AQP1-Driven Migration Is Independent of Other Known Adverse Factors but Requires a Hypoxic Undifferentiated Cell Profile in Neuroblastoma

Abstract

Neuroblastoma is a biologically very heterogeneous tumor with its clinical manifestation ranging from spontaneous regression to highly aggressive metastatic disease. Several adverse factors have been linked to oncogenesis, tumor progression and metastases of neuroblastoma including NMYC amplification, the neural adhesion molecule NCAM, as well as CXCR4 as a promoter of metastases. In this study, we investigate to what extent the expression of AQP1 in neuroblastoma correlates with changing cellular factors such as the hypoxic status, differentiation, expression of known adverse factors such as NMYC and NCAM, and CXCR4-related metastatic spread. Our results show that while AQP1 expression leads to an increased migratory behavior of neuroblastoma cells under hypoxic conditions, we find that hypoxia is associated with a reduction of NMYC in the same cells. A similar effect can be observed when using the tetracycline driven mechanism of SH-EP/Tet cells. When NMYC is not expressed, the expression of AQP1 is increased together with an increased expression of HIF-1α and HIF-2α. We furthermore show that when growing cells in different cell densities, they express AQP1, HIF-1α, HIF-2α, NMYC and NCAM to different degrees. AQP1 expression correlates with a hypoxic profile of these cells with increased HIF-1α and HIF-2α expression, as well as with NMYC and NCAM expression in two out of three neuroblastoma cell lines. When investigating cell properties of the cells that actually migrate, we find that the increased APQ1 expression in the migrated cells correlates with an increased NMYC and NCAM expression again in two out of three cell lines. Expression of the tumor cell homing marker CXCR4 varies between different tumor areas and between cell lines. While some migrated tumor cells highly express CXCR4, cells of other origin do not. In the initial phase of migration, we determined a dominant role of AQP1 expression of migrating cells in the scratch assay.

Perfusion-Based Bioreactor Culture and Isothermal Microcalorimetry for Preclinical Drug Testing with the Carbonic Anhydrase Inhibitor SLC-0111 in Patient-Derived Neuroblastoma

“…A classic example is the cell line SH-SY5Y that can be differentiated from ever-changing neuroblasts into mature neurons [ 31 ]. Our previous research has shown that even simple factors such as, e.g., cell density, can influence vital prognostic factors [ 28 ]. Moreover, hypoxia can significantly increase known adverse factors in neuroblastoma such as NMYC, CXCR4, PGK1 and AQP1, amongst others, leading to tumor progression and increased metastases [ 25 , 26 , 27 , 28 ].…”

“…Our previous research has shown that even simple factors such as, e.g., cell density, can influence vital prognostic factors [ 28 ]. Moreover, hypoxia can significantly increase known adverse factors in neuroblastoma such as NMYC, CXCR4, PGK1 and AQP1, amongst others, leading to tumor progression and increased metastases [ 25 , 26 , 27 , 28 ]. Neuroblastoma cells are characterized by changing biological behavior; a heterogeneous tumor microenvironment with stroma-rich or stroma-poor features; the presence or absence of immunomarkers such as GD2; adverse factors such as NMYC; or adhesion factors such as NCAM.…”

“…We have previously shown a poorer prognosis for CAIX-positive neuroblastoma patients in addition to the influence of hypoxia on progression and metastases in this disease, and were successfully able to inhibit neuroblastoma cell growth in vivo by applying the experimental CAIX inhibitors FC5-207A and FC8-325A [ 24 , 25 ]. We have identified further hypoxia-associated prognostic factors for neuroblastoma including CXCR4, PGK1 and AQP1 leading to increased metastases [ 26 , 27 , 28 ]. Especially, AQP1 furthers tumor cell migration and acts independently of other known adverse factors, such as NMYC or NCAM [ 27 , 28 ].…”

“…We have identified further hypoxia-associated prognostic factors for neuroblastoma including CXCR4, PGK1 and AQP1 leading to increased metastases [ 26 , 27 , 28 ]. Especially, AQP1 furthers tumor cell migration and acts independently of other known adverse factors, such as NMYC or NCAM [ 27 , 28 ].…”

AQP1 in the Gastrointestinal Tract of Mice: Expression Pattern and Impact of AQP1 Knockout on Colonic Function

“…Under hypoxic conditions, HIF-1α is upregulated and activates cellular responses to low oxygen levels [ 27 , 28 ]. Recent studies suggest that AQP1 expression is upregulated by a HIF-1α increase as a result of hypoxic conditions [ 29 , 30 , 31 , 32 , 33 ]. On the other hand, the upregulation of HIF-1α in the lungs under hypoxic conditions was inhibited in AQP1-knockout mice [ 34 ], suggesting a feedback mechanism between HIF-1α and AQP1 expression.…”