Genomic imbalance of HMMR/RHAMM regulates the sensitivity and response of malignant peripheral nerve sheath tumour cells to aurora kinase inhibition

Malignant peripheral nerve sheath tumours (MPNST) are rare, hereditary cancers associated with neurofibromatosis type I. MPNSTs lack effective treatment options as they often resist chemotherapies and have high rates of disease recurrence. Aurora kinase A (AURKA) is an emerging target in cancer and an aurora kinase inhibitor (AKI), termed MLN8237, shows promise against MPNST cell lines in vitro and in vivo. Here, we test MLN8237 against two primary human MPNST grown in vivo as xenotransplants and find that treatment results in tumour cells exiting the cell cycle and undergoing endoreduplication, which cumulates in stabilized disease. Targeted therapies can often fail in the clinic due to insufficient knowledge about factors that determine tumour susceptibilities, so we turned to three MPNST cell-lines to further study and modulate the cellular responses to AKI. We find that the sensitivity of cell-lines with amplification of AURKA depends upon the activity of the kinase, which correlates with the expression of the regulatory gene products TPX2 and HMMR/RHAMM. Silencing of HMMR/RHAMM, but not TPX2, augments AURKA activity and sensitizes MPNST cells to AKI. Furthermore, we find that AURKA activity is critical to the propagation and self-renewal of sphere-enriched MPNST cancer stem-like cells. AKI treatment significantly reduces the formation of spheroids, attenuates the self-renewal of spheroid forming cells, and promotes their differentiation. Moreover, silencing of HMMR/RHAMM is sufficient to endow MPNST cells with an ability to form and maintain sphere culture. Collectively, our data indicate that AURKA is a rationale therapeutic target for MPNST and tumour cell responses to AKI, which include differentiation, are modulated by the abundance of HMMR/RHAMM

Cancer Stem Cell-Like Cells Derived from Malignant Peripheral Nerve Sheath Tumors

This study aims to examine whether or not cancer stem cells exist in malignant peripheral nerve sheath tumors (MPNST). Cells of established lines, primary cultures and freshly dissected tumors were cultured in serum free conditions supplemented with epidermal and fibroblast growth factors. From one established human MPNST cell line, S462, cells meeting the criteria for cancer stem cells were isolated. Clonal spheres were obtained, which could be passaged multiple times. Enrichment of stem cell-like cells in these spheres was also supported by increased expression of stem cell markers such as CD133, Oct4, Nestin and NGFR, and decreased expression of mature cell markers such as CD90 and NCAM. Furthermore, cells of these clonal S462 spheres differentiated into Schwann cells, smooth muscle/fibroblast and neurons-like cells under specific differentiation-inducing cultural conditions. Finally, subcutaneous injection of the spheres into immunodeficient nude mice led to tumor formation at a higher rate compared to the parental adherent cells (66% versus 10% at 2.5×105). These results provide evidence for the existence of cancer stem cell-like cells in malignant peripheral nerve sheath tumors

Treatment of orthotopic malignant peripheral nerve sheath tumors with oncolytic herpes simplex virus

Malignant peripheral nerve sheath tumors (MPNSTs) are an aggressive and often lethal sarcoma that frequently develops in patients with neurofibromatosis type 1 (NF1). We developed new preclinical MPNST models and tested the efficacy of oncolytic herpes simplex viruses (oHSVs), a promising cancer therapeutic that selectively replicates in and kills cancer cells. Mouse NF1(-) MPNST cell lines and human NF1(-) MPNST stemlike cells (MSLCs) were implanted into the sciatic nerves of immunocompetent and athymic mice, respectively. Tumor growth was followed by external measurement and sciatic nerve deficit using a hind-limb scoring system. Oncolytic HSV G47 Delta as well as "armed" G47 Delta expressing platelet factor 4 (PF4) or interleukin (IL)-12 were injected intratumorally into established sciatic nerve tumors. Mouse MPNST cell lines formed tumors with varying growth kinetics. A single intratumoral injection of G47 Delta in sciatic nerve tumors derived from human S462 MSLCs in athymic mice or mouse M2 (37-3-18-4) cells in immunocompetent mice significantly inhibited tumor growth and prolonged survival. Local IL-12 expression significantly improved the efficacy of G47 Delta in syngeneic mice, while PF4 expression prolonged survival. Injection of G47 Delta directly into the sciatic nerve of athymic mice resulted in only mild symptoms that did not differ from phosphate buffered saline control. Two new orthotopic MPNST models are described, including in syngeneic mice, expanding the options for preclinical testing. Oncolytic HSV G47 Delta exhibited robust efficacy in both immunodeficient and immunocompetent MPNST models while maintaining safety. Interleukin-12 expression improved efficacy. These studies support the clinical translation of G47 Delta for patients with MPNST.N

Proliferation and self-renewal of MPNST spheres.

