and phalloidin (Invitrogen) fluorescence staining were performed on cells cultured on the gels after 2% paraformaldehyde fixation and 0.1% Triton X (Sigma) permeabilization. For experiments involving Ki67 staining, microscopy settings (exposure time, gain, etc.) remained constant to allow for qualitative comparisons in fluorescence signal across different conditions.
Analyses of variances were used to compare measurements of average neurite lengths per cell cluster on substrates of different rigidities (SPSS). Bonferroni post-hoc analyses were performed to compare each pair of populations.
RNA extraction and RT-PCR
RNA was extracted from the cells on gels by and converted to DNA using standard Trizol (Invitrogen) and reverse transcriptase (Applied Biosystems) protocols. Gene expression via RT-PCR using the ΔΔCt method was performed using primers for N-myc (Hs01014940_m1 from Applied Biosystems) and GAPDH as a control.
WST-1 cell proliferation assay
Cells were seeded at the same cell density for all experiments and cultured on ECMs of varying rigidities. After three days, WST-1 reagent (Roche) was added to the medium at 10% final volume and incubated for 2.5 hr. Supernatant was then analyzed per manufacturer's specifications.
The authors thank Dr. E. M. De Juan Pardo, T. A. Ulrich, and Dr. W. C. Gustafson for valuable technical guidance and discussions. This work was supported by an NIH National Research Service Award (F32HL078531 to W.A.L.), the Hammond Research Fellowship of the National Childhood Cancer Foundation/Children's Oncology Group (to W.A.L.), a Biomedical Research Fellowship from The Hartwell Foundation (to W.A.L.), NDSEG and NSF Graduate Research Fellowships (to A.J.K.), an Arnold and Mabel Beckman Foundation Young Investigator Award (to S.K.), an NIH Director's New Innovator Award (1DP2OD004213 to S.K., part of the NIH Roadmap for Medical Research), an NIH Physical Sciences in Oncology Center Grant (1U54CA143836 to S.K.) and a UC Berkeley Junior Faculty Research Grant (to S.K.).
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