Temozolomide resistance in glioblastoma

Temozolomide resistance in glioblastoma

Temozolomide resistance is considered to be one of the major reasons responsible for glioblastoma treatment failure.

TMZ is currently the only mono-chemotherapeutic agent for newly-diagnosed high-grade glioma patients and acquired resistance inevitably occurs in the majority of such patients, further limiting treatment options. Therefore, there is an urgent need to better understand the underlying mechanisms involved in TMZ resistance, a critical step to developing effective, targeted treatments. An emerging body of evidence suggests the intimate involvement of a novel class of nucleic acid, microRNA (miRNA), in tumorigenesis and disease progression for a number of human malignancies, including primary brain tumours. miRNA are short, single-stranded, non-coding RNA (∼22 nucleotides) that function as post-transcriptional regulators of gene expression 1).

At least 50% of TMZ treated patients do not respond to TMZ. This is due primarily to the over-expression of O6-methylguanine methyltransferase (MGMT) and/or lack of a DNA repair pathway in GBM cells. Multiple GBM cell lines are known to contain TMZ resistant cells and several acquired TMZ resistant GBM cell lines have been developed for use in experiments designed to define the mechanism of TMZ resistance and the testing of potential therapeutics. However, the characteristics of intrinsic and adaptive TMZ resistant GBM cells have not been systemically compared 2)


Many other molecular mechanisms have come to light in recent years. Key emerging mechanisms include the involvement of other DNA repair systems, aberrant signaling pathwaysautophagyepigenetic modificationsmicroRNAs, and extracellular vesicle production 3).


To date, aberrations in O6-methylguanine-DNA methyltransferase are the clear factor that determines drug susceptibility. Alterations of the other DNA damage repair genes such as DNA mismatch repair genes are also known to affect the drug effect. Together these genes have roles in the innate resistance, but are not sufficient for explaining the mechanism leading to acquired resistance. Recent identification of specific cellular subsets with features of stem-like cells may have role in this process. The glioma stem-like cells are known for its superior ability in withstanding the drug-induced cytotoxicity, and giving the chance to repopulate the tumor. The mechanism is complicated to administrate cellular protection, such as the enhancing ability against reactive oxygen species and altering energy metabolism, the important steps to survive 4).


Rabé et al. performed a longitudinal study, using a combination of mathematical models, RNA sequencing, single cell analyses, functional and drug assays in a human glioma cell line (U251). After an initial response characterized by cell death induction, cells entered a transient state defined by slow growth, a distinct morphology and a shift of metabolism. Specific genes expression associated to this population revealed chromatin remodeling. Indeed, the histone deacetylase inhibitor trichostatin (TSA), specifically eliminated this population and thus prevented the appearance of fast growing TMZ-resistant cells. In conclusion, they identified in glioblastoma a population with tolerant-like features, which could constitute a therapeutic target 5)


Ferroptosis, which is a new type of cell death discovered in recent years, has been reported to play an important role in tumor drug resistance. A study reviews the relationship between ferroptosis and glioma TMZ resistance, and highlights the role of ferroptosis in glioma TMZ resistance. Finally, the investigators discussed the future orientation for ferroptosis in glioma TMZ resistance, in order to promote the clinical use of ferroptosis induction in glioma treatment 6).


CUL4B has been shown to be upregulated and promotes progression and chemoresistance in several cancer types. However, its regulatory effect and mechanisms on TMZ resistance have not been elucidated. The aim of this study was to decipher the role and mechanism of CUL4B in TMZ resistance. Western blot and public datasets analysis showed that CUL4B was upregulated in glioma specimens. CUL4B elevation positively correlated with advanced pathological stage, tumor recurrence, malignant molecular subtype and poor survival in glioma patients receiving TMZ treatment. CUL4B expression was correlated with TMZ resistance in GBM cell lines. Knocking down CUL4B restored TMZ sensitivity, while upregulation of CUL4B promoted TMZ resistance in GBM cells. By employing senescence β-galactosidase staining, quantitative reverse transcription PCR and Chromatin immunoprecipitation experiments, we found that CUL4B coordinated histone deacetylase (HDAC) to co-occupy the CDKN1A promoter and epigenetically silenced CDKN1A transcription, leading to attenuation of TMZ-induced senescence and rendering the GBM cells TMZ resistance. Collectively, our findings identify a novel mechanism by which GBM cells develop resistance to TMZ and suggest that CUL4B inhibition may be beneficial for overcoming resistance 7).


