The tyrosine kinase c-Met promotes the formation and malignant progression of multiple cancers. It is well known that c-Met hyperactivation increases tumorigenicity and tumor cell resistance to DNA damaging agents, properties associated with tumor-initiating stem cells. However, a link between c-Met signaling and the formation and/or maintenance of neoplastic stem cells has not been previously identified. Here, we show that c-Met is activated and functional in glioblastoma (GBM) neurospheres enriched for glioblastoma tumor-initiating stem cells and that c-Met expression/function correlates with stem cell marker expression and the neoplastic stem cell phenotype in glioblastoma neurospheres and clinical glioblastoma specimens. c-Met activation was found to induce the expression of reprogramming transcription factors (RFs) known to support embryonic stem cells and induce differentiated cells to form pluripotent stem (iPS) cells, and c-Met activation counteracted the effects of forced differentiation in glioblastoma neurospheres. Expression of the reprogramming transcription factor Nanog by glioblastoma cells is shown to mediate the ability of c-Met to induce the stem cell characteristics of neurosphere formation and neurosphere cell self-renewal. These findings show that c-Met enhances the population of glioblastoma stem cells (GBM SCs) via a mechanism requiring Nanog and potentially other c-Met–responsive reprogramming transcription factors.

Current glioblastoma therapies are insufficient to prevent tumor recurrence and eventual death. Here, we describe a method to treat malignant glioma by nonviral DNA delivery using biodegradable poly(β-amino ester)s (PBAEs), with a focus on the brain tumor initiating cells (BTICs), the tumor cell population believed to be responsible for the formation of new tumors and resistance to many conventional therapies. We show transfection efficacy of >60% and low biomaterial-mediated cytotoxicity in primary human BTICs in vitro even when the BTICs are grown as 3-D oncospheres. Intriguingly, we find that these polymeric nanoparticles show intrinsic specificity for nonviral transfection of primary human BTICs over primary healthy human neural progenitor cells and that this specificity is not due to differences in cellular growth rate or total cellular uptake of nanoparticles. Moreover, we demonstrate that biodegradable PBAE/DNA nanoparticles can be fabricated, lyophilized, and then stored for at least 2 years without losing efficacy, increasing the translational relevance of this technology. Using lyophilized nanoparticles, we show transgene expression by tumor cells after intratumoral injection into an orthotopic murine model of human glioblastoma. PBAE/DNA nanoparticles were more effective than naked DNA at exogenous gene expression in vivo, and tumor cells were transfected more effectively than noninvaded brain parenchyma in vivo. This work shows the potential of nonviral gene delivery tools to target human brain tumors.

It is necessary to understand mechanisms by which differentiating agents influence tumor-initiating cancer stem cells. Toward this end, we investigated the cellular and molecular responses of glioblastoma stem-like cells (GBM-SCs) to all-trans retinoic acid (RA). GBM-SCs were grown as non-adherent neurospheres in growth factor supplemented serum-free medium. RA treatment rapidly induced morphology changes, induced growth arrest at G1/G0 to S transition, decreased cyclin D1 expression and increased p27 expression. Immunofluorescence and western blot analysis indicated that RA induced the expression of lineage-specific differentiation markers Tuj1 and GFAP and reduced the expression of neural stem cell markers such as CD133, Msi-1, nestin and Sox-2. RA treatment dramatically decreased neurosphere-forming capacity, inhibited the ability of neurospheres to form colonies in soft agar and inhibited their capacity to propagate subcutaneous and intracranial xenografts. Expression microarray analysis identified ∼350 genes that were altered within 48 h of RA treatment. Affected pathways included retinoid signaling and metabolism, cell-cycle regulation, lineage determination, cell adhesion, cell–matrix interaction and cytoskeleton remodeling. Notch signaling was the most prominent of these RA-responsive pathways. Notch pathway downregulation was confirmed based on the downregulation of HES and HEY family members. Constitutive activation of Notch signaling with the Notch intracellular domain rescued GBM neurospheres from the RA-induced differentiation and stem cell depletion. Our findings identify mechanisms by which RA targets GBM-derived stem-like tumor-initiating cells and novel targets applicable to differentiation therapies for glioblastoma.

Sex differences have been well identified in many brain tumors. Even though glioblastoma (GBM) is the most common primary malignant brain tumor in adults and has the worst outcome, well-established differences between men and women are limited to incidence and outcome. Little is known about sex differences in GBM at the disease phenotype and genetical/molecular level. This review focuses on a deep understanding of the pathophysiology of GBM, including hormones, metabolic pathways, the immune system, and molecular changes, along with differences between men and women and how these dimorphisms affect disease outcome. The information analyzed in this review shows a greater incidence and worse outcome in male patients with GBM compared with female patients. We highlight the protective role of estrogen and the upregulation of androgen receptors and testosterone having detrimental effects on GBM. Moreover, hormones and the immune system work in synergy to directly affect the GBM microenvironment. Genetic and molecular differences have also recently been identified. Specific genes and molecular pathways, either upregulated or downregulated depending on sex, could potentially directly dictate GBM outcome differences. It appears that sexual dimorphism in GBM affects patient outcome and requires an individualized approach to management considering the sex of the patient, especially in relation to differences at the molecular level.

Glioblastoma (GB) is a highly invasive and lethal brain tumor due to its universal recurrence. Although it has been suggested that the electroneutral Na⁺-K⁺-Cl⁻ cotransporter 1 (NKCC1) can play a role in glioma cell migration, the precise mechanism by which this ion transporter contributes to GB aggressiveness remains poorly understood. Here, we focused on the role of NKCC1 in the invasion of human primary glioma cells in vitro and in vivo. NKCC1 expression levels were significantly higher in GB and anaplastic astrocytoma tissues than in grade II glioma and normal cortex. Pharmacological inhibition and shRNA-mediated knockdown of NKCC1 expression led to decreased cell migration and invasion in vitro and in vivo. Surprisingly, knockdown of NKCC1 in glioma cells resulted in the formation of significantly larger focal adhesions and cell traction forces that were approximately 40% lower than control cells. Epidermal growth factor (EGF), which promotes migration of glioma cells, increased the phosphorylation of NKCC1 through a PI3K-dependant mechanism. This finding is potentially related to WNK kinases. Taken together, our findings suggest that NKCC1 modulates migration of glioma cells by two distinct mechanisms: (1) through the regulation of focal adhesion dynamics and cell contractility and (2) through regulation of cell volume through ion transport. Due to the ubiquitous expression of NKCC1 in mammalian tissues, its regulation by WNK kinases may serve as new therapeutic targets for GB aggressiveness and can be exploited by other highly invasive neoplasms. Treatment of many cancers has been hampered by the invasive ability of tumor cells. A notable example is brain cancer, which is incurable due to its invasiveness and resulting high tumor recurrence after surgical resection. Here, we analyze further the function of NKCC1, an ion transporter that is known to regulate cell volume and intracellular chloride concentration, and to play an important role in brain tumor cell invasion. Our findings suggest that in addition to its conventional function as an ion transporter, NKCC1 may also interact with the cytoskeleton and affect brain tumor cell migration by acting as an anchor that transduces contractile forces from the plasma membrane to the extracellular matrix en route to cell migration. Moreover, we show that regulation of NKCC1 by a family of unconventional enzymes, the WNK kinases, is an important factor that affects the activity of NKCC1 and may determine the invasive ability of brain tumor cells. We postulate that NKCC1 has multiple functions in brain tumor cell migration and that together with its regulatory enzymes may be therapeutic targets in the treatment of brain tumors or other types of cancer, given the wide expression of these proteins throughout the body.