Malignant gliomas remain an almost invariably destructive and rapidly lethal form of cancer despite substantial progress in the development of new diagnostic and treatment modalities. It is clear that a more detailed understanding of the underlying molecular and cellular basis of this disease is required to identify critical new targets that might be amenable to therapeutic intervention.
The impact of the cyclin D1-cdk4 axis on tumor development and progression resides in its ability to phosphorylate the Rb tumor suppressor, which pushes cells through the G1-S phase boundary (13
). Deregulation of the cyclin D1-cdk4 cell-cycle axis is a common feature in glioma, with levels of proliferation increasing as tumors progress towards higher states of malignancy (45
). However, direct evidence that cyclin D1 and cdk4 can contribute to gliomagenesis has been lacking.
Using the RCAS-PDGF/nestin-tvA model of glioma, we found that both cyclin D1 and cdk4 are necessary for glioma development and progression. Whereas cdk4 knockout mice were completely refractory to glioma, tumors formed in cyclin D1 knockout animals. Reconstituting cyclin D1 or cdk4 expression in the nestin-positive progenitors that give rise to the tumors could increase the proliferation of the tumor cells in low-grade lesions; however, this was insufficient to support progression to higher-grade. Likewise, the stroma of cyclin D1 deficient mice was unable to support progression of transplanted tumor cells. This tumor cell independent stromal effect was correlated with a failure of the tumor associated microglia to enter a strongly activated state, both by morphologic and immunohistochemical criteria. On the basis of these observations, we concluded that cyclin D1 and cdk4 have roles in both the tumor cell and in the development of the surrounding stroma.
The traditional view of cancer as an autonomously growing aggregation of mutant cells has been superseded by one in which the tumor acts more insidiously, actively subverting the surrounding tissue to support its growth and proliferation. This is especially true in glial tumors, where development, progression and ultimately, invasion, relies on extracellular cues derived from stromal cells, including astrocytes, neurons and brain macrophages, recruited into the growing tumor mass, (29
). This dynamic interplay between tumor and stroma, coordinated by the tumor cells themselves, creates a permissive environment in which progression to malignancy is favored. For example, brain microglia stimulated by exposure to glioma cell conditioned media or infiltrating into tumor sites have been shown to actively secrete numerous factors, including immunosuppressive cytokines such as TGF-β, mitogens, cathepsins and metalloproteinases (41
). Such microglia are polarized toward a phenotype that is pro-growth and immunosuppressive, leading to a state in which tumor cell growth and diffusion throughout the brain parenchyma becomes more likely (49
). All of these processes can contribute to the ability of glial tumor cells to grow and invade into surrounding tissue, while simultaneously avoiding clearance by immunological sentinels in the brain (38
). With TAMs comprising an active component of the tumor microenvironment, understanding the mechanisms that govern the responsiveness of these cells to external cues is important.
Our results suggest that cyclinD1-cdk4 can play two roles in the development of glioma: one, a tumor cell-autonomous role driving proliferation and the other, regulating the activity of non-tumor cells in the microenvironment, which can also affect malignant progression. Microglia are known to show a burst of cyclin D1 and cdk4 expression that precedes activation and correlates with proliferation and migration into regions exhibiting damage to neural integrity (52
). Consistent with this, we found that cyclin D1 and cdk4 knockout TAMs exhibited a ramified morphology (characterized by multiple processes extending from the central cell body into the adjacent tissue) associated with a reduced state of activation. Furthermore, other markers of TAM activation, including cathepsin X, H and S and CSF1R expression were reduced in knockout tumors relative to wild-type gliomas. Hence, in the absence of cyclin D1 or cdk4, TAMs may be unable to evolve into fully activated cells, which impedes progression to increased malignancy. How this occurs remains unclear. It is possible that cyclin D1 and cdk4 are required in TAMs or alternatively, are required in other stromal cells which facilitate activation of TAMs. In vitro
experiments on isolated cells and the use of specific genetically engineered mouse models may clarify this in the future.
Toogood, Chin, and Waldman independently demosntrated that the cdk4 inhibitor drug, PD0332991, currently in phase II clinical trials, may be effective in halting the progression of glioma cell lines and xenografts. We are in the process of addressing whether PD0332991 can inhibit disease progression or reduce tumor burden in mice that have developed oligodendroglioma in situ and whether this is due to effects in the tumor cell, the microglia, or both. Current therapeutic strategies typically focus on direct inhibition of glial tumor proliferation and growth, even though compromising macrophage activity can also enhance chemotherapeutic efficiencies in other systems (reviewed in Nature 272: 303–4, 2011). Thus, cdk4 inhibitor therapies may be useful in targeting both the tumor cell and modulating the activity of a stromal cell-type that is critical to supporting malignant progression.