CXCL12/CXCR4 has been demonstrated to be involved in cell proliferationcell migrationcell invasionangiogenesis, and radioresistance in glioblastoma (GBM). However, its role in TMZ resistance in GBM is unknown. Wang et al. aimed to evaluate the role of CXCL12/CXCR4 in mediating the TMZ resistance to GBM cells and explore the underlying mechanisms. They found that the CXCL12/CXCR4 axis enhanced TMZ resistance in GBM cells. Further study showed that CXCL12/CXCR4 conferred TMZ resistance and promoted the migration and invasion of GBM cells by up-regulating FOXM1. This resistance was partially reversed by suppressing CXCL12/CXCR4 and FOXM1 silencing. This study revealed the vital role of CXCL12/CXCR4 in mediating the resistance of GBM cells to TMZ, and suggested that targeting CXCL12/CXCR4 axis may attenuate the resistance to TMZ in GBM 8).


The YTHDF2 expression in TMZ-resistant tissues and cells was detected. Kaplan-Meier analysis was employed to evaluate the prognostic value of YTHDF2 in GBM. Effect of YTHDF2 in TMZ resistance in GBM was explored via corresponding experiments. RNA sequence, FISH in conjugation with fluorescent immunostaining, RNA immunoprecipitation, dual-luciferase reporter gene and immunofluorescence were applied to investigate the mechanism of YTHDF2 that boosted TMZ resistance in GBM.

YTHDF2 was up-regulated in TMZ-resistant tissues and cells, and patients with high expression of YTHDF2 showed lower survival rate than the patients with low expression of YTHDF2. The elevated YTHDF2 expression boosted TMZ resistance in GBM cells, and the decreased YTHDF2 expression enhanced TMZ sensitivity in TMZ-resistant GBM cells. Mechanically, YTHDF2 bound to the N6-methyladenosine (m6A) sites in the 3’UTR of EPHB3 and TNFAIP3 to decrease the mRNA stability. YTHDF2 activated the PI3K/Akt and NF-κB signals through inhibiting expression of EPHB3 and TNFAIP3, and the inhibition of the two pathways attenuated YTHDF2-mediated TMZ resistance.

YTHDF2 enhanced TMZ resistance in GBM by activation of the PI3K/Akt and NF-κB signalling pathways via inhibition of EPHB3 and TNFAIP3 9).


