Figure S8 from Elevated Heme Synthesis and Uptake Underpin Intensified Oxidative Metabolism and Tumorigenic Functions in Non–Small Cell Lung Cancer Cells

Sohoni, Sagar; Ghosh, Poorva; Wang, Tianyuan; Kalainayakan, Sarada Preeta; Vidal, Chantal; Dey, Sanchareeka; Konduri, Purna Chaitanya; Zhang, Li

doi:10.1158/0008-5472.22419138.v1

00085472can182156-sup-205166_3_supp_5401680_ppdhpp.pdf (294.93 kB)

Figure S8 from Elevated Heme Synthesis and Uptake Underpin Intensified Oxidative Metabolism and Tumorigenic Functions in Non–Small Cell Lung Cancer Cells

journal contribution

posted on 2023-03-31, 01:47 authored by Sagar Sohoni, Poorva Ghosh, Tianyuan Wang, Sarada Preeta Kalainayakan, Chantal Vidal, Sanchareeka Dey, Purna Chaitanya Konduri, Li Zhang

Fig. S8 The effect of HSP2 on the growth and progression of H1299 NSCLC lung tumor xenografts and on blood and liver functions in mice. (A) The quantified luminescence signals representing tumor volumes. Treatment started at week 3 when tumors have grown significantly and BLI signals were about 5x107 photons/seconds. Data are plotted as mean {plus minus} SD. For statistical analysis, the levels in treated tumors were compared to the levels in untreated tumors with a Welch 2-sample t-test. *, p-value < 0.05. (B) Representative H&E images of control tumors and tumors treated with HSP2 or control saline. Tumors are marked with light blue outlines. Scale bar: 2 mm. (C) Average red blood cell count in mice bearing orthotopic lung tumor xenografts treated with or without HSP2. (D) Average hemoglobin levels in mice bearing orthotopic lung tumor xenografts treated with or without HSP2. (E) Average serum ALT (alanine transaminase) levels in mice bearing orthotopic lung tumor xenografts treated with or without HSP2. (F) Western blots showing the levels of ALAS1 in H1299 cells bearing the control or overexpression vector for ALAS1. (G) Western blots showing the levels of SLC48A1 in H1299 cells bearing the control or overexpression vector for ALAS1. (H) Oxygen consumption rates are increased in cells overexpressing ALAS1 or SLC48A1. (I) overexpression of ALAS1 or SLC48A1 promotes colony formation by H1299 NSCLC cells. For statistical analysis, the levels in cells overexpressing ALAS1 or SLC48A1 were compared to the levels in control cells with a Welch 2-sample t-test. **, p-value < 0.005. (J) The levels of heme synthesis in H1299 cells cultured in normal medium and heme-depleted medium (H dep), respectively. The level of heme synthesis in heme-depleted medium presumably represents the total cellular heme levels needed for the cells. The data show that de novo heme synthesis accounts for approximately 68% of total cellular heme levels in H1299 cells, indicating that heme uptake accounts for 32%. Data are plotted as mean {plus minus} SD. For statistical analysis, the levels in heme-depleted medium were compared to the levels in normal medium with a Welch 2-sample t-test.*, p-value, 0.05, **, p-value < 0.005.

Funding

Cancer Prevention and Research Institute of Texas

History

ARTICLE ABSTRACT

Tumors of human non–small cell lung cancer (NSCLC) are heterogeneous but exhibit elevated glycolysis and glucose oxidation relative to benign lung tissues. Heme is a central molecule for oxidative metabolism and ATP generation via mitochondrial oxidative phosphorylation (OXPHOS). Here, we showed that levels of heme synthesis and uptake, mitochondrial heme, oxygen-utilizing hemoproteins, oxygen consumption, ATP generation, and key mitochondrial biogenesis regulators were enhanced in NSCLC cells relative to nontumorigenic cells. Likewise, proteins and enzymes relating to heme and mitochondrial functions were upregulated in human NSCLC tissues relative to normal tissues. Engineered heme-sequestering peptides (HSP) reduced heme uptake, intracellular heme levels, and tumorigenic functions of NSCLC cells. Addition of heme largely reversed the effect of HSPs on tumorigenic functions. Furthermore, HSP2 significantly suppressed the growth of human NSCLC xenograft tumors in mice. HSP2-treated tumors exhibited reduced oxygen consumption rates (OCR) and ATP levels. To further verify the importance of heme in promoting tumorigenicity, we generated NSCLC cell lines with increased heme synthesis or uptake by overexpressing either the rate-limiting heme synthesis enzyme ALAS1 or uptake protein SLC48A1, respectively. These cells exhibited enhanced migration and invasion and accelerated tumor growth in mice. Notably, tumors formed by cells with increased heme synthesis or uptake also displayed elevated OCRs and ATP levels. These data show that elevated heme flux and function underlie enhanced OXPHOS and tumorigenicity of NSCLC cells. Targeting heme flux and function offers a potential strategy for developing therapies for lung cancer. These findings show that elevated heme availability due to increased heme synthesis and uptake causes intensified oxygen consumption and ATP generation, promoting tumorigenic functions and tumor growth in NSCLC.