Acquired Drug Resistance

The acquisition of increased resistance to chemotherapeutic agents by cancer cells is a complex matter, involving multiple molecules and pathways, even in response to a single agent. Furthermore, the details of this adaptation will differ for each different therapeutic agent, though there may be significant overlap in the survival pathways used in response to various agents.

In this section, I’ll focus on how glioma cells adapt and survive following temozolomide treatment, as a representative example of cytotoxic DNA alkylating therapy.

Temozolomide (Temodar, TMZ) Resistance

Several studies have elucidated the mechanisms by which glioma cells acquire resistance to the DNA methylating agent temozolomide (TMZ). The main lesion by which TMZ causes cytotoxicity is the addition of a methyl group to guanine, which is one of the bases which makes up DNA. The chief molecule involved in resistance to DNA alkylating agents such as temozolomide and the nitrosoureas is MGMT (O-6-methylguanine-DNA methyltransferase). MGMT is a DNA repair enzyme which removes the methyl group from methylguanine, thereby undoing the initial damage caused by TMZ.

MGMT and TMZ resistance

In one study, three different glioblastoma cell lines were repeatedly exposed to TMZ, eventually resulting in TMZ-resistant sub-lines (1). In a cell line which had an unmethylated MGMT promoter, and active MGMT, exposure to temozolomide caused a strong increase in the MGMT protein. This increase in MGMT expression is likely the main mechanism of TMZ resistance in tumours which have active MGMT expression to begin with.

On the other hand, a cell line with a hypermethylated MGMT promoter, and lack of MGMT activity, developed TMZ resistance by other means. These lines retained a hypermethylated MGMT promoter (which silences the MGMT gene) even after acquiring TMZ resistance.

Mismatch repair alterations and TMZ resistance

Several studies show that the DNA mismatch repair system (MMR) is necessary for the cytotoxic effects of TMZ, as futile cycles of mismatch repair eventually creates breaks in the DNA. Studies of cultured glioblastoma cells as well as comparative analysis of malignant glioma tissue samples taken from both initial surgery and recurrence, show that recurrent tumours have significantly decreased incidence of mismatch repair proteins such as MSH6, MLH1 and PMS2 (2). Further, 5 of 19 (26%) recurrent alkylating agent-treated glioblastomas included in the Cancer Genome Atlas were found to have an inactivating mutation in the MSH6 mismatch repair gene (3). These tumours all showed the hypermutation phenotype, due to the disabling of the mismatch repair system. A potential danger of long-term use of temozolomide is therefore the evolution of cells with mutations in mismatch repair genes, leading to increased genetic instability and further rapid tumour evolution (4).

Akt (protein kinase B), NF-KB, and TMZ resistance

The PI3K/Akt pathway is well-known as one of the most prevalent pathways upregulated in cancer cells of various types. It is a pro-growth, prosurvival pathway. Activity of this pathway is suppressed by the PTEN tumour suppressor gene, which is frequently mutated or deleted in glioblastomas. While PTEN is not frequently mutated in lower grade IDH-mutant astrocytomas, the promoter region of this gene may be hypermethylated in these cases, resulting in the silencing of PTEN, and consequently, an increase in the PI3K/Akt pathway.

A laboratory study with glioblastoma cells showed that Akt (also called protein kinase B) is able to not only allow cells to avoid cell cycle arrest (which gives cells time to repair DNA damage), but also to allow cells to avoid the fate of senescence (removal from the cell cycle) or mitotic catastrophe and cell death, in spite of DNA damage caused by temozolomide (10). In short, Akt allows cells to continue proliferating in spite of DNA damage caused by chemotherapy. This ability to survive in spite of DNA damage may also lead to increased mutagenicity and the accumulation of mutations caused by agents which target DNA, such as temozolomide (11).

One of the effects of increased Akt activity is an increase in transcriptional activity of nuclear factor kappa beta (NF-KB), which is discussed further in the Targeting Invasion section. In a laboratory study, Akt-dependent NF-KB activity provided protection against temozolomide-induced growth suppression (12).

A novel, oral PI3K/Akt/mTOR inhibitor, NVP-BEZ235, was alone able to dose-dependently increase survival time in a mouse model using human glioblastoma cells without causing obvious toxicity (13). This potentially helpful drug is currently in various early phase trials for solid cancers.