1) 
Low SY, Ho YK, Too HP, Yap CT, Ng WH. MicroRNA as potential modulators in chemoresistant high-grade gliomas. J Clin Neurosci. 2013 Oct 6. pii: S0967-5868(13)00518-3. doi: 10.1016/j.jocn.2013.07.033. [Epub ahead of print] PubMed PMID: 24411131.
2) 
Lee SY. Temozolomide resistance in glioblastoma multiforme. Genes Dis. 2016 May 11;3(3):198-210. doi: 10.1016/j.gendis.2016.04.007. PMID: 30258889; PMCID: PMC6150109.
3) 
Singh N, Miner A, Hennis L, Mittal S. Mechanisms of temozolomide resistance in glioblastoma – a comprehensive review. Cancer Drug Resist. 2021;4(1):17-43. doi: 10.20517/cdr.2020.79. Epub 2021 Mar 19. PMID: 34337348; PMCID: PMC8319838.
4) 
Chien CH, Hsueh WT, Chuang JY, Chang KY. Dissecting the mechanism of temozolomide resistance and its association with the regulatory roles of intracellular reactive oxygen species in glioblastoma. J Biomed Sci. 2021 Mar 8;28(1):18. doi: 10.1186/s12929-021-00717-7. PMID: 33685470; PMCID: PMC7938520.
5) 
Rabé M, Dumont S, Álvarez-Arenas A, Janati H, Belmonte-Beitia J, Calvo GF, Thibault-Carpentier C, Séry Q, Chauvin C, Joalland N, Briand F, Blandin S, Scotet E, Pecqueur C, Clairambault J, Oliver L, Perez-Garcia V, Nadaradjane A, Cartron PF, Gratas C, Vallette FM. Identification of a transient state during the acquisition of temozolomide resistance in glioblastoma. Cell Death Dis. 2020 Jan 6;11(1):19. doi: 10.1038/s41419-019-2200-2. PMID: 31907355; PMCID: PMC6944699.
6) 
Hu Z, Mi Y, Qian H, Guo N, Yan A, Zhang Y, Gao X. A Potential Mechanism of Temozolomide Resistance in Glioma-Ferroptosis. Front Oncol. 2020 Jun 23;10:897. doi: 10.3389/fonc.2020.00897. PMID: 32656078; PMCID: PMC7324762.
7) 
Ye X, Liu X, Gao M, Gong L, Tian F, Shen Y, Hu H, Sun G, Zou Y, Gong Y. CUL4B Promotes Temozolomide Resistance in Gliomas by Epigenetically Repressing CDNK1A Transcription. Front Oncol. 2021 Apr 2;11:638802. doi: 10.3389/fonc.2021.638802. PMID: 33869025; PMCID: PMC8050354.
8) 
Wang S, Chen C, Li J, Xu X, Chen W, Li F. The CXCL12/CXCR4 axis confers temozolomide resistance to human glioblastoma cells via up-regulation of FOXM1. J Neurol Sci. 2020 Apr 14;414:116837. doi: 10.1016/j.jns.2020.116837. [Epub ahead of print] PubMed PMID: 32334273.
9) 
Chen Y, Wang YL, Qiu K, Cao YQ, Zhang FJ, Zhao HB, Liu XZ. YTHDF2 promotes temozolomide resistance in glioblastoma by activation of the Akt and NF-κB signalling pathways via inhibiting EPHB3 and TNFAIP3. Clin Transl Immunology. 2022 May 9;11(5):e1393. doi: 10.1002/cti2.1393. PMID: 35582627; PMCID: PMC9082891.

Cilostazol

Cilostazol

Cilostazol, is a antiplatelet drug that inhibits phosphodiesterase 3.

Application of cilostazol was reported to ameliorate vasospasm and improve outcomes in series and clinical trials. But the effectiveness and feasibility of cilostazol on aneurysmal subarachnoid hemorrhage remained controversial.


Kim et al. from the Asan Medical Center retrospectively analyzed the data of 427 patients with unruptured intracranial aneurysms who underwent endovascular treatment between July 2011 and June 2014. When clopidogrel resistance was confirmed via platelet reactivity unit (PRU) assay after dual antiplatelet therapy (aspirin plus clopidogrel) administration for 5 days, triple antiplatelet therapy with cilostazol was administered (Group I, 274 patients). The other group was placed on standard dual antiplatelet therapy (Group II, 153 patients). All patients underwent magnetic resonance diffusion-weighted imaging within 2 days after endovascular coiling.

No significant associations with the occurrence of a thromboembolic event and microembolic event were found between the groups. The occurrence of thromboembolic and microembolic events showed no statistical difference between groups I and II (p = 0.725 for thromboembolic events and p = 0.109 for microembolic events). Also, the PRU value and the occurrence of microembolic events, using a PRU cutoff value of 240, showed no statistical difference (p = 0.114 in group I and 0.064 in group II). There was significant increase in microembolic events after the use of a stent-assisted endovascular procedure. As the PRU value increased, there was a trend toward an increase in the mean number of microembolic lesions without statistical significance.

Even though there is a presumed anti-thromboembolic effect for clopidogrel resistance in other literature, the clinical efficacy of adjustment of additional cilostazol for endovascular coiling of unruptured aneurysms may be limited due to the unspecified cutoff value of the PRU assay for evaluating the resistance 1).

