**5. Concept 4: Tumor initiating cells**

Another advance that may profoundly change our thinking about solid tumors including glioblastoma involves the concept of tumor initiating cells. The experimental observation is

Emerging literature suggests an alternative strategy to the multi-target approach. These studies reveal that oncogene activation introduces secondary physiologic changes that stress cellular capacity for survival. Consequently, tumor cells become hyper-dependent on processes required to compensate for these stressful conditions 26, 27. This phenomenon is termed "non-oncogene addiction" since the compensatory processes required for tumor survival do not directly contribute to the cancer formation. In other words, even genes that are not themselves targeted by tumorigenic mutations may well become essential for the tumor to survive the stressful environment and fuel the demanding process of tumor progression. Consequently, interference with the function of such genes can be rate-limiting to the particular mechanism in the tumor, but not as much in the normal counterpart cells. Importantly, such adaptively essential genes that underlie the 'non-oncogene addiction' 26, 27 of cancer cells can be therapeutically targeted if suitable drugs or other approaches are available. There are several examples of such critical non-oncogenic pro-survival functions required for maintenance of the tumorigenic state in glioblastoma. EGFR is a critical proto-oncogene in glioblastoma pathogenesis 10, 28. Our laboratory has demonstrated that EGFR hyperactivation results in increased accumulation of reactive oxygen species (ROS), which in turn cause cytotoxic DNA damage. To compensate for the deleterious effect of ROS, EGFR hyperactive glioblastomas exhibit increased reliance on DNA repair process that repair ROS related DNA damage 29. Selective targeting of EGFR hyperactive glioblastomas can, thus, be achieved by inhibition of these repair process. Other groups have demonstrated that EGFR hyperactivation in glioblastoma cell lines heightens requirement for lipogenesis 30, 31. Other examples of such critical non-oncogenic pro-survival functions required for maintenance of the tumorigenic state include dependency on mechanism for compensating mitotic and proteotoxic stress and interplay with the tumor microenvironment including the immune system 26. While illustrative examples of strategies based on these "non-oncogene" addiction paradigms have been established in other cancers,

The principle of non-oncogene addiction suggests that there is a wider spectrum of therapeutic options than afforded under the paradigm of "oncogene addiction". In many cases, compensatory processes involved in "non-oncogene addiction" are the same as those that basic scientists have studied for years (for instance, DNA repair). Mechanistic investigations into these biologic processes by the basic scientists have yielded a rich database of inhibitors. Thus, identifying gene functions that compensate for oncogene induced cellular stress should afford opportunities to tap into this rich database and expand the denominator of drugs available for combinatorial therapy. Identifying genes that are

It is important to note that effects of therapies designed based on the principles of "oncogene addiction" and of "non-oncogene addiction" are inherently antagonistic. For instance, EGFR inhibition leads to a reduction of ROS, obviating the need for DNA repair 29. In this context, combination of DNA repair inhibition and EGFR inhibition would not be desirable. Rational strategies for synthesizing the two therapeutic paradigms remains a

Another advance that may profoundly change our thinking about solid tumors including glioblastoma involves the concept of tumor initiating cells. The experimental observation is

synthetically lethal with oncogenes constitute an attractive means to this end.

**4. Concept 3: Non-oncogene addiction**

the pertinence to glioblastoma awaits rigorous interrogation.

major intellectual challenge.

**5. Concept 4: Tumor initiating cells** 

that within a total population of glioblastoma cells, there appears to be a small subpopulation of cells that are highly tumorigenic (hence the term "tumor initiating cells" or "TICs") with tremendous capacity for self-renewal 32, 33. To the extent that glioblastoma tumor initiating cells share many common properties when compared to neural stem cells, it is proposed that the TICs originated from stem cells. While there are some data supporting this hypothesis 5, the universality of this hypothesis remain controversial.