STAT3 and TMZ resistance

The transcription factor STAT3, which frequently cooperates with NF-KB, is also found to be highly upregulated in TMZ resistant glioma cell lines (14). I discuss STAT3 and specific inhibitors such as curcumin in the Targeting Invasion section.

Proliferation versus Invasion (Grow versus Go)

Multiple studies document a dichotomy between cellular programs for proliferation versus migration and invasion. In other words, highly proliferative cells tend to be stationary, while migratory cells temporarily defer proliferation. As migrating cells are both less proliferative and have reduced expression of proapoptotic proteins (necessary for programmed cell death), these cells are likely highly resistant to chemotherapy (5, 6, 7). Some of the more effective anti-proliferative drug regimes result in a disturbing increase in distant or multifocal tumour recurrences.

While multifocal recurrences of tumour outside the primary site have previously been found in 4-14% of cases, a phase II trial of tamoxifen and carboplatin plus radiation for glioblastoma patients resulted in 16 of 49 patients (33%) eventually experiencing a multifocal recurrence (8). In vitro testing to explain this phenomenon revealed that cells which had acquired resistance to tamoxifen through prolonged exposure had higher rates of proliferation, faster migration and increased adhesion to matrix proteins (necessary for cell migration).

Another trial, with low-dose temozolomide and celecoxib (Celebrex) for 32 glioblastoma patients, a “presumably anti-angiogenic therapy” resulted in a high rate of distant recurrences (62.5%), several centimetres away from the primary site (9).

This increase in migratory, invasive behaviour is most commonly associated with the tumour response to anti-angiogenic therapy, which may decrease tumour blood perfusion and oxygenation, thus provoking an increase in motility and invasion into new territory. I will discuss this phenomenon specifically as a response to the anti-angiogenic agent bevacizumab (Avastin), which is commonly used to treat recurrent glioblastomas. It is clear that a single-pronged approach to glioma therapy, focused only on hindering cell proliferation, will ultimately fail due to an increase in tumour cell migration and invasion. Any effective therapeutic regime must consider an anti-invasion strategy as being a counterpart of equal importance to anti-proliferative therapy.

References
  1. Distinct molecular mechanisms of acquired resistance to temozolomide in glioblastoma cells. Happold et al. 2012.
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  2. Reduction of MLH1 and PMS2 confers temozolomide resistance and is associated with recurrence of glioblastoma. Shinsato et al. 2013.
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  3. MSH6 mutations arise in glioblastomas during temozolomide therapy and mediate temozolomide resistance. Yip et al. 2009.
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  4. A hypermutation phenotype and somatic MSH6 mutations in recurrent human malignant gliomas after alkylator chemotherapy. Hunter et al. 2006.
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  5. Glioma cell motility is associated with reduced transcription of proapoptotic and proliferation genes: a cDNA microarray analysis. Mariani et al. 2001.
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  6. Cost of migration: invasion of malignant gliomas and implications for treatment. Giese et al. 2003.
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  7. Reciprocal activation of transcription factors underlies the dichotomy between proliferation and invasion of glioma cells. Dhruv et al. 2013.
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  8. High-dose tamoxifen treatment increases the incidence of multifocal tumor recurrences in glioblastoma patients. Puchner et al. 2004.
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  9. Recurrence pattern in glioblastoma multiforme patients treated with anti-angiogenic chemotherapy. Tuettenberg et al. 2009.
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  10. Akt activation suppresses Chk2-mediated, methylating agent-induced G2 arrest and protects from temozolomide-induced mitotic catastrophe and cellular senescence. Hirose et al. 2005.
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  11. Activation of Akt/Protein Kinase B overcomes a G2/M cell cycle checkpoint induced by DNA damage. Kandel et al. 2002.
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  12. NF-KB is activated in response to temozolomide in an AKT-dependent manner and confers protection against the growth suppressive effect of the drug. Caporali et al. 2012.
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  13. NVP-BEZ235, a novel dual phosphatidylinositol 3-kinase / mammalian target of rapamycin inhibitor, elicits multifaceted antitumor activities in human gliomas. Liu et al. 2009.
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  14. Inhibition of STAT3 reverses drug resistance acquired in temozolomide-resistant human glioma cells. Lee et al. 2011.
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