A total of 454 articles were identified using the search criteria. Six articles were selected for systematic review and the 4 randomized controlled trials were included in the meta-analysis. The pooled odds ratio for symptomatic vasospasm, new-onset infarct, and angiographic vasospasm was 0.35 (95% confidence interval [CI], 0.21-0.59; P < 0.0001), 0.38 (95% CI, 0.21-0.66; P = 0.0007) and 0.49 (95% CI, 0.31-0.80; P = 0.004), respectively. The pooled risk ratio for unfavorable outcome was 0.52 (95% CI, 0.37-0.74; P = 0.0003).

Cilostazol decreases the prevalence of symptomatic vasospasm, new-onset infarct, and angiographic vasospasm when administered after aSAH. Trial sequential analysis increased the precision of our results because the defined thresholds of effect were met by the available studies. However, further studies involving patients from other geographic areas are required to confirm the generalization of the results 2)

Shan et al., performed a systematic review to clarify this issue.

PubMed, Ovid and Cochrane library database were systematically searched up to May 2018 for eligible publications in English. Quality assessment was conducted for included studies. Meta-analysis was conducted to evaluate the overall effect on events of interest. Subgroup analyses and sensitivity analyses were used to check whether the results were robust. Publication bias was evaluated with the funnel plot.

Pooled analyses found cilostazol significantly reduced incidences of severe angiographic vasospasm (p = 0.0001), symptomatic vasospasm (p < 0.00001), new cerebral infarction (p < 0.00001) and the poor outcome (p < 0.0001). Subgroup and sensitivity analyses achieved consistent results. There was no statistical difference between cilostazol and the control group in reducing mortality (p = 0.07). But sensitivity analysis changed the result after excluding one study. Under the prescribed dosage, complication was few and non-lethal.

Cilostazol was effective and safe to reduce incidences of severe angiographic vasospasm, symptomatic vasospasm, new cerebral infarction and poor outcome in patients after aneurysmal subarachnoid hemorrhage. However, its effect on mortality and the interactive effect with nimodipine warranted further research 3).

Beneficial for patients with atherothrombosis. In contrast to other anti-platelet drugs such as aspirin and thienopyridines, little information is available on the relationship between platelet responses to cilostazol and clinical outcomes.

Ikeda et al. from the Ehime University Graduate School of Medicine in Japan, conducted a prospective study on patients with cerebral infarction who were treated with cilostazol. The platelet response to cilostazol was assessed with a new assay for the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) subsequent to the pharmacological action of cilostazol. Patients were followed up for 2 years and the relationship between VASP assay results and the recurrence of thrombotic events was examined. We also investigated the effects of CYP3A5 and CYP2C19 genotypes involved in the metabolism of cilostazol on the platelet response to cilostazol.

Among the 142 patients enrolled, 130 completed the 2-year follow-up and the recurrence of thrombotic events was noted in 8 (6.2%). VASP phosphorylation levels were significantly lower in patients with than in those without recurrence. The combined genotype of CYP3A51/3 and CYP2C191/1 was associated with a low level of VASP phosphorylation, while either genotype was not. A multivariate analysis showed that high residual platelet reactivity during the cilostazol treatment, which was defined by a low response of platelet VASP phosphorylation to cilostazol, was an independent risk factor for the recurrence of thrombotic events.

A low platelet response to cilostazol determined by a new platelet assay was associated with the recurrence of thrombotic events in patients with cerebral infarction 4).

established an experimental model using normal and diabetic rats at 12 months of age. The diabetic rats were assigned to 4 different diet groups, distinguished by whether they were fed plain rat feed, or the same feed supplemented by 1 of 3 antiplatelet drugs (cilostazol, aspirin, or clopidogrel: all 0.1%) for 2 weeks, and the carotid artery was perforated by an embolization coil (“carotid coil model”). We monitored the process by which vascular endothelial cells formed the new endothelium on the surface of the coil by sampling and evaluating the region at 1, 2, and 4 weeks after placement. This repair process was also compared among 3 groups treated with different antiplatelet drugs (i.e. aspirin, clopidogrel, and cilostazol). One-way analysis of variance tests were performed to evaluate the differences in vascular thickness between groups, and P < .05 was considered statistically significant.