<p>Bromodeoxyuridine incorporation assay revealed proliferation of S462 cells in the clonal spheres (secondary spheres). The increase in cell absorbance was significant for 8, 10, and 15 days in SCM versus 3 days (p<0.01 **). Changes in the frequency of sphere formation in wells containing single cells originally from adherent S462, then from dissociated spheres plated as single cells and passaged consecutively. The increase in frequency of sphere formation with increasing passage was significant (passage 1 (primary spheres) versus passages 10 (10^th spheres) and 12 (12^th spheres), P<0.01 **, passage 2 (secondary spheres) versus passages 10 (10^th spheres) and 12^th spheres), P<0.05*).</p

Co-expression of NGFR and CD133 in adherent and S462 spheres.

<p>Side and forward scatter analysis of adherent cells (left column) and spheres (at passage 8, right column) showed different types of cells. Live cells were gated in R1 (upper panels), and gatings for NGFR (R2) positive cells (middle panels) are shown in orange. NGFR-positive cells (represented in orange) within the CD133-positive cells show co-expression of CD133/NGFR (lower panels). NGFR cells co-expressing CD133 are infrequent in the adherent cell population and increased in spheres. Isotype controls for all the antigens were used to set up the quadrants for negative populations. NGFR, nerve growth factor receptor.</p

Comparison of MPNST adherent and sphere cultures.

<p>(A) Parental S462 cells under standard culture conditions with serum grow adherently (passage 35). (B) Floating secondary sphere after two weeks under stem cell conditions without serum. The sphere was derived from a single cell and thus was clonal. Bars = 20 µm.</p

Tumor formation in immunodeficient nude mice.

<p>(A) Frequency and time of tumor formation with 2.5×10⁵ adherent (n = 10, passage 35) or clonal sphere S462 (n = 9), CD133⁺ (n = 9) and CD133⁻ (n = 5) cells injected subcutaneously. All sphere cells were obtained between passages 8 and 13. Adherent versus spheres and adherent versus CD133+ spheres p<0.05 *. (B) Histology of tumors derived from adherent cells (left panel) and sphere cells showed that these tumors resemble MPNSTs. H&E shows typical spindle shape cells of MPNST (arrows, top panel), which are highly proliferative by Ki-67 immunstaining (arrows, middle panel) and negative for S100 (bottom panel). Bar = 200 µm, applies for all the micrographs. (C) Cells dissociated from mice tumors formed secondary spheres <i>in vitro</i> (representative secondary sphere 3 weeks after plating single cells). Bar = 20 µm.</p

Multi-lineage differentiation of cells of clonal S462 spheres.

<p>Adherent cells (left column) and dissociated sphere cells (right columns) were cultured with growth factors inducing differentiation into cells resembling (A) Schwann cells, (B) SM/Fb and (C) neurons, which are positively stained for S100/NGFR/neurofilament (Schwann cells), SMA (SM/Fb), and MAP-2 (neurons), respectively. Insert in A illustrates S100+ Schwann cells derived from a plexiform neurofibroma culture. (D) phase contrast micrographs from differentiated cells under 3 culture conditions to generate Schwann cell, SM/Fb and neuron-like cells. Differentiated cells are indicated by arrows and non-differentiated cells by arrowheads. Bars = 20 µm. NGFR, nerve growth factor; SMA, smooth muscle actin; MAP-2, microtubule associated protein-2; PNF, plexiform neurofibroma; LC, like-cells.</p

Genomic imbalance of HMMR/RHAMM regulates the sensitivity and response of malignant peripheral nerve sheath tumour cells to aurora kinase inhibition

Malignant peripheral nerve sheath tumours (MPNST) are rare, hereditary cancers associated with neurofibromatosis type I. MPNSTs lack effective treatment options as they often resist chemotherapies and have high rates of disease recurrence. Aurora kinase A (AURKA) is an emerging target in cancer and an aurora kinase inhibitor (AKI), termed MLN8237, shows promise against MPNST cell lines in vitro and in vivo. Here, we test MLN8237 against two primary human MPNST grown in vivo as xenotransplants and find that treatment results in tumour cells exiting the cell cycle and undergoing endoreduplication, which cumulates in stabilized disease. Targeted therapies can often fail in the clinic due to insufficient knowledge about factors that determine tumour susceptibilities, so we turned to three MPNST cell-lines to further study and modulate the cellular responses to AKI. We find that the sensitivity of cell-lines with amplification of AURKA depends upon the activity of the kinase, which correlates with the expression of the regulatory gene products TPX2 and HMMR/RHAMM. Silencing of HMMR/RHAMM, but not TPX2, augments AURKA activity and sensitizes MPNST cells to AKI. Furthermore, we find that AURKA activity is critical to the propagation and self-renewal of sphere-enriched MPNST cancer stem-like cells. AKI treatment significantly reduces the formation of spheroids, attenuates the self-renewal of spheroid forming cells, and promotes their differentiation. Moreover, silencing of HMMR/RHAMM is sufficient to endow MPNST cells with an ability to form and maintain sphere culture. Collectively, our data indicate that AURKA is a rationale therapeutic target for MPNST and tumour cell responses to AKI, which include differentiation, are modulated by the abundance of HMMR/RHAMM