NMDA RECEPTOR INHIBITION REDUCES SURVIVAL OF MUTANT P53 BREAST CANCER CELL LINES

INTRODUCTION

• Thirty years ago, p53 was discovered as a cellular partner of simian virus 40 large T-antigen, the oncoprotein of this tumor virus (Levine and Oren, 2009). p53 is a transcription factor induced by stress that promotes cell cycle arrest, apoptosis, and senescence, and is undoubtedly among the most extensively studied genes and proteins (Levine and Oren, 2009).

• The p53 tumor suppressor provides a powerful intrinsic defense against cancer (Levine and Oren, 2009). Hence, mutations in the TP53 gene are the most frequent genetic alteration in human cancers. Some frequently observed p53 mutations also contribute actively to cancer development through gain-of-function (GOF) activities (Brosh and Rotter, 2009).This may involve enhancement of invasive properties, attenuation of apoptosis, and increased genomic instability.

• N-methyl D-aspartate receptors (NMDARs) are glutamate-gated ionotropic receptors/channels that are central to many physiological processes, including learning and memory, and are involved in neurotoxicity and psychiatric disorders (Paoletti, Bellone, and Zhou, 2013). NMDAR expression has been implicated in multiple tumor cell types, including neuroblastoma cells, lung cancer cells, and breast cancer cells (North, Gao, Memoli, and Du, 2010). North et al. report that most breast tumor cell lines necessarily express high levels of NMDAR1 and NMDAR2 (North, Gao, Memoli. Pang, and Lynch, 2010).

• NMDARs regulate mTOR signaling activity; their inappropriate expression on several human cancer cell lines represents a potential therapeutic avenue to control tumor development and progression (Figure 1) (Deutsch, Tang, Burket, and Benson, 2014). NMDAR antagonists, such as MK-801 (uncomp-etitive antagonist) were shown to possess anti-proliferative and anti-invasive effects in human lung adenocarcinoma cells (Lafon-Cazal, Perez, Bockaert, and Marin, 2002). These properties conflict with prevalent hypotheses that suggest promoting NMDA receptor activation as a cancer chemotherapeutic strategy (Zheng and Quirion, 2009).

• Determining the effect of NMDAR on specific tumor cell lines is therefore a critical step in developing potential NMDAR chemotherapeutics. We studied these effects in breast cancer by inhibiting NMDAR activity in three mutant p53 breast cancer cell lines

REFERENCES

METHODS & RESULTS

Donald Gosife Okoye2, & Luis Martinez PhD1

CONCLUSIONS

• These findings indicate that NMDAR activity increases cell survival via an mTOR-dependent mechanism that can be abrogated by NMDAR antagonists

• They also present the inhibition of the NMDAR pathway as a potentially less risky and less harmful chemotherapeutic target for mutant p53-based breast cancer treatment.

• Irrespective of whether activation or antagonism is associated with anti-proliferative and anti-invasive effects for specific types of cancer, emerging data support the exploration of targeting NMDA receptors expressed on the surface of cancer cells as a therapeutic strategy.

Future Work: • Further elucidation of the specific mechanism of activity is required.• Comparative experiments with other inhibitors and/or activators of NMDAR will be

helpful in confirming results.

1Biochemistry Dept. University of Mississippi Medical Center, 2Sewanee: The University of the South

ACKNOWLEDGEMENTS

My sincerest thanks to Drs. Luis Martinez & Madhu Kollaredy for their guidance and mentorship. My gratitude as well to Zunamys Carrerro, Krishna Chauhan, and Gopal Ramakrishnan for all of their help and advice during my stay. Finally, thanks to Mary Canterbury and the University of Mississippi Medical Center School of Graduate Studies in the Health Sciences for making my research experience a successful one.

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CELL LINES: We used 3 different cancer cell lines for this experiment: HCC 38 (R273L); BT 549 (R249S); MDA-MB-231 (R280K)

SEEDING: We seeded 5000 cells per well

DRUG TREATMENT: We added the appropriate concentration of the MK-801 drug in DMSO to the experimental plates (then incubated for 72 hours) normalizing for DMSO toxicity

MTT : We prepared and added 10 microliters of 5mg/ml MTT to each well and incubated for 3 hours noting color changes; then we added lysis buffer and incubated for another 24 hours

ABSORBANCE AND CONCENTRATION READING (CELL COUNT): We obtained absorbance and thus concentration readings of the experimental wells and compared them to the absorbance/concentration of the control wells in order to determine the IC50 of MK-801 for different cell lines.