Results: The diabetic rats showed delayed neoendothelialization and marked intimal hyperplasia. Cilostazol and clopidogrel effectively counteracted this delayed endothelial repair process. Flk1 immunostaining revealed greater expression in the diabetic rats administered cilostazol, second only to normal rats, suggesting that this agent acted to recruit EPCs.

Conclusion: Neoendothelialization is delayed when vascular endothelial cells fail to function normally, which consequently leads to the formation of hyperplastic tissue. Cilostazol may remedy this dysfunction by recruiting EPCs to the site of injury 5).


1)

Kim GJ, Heo Y, Moon EJ, Park W, Ahn JS, Lee DH, Park JC. Thromboembolic events during endovascular coiling for unruptured intracranial aneurysms: Clinical significance of platelet reactivity unit and adjunctive cilostazol. Clin Neurol Neurosurg. 2022 Jan 15;213:107133. doi: 10.1016/j.clineuro.2022.107133. Epub ahead of print. PMID: 35065532.
2)

Bohara S, Garg K, Singh Rajpal PM, Kasliwal M. Role of Cilostazol in Prevention of Vasospasm After Aneurysmal Subarachnoid Hemorrhage-A Systematic Review, Meta-Analysis, and Trial Sequential Analysis. World Neurosurg. 2021 Jun;150:161-170. doi: 10.1016/j.wneu.2021.02.069. Epub 2021 Feb 23. PMID: 33631387.
3)

Shan T, Zhang T, Qian W, Ma L, Li H, You C, Xie X. Effectiveness and feasibility of cilostazol in patients with aneurysmal subarachnoid hemorrhage: a systematic review and meta-analysis. J Neurol. 2019 Feb 9. doi: 10.1007/s00415-019-09198-z. [Epub ahead of print] Review. PubMed PMID: 30739182.
4)

Ikeda Y, Yamanouchi J, Kumon Y, Yasukawa M, Hato T. Association of platelet response to cilostazol with clinical outcome and CYP genotype in patients with cerebral infarction. Thromb Res. 2018 Oct 10;172:14-20. doi: 10.1016/j.thromres.2018.10.003. [Epub ahead of print] PubMed PMID: 30342278.
5)

Fukawa N, Ueda T, Ogoshi T, Kitazawa Y, Takahashi J. Vascular Endothelial Repair and the Influence of Circulating Antiplatelet Drugs in a Carotid Coil Model. J Cent Nerv Syst Dis. 2021 May 20;13:11795735211011786. doi: 10.1177/11795735211011786. PMID: 34104032; PMCID: PMC8145582.

Somatostatin Analogs in Acromegaly

Somatostatin Analogs in Acromegaly

In vitro, native somatostatin retains its inhibitory effect on GH secretion in many GH-secreting tumors, and this led to the development of analogs of somatostatin for clinical use in the treatment of acromegaly 1).

The two analogs of somatostatin available for clinical use are the cyclic octapeptides octreotide (Dphe-cys-phe-Dtrp-lys-thr-cys-thr-ol) and lanreotide (Dnal-cys-tyr-Dtrp-lys-val-cys-thr) (1, 5–7). Octreotide is the only analog currently available for clinical use in the treatment of acromegaly in the United States.


Clinically available somatostatin analogs control GH or IGF-I excess in about 50–60% of patients whether used as primary or secondary therapy. Signs and symptoms of the disease improve in most patients. Tumor shrinkage occurs with somatostatin analogs used as adjunctive therapy in about 30% of patients and with their use as primary therapy in about 48% of patients. The shrinkage in most patients is greater than 20%, but less than 50% of tumor size 2).


Current data suggest that response to these drugs is better analyzed by taking together biochemical and tumoral effects because only the absence of both responses might be considered as a poor response or resistance. This latter evidence seems to occur in 25% of treated patients after 12 months of currently available long-acting SA 3).