Protein markers to prospectively identify and isolate these putative TICs such as the transmembrane glycoprotein CD133 (prominin-1) in glioblastomas have been identified 5. However, the value of CD133 as a single marker of glioblastoma TICs remains controversial, partly because also CD133-negative glioblastoma cells could give rise to tumors in an intracranial mouse xenograft model 34-36. These uncertainties motivate an ongoing search for additional candidate TIC markers. Candidate cell surface molecules suggested in this context include the adhesion glycoprotein L1CAM 37, surface carbohydrate antigen CD15 (SSEA-1) 38, surface marker A2B5 39, and integrin 6 40. Currently, there are no generally accepted cell surface markers for defining TIC. The definition of TICs remains a functional one as defined by the ability of a tumor cell to sustain self-renewal and initiate glioblastoma formation in immuno-compromised xenograft models.

Arguably, the most important aspect of the concept of TICs is that this population appeared particularly resistant to conventional radiation and chemotherapy 32. In this context, TICs may be responsible for glioblastoma recurrence after conventional therapy. Given such properties, it is understandable that glioblastoma research has recently focused on identification and development of potential anti-TIC therapies. Two of these strategies, namely targeting the TICs as part of a vascular niche, and attempts to overcome their therapeutic resistance, will be discussed in the following sections on glioblastoma angiogenesis and the role of DNA damage response pathways, respectively. Here, we briefly consider strategies that are emerging as potentially fruitful approaches to treat glioblastoma through targeting TICs.

The first strategy reflects the efforts to identify suitable cell surface markers to reliably identify glioblastoma TICs – with the hope of conjugating the corresponding antibody to cytotoxic compounds as therapeutic agents. The second strategy is based on observations that some TICs, like neural stem cells, can be induced into a differentiated state whereby the self-renewal properties are lost. Among the suggested agents to induce such TIC differentiation, the bone morphogenetic proteins (BMPs) appear promising 41. The third strategy involves modulating specific signaling pathways required for maintaining the TIC state. Pathways targeted include those mediated by EGFR, Wnt-beta catenin, STAT3, Sonic Hedgehog-Gli, and Notch pathways 42. To the extent that these pathways are also regulated by miRNAs such as miR-21 43, such miRNA constitute therapeutic targets in this strategy. Finally, normal neural stem cells have been shown to migrate toward and track TICs. Based on this principle, neural stem cells have been as delivery vehicles to increase local concentration of therapeutic agents in the vicinity of TICs 44.

#### **6. Summary**

In this chapter, we have discussed key principles underlying current development of glioblastoma therapeutics. Emphasis was placed on conceptual framework rather than specific drugs or targets. These frameworks should serve as the basis for translating fundamental biologic tenets into clinically useful therapeutic strategies.

Key Principles in Glioblastoma Therapy 59

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**4** 

*Japan* 

**Biological Responses of Glioma Cell** 

*Department of Neurosurgery, Fujita Health University, Toyoake Department of Neurosurgery, Ashikaga Red Cross Hospital, Ashikaga* 

Because gliomas are not curable surgically, development of effective adjuvant therapies is warranted. A chemotherapeutic agent temozolimide (TMZ) has been widely used not only because it is well tolerated and easily administrated orally but because various clinical trials had revealed that high grade gliomas could show objective response or stable disease to this compound (Stupp et al., 2005). The action of TMZ had been extensively studied primarily in leukemia and lymphoma cells. TMZ spontaneously decomposes in aqueous solution to form the cytotoxic methylating agent, and the cytotoxicity of TMZ appears to be mediated mainly through adduction of a methyl group to O6 position of guanine (G) in genomic DNA. The methyl group can be removed from O6-methylguanine by O6-methylguanine-DNA methyltransferase (MGMT). If MGMT is deficient in the cell, however, O6-methylguanine is not repaired, and incorporation of a thymine (T) rather than a cytosine opposite the O6 methylguanine during the next cycle of DNA replication leads to the formation of GT mismatches in DNA. This triggers the DNA mismatch repair (MMR) system which removes the T, only to have the T reinserted during repair synthesis. Futile cycles of MMR triggered

by GT mismatches can lead to a variety of outcomes in TMZ-treated cells (Figure 1).

Fig. 1. Mechanism of temozolomide-induced DNA damage creation

**1. Introduction** 

**to Chemotherapeutic Agents** 

Yuichi Hirose and Shigeo Ohba

