Activated Erk 1/2 Kinases Decrease Cell Viability Caused by Erastin in HNSCC

Head and neck cancers originating from the mucosal epithelium in the oral cavity, larynx, and pharynx are collectively referred to as neck and head squamous cell carcinoma (HNSCC). As one of the sixth most common cancers worldwide, HNSCC tumors have an extremely negative impact on global health. 1 In 2020 around 377,700 new cases were documented, causing 177,800 deaths.2 At present, cases of HNSCC are still on the rise and scientists predict a 30% increase in cases by 2030, which equivalates to 1.08 million new cases per year.1 With a high percentage of HNSCC patients having undesired clinical outcomes, the development of novel therapeutic strategies is essential.  

Despite innovation in the realm of HNSCC therapeutics, treatments for the disease have remained almost unchanged over the last few decades.1 Thus, novel therapeutic strategies are necessary to increase survival rates and improve therapy results for HNSCC patients.

One of the ways to see an increase in survival rates is to increase the number of patients that have a positive response to therapy. However, as HNSCC tumor cells are highly heterogenous, differences in survival rates can also depend on the anatomical site and or stage of the cancer, and whether the patient is infected with human papillomavirus (HPV).3

Tumors like HNSCC comprise different cells that interact with each other via signaling pathways and to study them it is important to know their quantity and spatial distribution. Subpopulations of tumor cells can be localized and analyzed by expression of biomarkers such as xCT, which can be automatically imaged with the TissueFAXS platform and measured with HistoQuest (TissueGnostics) single cell analysis software.

Ferroptosis cell death

In the last decade, a unique type of non-apoptotic, iron-dependent cell death termed ferroptosis has been described. This cell death is induced by erastin, which prevents the uptake of cystine by the cystine/glutamate antiporter system xCT, inhibiting the cell’s antioxidant defense mechanism.4 This results in increased iron uptake, which has been well demonstrated in HNSCC cells to cause intracellular augmentation. There is still much research to be conducted to fully understand the ferroptotic pathway, but xCT inhibition can cause the abrogation of cancer cells that are resistant to HNSCC chemo-radiotherapy, making it a potential treatment target.3

An increase in reactive oxygen species (ROS) within the cell accompanies this process, to which cells may respond by upregulating the antioxidant system. Numerous MAP kinases, including Erk1/2, regulate oxidative stress within the cell, and inhibition of MAP kinases has been shown to protect cells from ferroptosis.5 xCT regulation by Erk1/2 could therefore be a therapeutic target for anti-tumor therapies. This was first recognized by Dr. Dragana Savic and her team, who investigated whether Erk1/2 activation made HNSCC cells more vulnerable to ferroptosis and if the development of ferroptosis can be modulated through inhibiting the expression and phosphorylation of Erk1/2 through erastin.

Using TissueFAXS and HistoQuest to assess xCT regulation by Erk1/2

The study correlated xCT (encoded by SLC7A11) activity within the cell with patient survival rates to determine whether this molecule can serve as a potential therapeutic target in HNSCC patients. TissueFAXS was used to automatedly scan slides with tissues stained for hematoxylin and xCT. HistoQuest was utilized to quantify xCT expression within single cells based on its signal staining intensity.

Cells are recognized as objects based on nuclear hematoxylin staining, and marker-positive cells should express xCT above the threshold level. The HistoQuest software quantified positive cells in relation to negative cells based on the staining intensity to assess the effect of Erk1/2 activation on cell ferroptosis. This assessment was performed in both normal mucosa and HNSCC samples to compare the difference in xCT expression. Tumor cells were then treated with erastin to investigate the role of Erk1/2 in decreased viability of cells overexpressing xCT.

HNSCC patients with high cellular levels of xCT were found to have significantly decreased survival rates in comparison to patients with low levels. The xCT molecule was identified as a significant negative regulator of ferroptosis, and xCT overexpression in cancerous cells indicated its potential as a target molecule in HNSCC treatment.3 This is in conjunction with previous studies that have reported a reduction in the overall survival time of oncology patients that displayed overexpression of xCT.6,7

Inhibition of the Erk1/2 signaling pathway resulted in a decrease in the efficacy of erastin due to its effects on the production of ROS. Thus, researchers suggest that carcinoma cells which express phosphorylated Erk1/2 are more sensitive to ferroptosis.3 As erastin-induced ferroptosis seems to depend strongly on Erk1/2 expression; it can be implied that cellular levels of phosphorylated Erk1/2 can be used to predict the response of cancerous cells to erastin, improving current oncology treatments.

TissueFAXS and HistoQuest continue to provide state-of-the-art immunohistochemistry technologies that effectively assess potential therapeutic targets and predictor molecules/pathways to advance oncology treatment and improve patient outcomes and survival rates. Contact our team to know more about our solutions.

References and Further Reading

  1. Johnson, D.E., Burtness, B., Leemans, C.R., Lui, V.W.Y., Bauman, J.E. and Grandis, J.R. 2020. Head and neck squamous cell carcinoma. Nature reviews Disease primers. 6(1), p.92.
  2. Sung, H., Ferlay, J., Siegel, R.L., Laversanne, M., Soerjomataram, I., Jemal, A. and Bray, F. 2021. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: a cancer journal for clinicians. 71(3), pp.209-249.
  3. Savic, D., Steinbichler, T.B., Ingruber, J., Negro, G., Aschenbrenner, B., Riechelmann, H., Ganswindt, U., Skvortsov, S., Dudás, J. and Skvortsova, I.I. 2023. Erk1/2-Dependent HNSCC Cell Susceptibility to Erastin-Induced Ferroptosis. Cells. 12(2), p.336.
  4. Dixon, S.J., Lemberg, K.M., Lamprecht, M.R., Skouta, R., Zaitsev, E.M., Gleason, C.E., Patel, D.N., Bauer, A.J., Cantley, A.M., Yang, W.S. and Morrison, B. 2012. Ferroptosis: an iron-dependent form of nonapoptotic cell death. 149(5), pp.1060-1072.
  5. Poursaitidis, I., Wang, X., Crighton, T., Labuschagne, C., Mason, D., Cramer, S.L., Triplett, K., Roy, R., Pardo, O.E., Seckl, M.J. and Rowlinson, S.W. 2017. Oncogene-selective sensitivity to synchronous cell death following modulation of the amino acid nutrient cystine. Cell reports. 18(11), pp.2547-2556.
  6. Shi, Z.Z., Tao, H., Fan, Z.W., Song, S.J. and Bai, J. 2021. Prognostic and immunological role of key genes of ferroptosis in pan-cancer. Frontiers in Cell and Developmental Biology. 9, p.748925.
  7. Lee, J.R., Roh, J.L., Lee, S.M., Park, Y., Cho, K.J., Choi, S.H., Nam, S.Y. and Kim, S.Y. 2018. Overexpression of cysteine‐glutamate transporter and CD44 for prediction of recurrence and survival in patients with oral cavity squamous cell carcinoma. Head & neck. 40(11), pp.2340-2346.

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