Somatostatin analogues may be used when complete recovery cannot be achieved by surgical excision of GH-secreting pituitary adenomas or the patient declines surgery. This position statement is established based on the consensus of opinion among experts and evidence from published data regarding the use of somatostatin analogs in patients with acromegaly. However, this position statement cannot be considered as complete, given the clinical characteristics of acromegaly and the absence of large-scale clinical data in Korea; at this time, the clinical judgment of the physician should take precedence over this statement. This position statement will be revised as needed when additional data for Korean patients become available 4).


Shao et al. retrospectively analyzed the effects of SSAs on lipid profiles and associated cardiovascular risk factors in a cohort of 120 newly diagnosed acromegaly patients. In this study, 69 females and 51 males were included. These patients were treated with either octreotide LAR (OCT) or lanreotide SR (LAN) for 3 months. After Somatostatin Analogs treatment, both GH and IGF-1 significantly decreased (p<0.001). Triglyceride (TG), total to high-density lipoprotein cholesterol (HDL-C) ratio, and lipoprotein (a) [Lp(a)] levels were significantly decreased, while HDL-C levels were increased (p<0.05). The reduction of mean serum GH (GHm) was positively associated with the decrease of TG (r=0.305, p=0.001) and Lp(a) (r=0.257, p=0.005), as well as the increase of HDL-C (r=-0.355, p<0.001). The changes of lipid profiles were observed only in OCT group, but not in LAN group. In addition, systolic blood pressure (SBP) had significantly declined after SSAs treatment, with an average reduction of 4.4 mmHg (126.7±1.28 vs. 122.3±1.44 mmHg, p=0.003), while no change was observed regarding diastolic blood pressure (DBP) (p>0.05). Fasting insulin, fasting C-peptide, and HOMA-IR were significantly decreased after SSAs treatment. In conclusion, the study revealed that short-term SSAs treatment improves lipid profiles and other cardiovascular risk factors in patients with acromegaly 5).

Lanreotide for Acromegaly.

Octreotide for Acromegaly.


1)

Lamberts SW. The role of somatostatin in the regulation of anterior pituitary hormone secretion and the use of its analogs in the treatment of human pituitary tumors. Endocr Rev. 1988 Nov;9(4):417-36. doi: 10.1210/edrv-9-4-417. PMID: 2905987.
2)

Freda PU. Somatostatin analogs in acromegaly. J Clin Endocrinol Metab. 2002 Jul;87(7):3013-8. doi: 10.1210/jcem.87.7.8665. PMID: 12107192.
3)

Colao A, Auriemma RS, Lombardi G, Pivonello R. Resistance to somatostatin analogs in acromegaly. Endocr Rev. 2011 Apr;32(2):247-71. doi: 10.1210/er.2010-0002. Epub 2010 Dec 1. PMID: 21123741.
4)

Chin SO, Ku CR, Kim BJ, Kim SW, Park KH, Song KH, Oh S, Yoon HK, Lee EJ, Lee JM, Lim JS, Kim JH, Kim KJ, Jin HY, Kim DJ, Lee KA, Moon SS, Lim DJ, Shin DY, Kim SH, Kwon MJ, Kim HY, Kim JH, Kim DS, Kim CH. Medical Treatment with Somatostatin Analogues in Acromegaly: Position Statement. Endocrinol Metab (Seoul). 2019 Mar;34(1):53-62. doi: 10.3803/EnM.2019.34.1.53. PMID: 30912339; PMCID: PMC6435847.
5)

Shao XQ, Chen ZY, Wang M, Yang YP, Yu YF, Liu WJ, Wang Y, Zeng FF, Gong W, Ye HY, Wang YF, Zhao Y, Zhang L, Zhang ZY, He M, Li YM. Effects of Long-Acting Somatostatin Analogues on Lipid Metabolism in Patients with Newly Diagnosed Acromegaly: A Retrospective Study of 120 Cases. Horm Metab Res. 2022 Jan;54(1):25-32. doi: 10.1055/a-1717-9332. Epub 2022 Jan 5. PMID: 34986497.
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