WESTERN BLOT ANALYSIS

Method: Probing for the presence of phospho-Akt. [Akt has been reported to mediate the anti-apoptotic effects of NMDAR activity when phosphorylated by kinases upstream of the mTOR pathway (Lafon-Cazal, Perez, Bockhaert, and Marin, 2002)].Results: Treatment of the cell lines with MK-801 reduced the levels of phospho-AKT present in all cell lines

Fig. 1. Regulation of mTOR signaling activity through NMDA receptor activation. The figure depicts NMDA receptor activation leading to both diminished transport of arginine via cationic amino acid transporters (CAT) into the cell, and shortening of the duration of signaling by the phosphorylated form of extracellular signal-regulated kinase (ERK) ½ (Originally published by Deutsch, Tang, Burket, and Benson, 2014).

MK-801 concentration

Fig 2. Colorimetric measurement of amount of viable mutant p53 breast cancer cells after drug treatment. Increasing concentration of MK-801 treatment resulted in fewer concentration of viable cells in the wells.

Fig. 3. Determination of IC50 values for MK-801 effect on mutant p53 breast cancer cell lines. The calculations, plot and subsequent extrapolation was done using Graphpad Prism Version 5.

24 hrs treatment

Fig. 4. Western Blot analysis for phosphor-Akt expression in MK-801-treated mutant p53 breast cancer cell lines. *Con refers to control (untreated cell lines). Native Akt or inactive Akt probed to ensure that Akt expression levels are unaffected. Actin is used as endogenous control.

Cell MembraneNMDAR

NMDA

AKT-p

mTOR pathway

Cell Proliferation

Cell Growth

Cell Motility Cell survival

Protein synthesis and transcription

Apoptosis

MK-801Synaptic Plasticity, Learning, and Memory

Fig. 5. Regulation of mTOR signaling activity through NMDA receptor activation. The figure depicts NMDA receptor activation leading to phosphorylation of Akt and a resulting activation of the mTOR pathway. MK-801 activity prevents the activation of NMDAR.

• Levine, A. J., Oren, M. (2009). The first 30 years of p53: growing even more complex. Nat. Rev. Cancer 9, 749-758.• Brosh, R., Rotter, V. (2009). When mutants gain new powers: news from the mutant p53 field. Nat. Rev. Cancer 9, 701-713.• Paoletti, P., Bellone, C., Zhou Q. (2013). NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity

and disease. Nat. Rev. Neurosci. 14, 383–400.• North, G.W., Gao, G., Jensen, A., Memoli, V.A., Du, J. (2010). NMDA receptors are expressed by small-cell lung cancer and are

potential targets for effective treatment. Clin Pharmacol. 2, 31–40.• North, G.W., Gao, G., Memoli, V.M., Pang, R.H.L., Lynch, L. (2010). Breast Cancer Expresses Functional NMDA Receptors.

Breast Cancer Res Treat. 122(2), 307–314.• Deutsch, S.I., Tang, A.H., Burket, A.J., Benson, A.D. (2014). NMDA receptors on the surface of cancer cells: Target for

chemotherapy? Biomedicine & Pharmacotherapy 68, 493–496.• Lafon-Cazal, M., Perez, V., Bockhaert, J., Marin, P. (2002). Akt mediates the anti-apoptotic effect of NMDA but not that

induced by potassium depolarization in cultured cerebellar granule cells. European Journal of Neuroscience 10(1046), 1460-9568.

• Zheng, W., Quirion, R. (2009). Glutamate Acting on N-Methyl-D-aspartate Receptors Attenuates Insulin-like Growth Factor-1 Receptor Tyrosine Phosphorylation and Its Survival Signaling Properties in Rat Hippocampal Neurons. The Journ. Of Biol. Chem. 284(2), 855–861.