The role of tumor cell heterogeneity in the targeted therapy of ALK-positive neuroblastoma
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AbstractNeuroblastoma (NB) is the most common extracranial solid tumor of childhood. One of its hallmarks is high histological heterogeneity. The primary tumors are composed of a mixture of neuroblastic cancer cells, Schwannian stroma and some infiltrating immune cells. The origin and functions of Schwannian stroma still remain controversial. It is suggested that they are tumor-infiltrating normal cells. Studies using Bone Marrow Stromal Cells (BMSCs) or Schwann cells to mimic Schwannian stroma have shown both tumor promoting or suppression effects. An alternative hypothesis is that these Schwannian cells are derived from cancer stem-like cells since they shared the same genetic lesions with neuroblastic tumor cells. Our laboratory had previously reported that in the SK-N-SH cell line, both N-type cells (neuroblastic cells) and S-type cells (Schwann-like cells) shared almost identical genetic background. These cell lines may serve as relevant models to study NB tumor heterogeneity.

Genome-wide sequencing for driver oncogenes in familial NB uncovered mutations in Anaplastic Lymphoma Kinase (ALK) gene. ALK point mutations have been found in 8-12% of all NB patients, and are restricted to the kinase domain. Aberrant activation of ALK signaling, and its downstream target, PI3K/AKT, STAT3 and ERK1/2, can contribute to NB tumorigenesis. Thus, ALK is an intensely studied therapeutic target.

Sublines of N-type and S-type cells were isolated from an early passage of SK-N-SH. Surprisingly, over 90% have cell morphology that resembled S-type cells. Sequencing analysis revealed that all harbored the same ALK F1174L mutation, indicating they were tumor-derived. Western blotting analysis showed that N-type cells, but not S-type cells, expressed ALK protein. This may explain the insensitivity of S-type cells towards ALK inhibitor, TAE684. Next, co-culture experiments showed that S-type cells protect neuroblastic N-type cells from apoptosis induced by TAE684. Western blotting analysis showed that both ALK, AKT and STAT3 signaling were stimulated in the co-cultures. Furthermore, conditioned medium (CM) from Schwann-like cells activated these downstream signaling molecules in N-type cells, indicating secreted factors from S-type cells contributed to the pathway stimulations. As AKT and STAT3 were ALK downstream targets, ALK ligand could be a potential factor. We further confirmed these results in two additional N-type cell lines, IMR 32 and BE (2)-M17, which were ALK WT cell lines. Co-cultivation and CM experiments had demonstrated robust STAT3 activation even without ALK stimulation.

Overall, tumor-derived S-type cells could prevent N-type cells from apoptosis via secreted factors. The signaling involved could be separated into an ALK-independent STAT3 activation and a ligand-activated ALK-AKT pathway. Future work will focus on identifying STAT3 stimulators and ALK ligands in CM of tumor-derived S-type cells.

This work was supported by a General Research Fund grant (#14100514) from the Hong Kong University Grants Committee to A.C.
All Author(s) ListJing LI, Penelope M.Y. OR and Andrew M. CHAN
Name of ConferenceAmerican Association for Cancer Research Annual Meeting 2017
Start Date of Conference01/04/2017
End Date of Conference05/04/2017
Place of ConferenceWashington, D.C.
Country/Region of ConferenceUnited States of America
Year2017
LanguagesEnglish-United States

Last updated on 2018-18-01 at 11:20