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Supplementary Fig 1 from Heme Sequestration Effectively Suppresses the Development and Progression of Both Lung Adenocarcinoma and Squamous Cell Carcinoma

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posted on 2023-04-03, 19:40 authored by Sanchareeka Dey, Adnin Ashrafi, Chantal Vidal, Nivesh Jain, Sarada Preeta Kalainayakan, Poorva Ghosh, Parinaz Sadat Alemi, Narges Salamat, Purna Chaitanya Konduri, Jung-whan Kim, Li Zhang

Supplementary Figure 1. The levels of proteins and enzyme subunits involved in OXPHOS and glutamine metabolism increase with lung tumor progression. A, Representative H&E and IHC images of lung tissue sections from KLLuc mice showing levels of the OXPHOS protein HCCS. B, Representative H&E and IHC images of lung tissue sections from KLLuc mice showing levels of the glutamine transporter SLC1A5. C, Representative H&E and IHC images of lung tissue sections from KLLuc mice showing levels of GLS. Shown are montage images of lung tissue sections stained with H&E (row 1), montage images stained with DAPI (row 2) or antibodies against the indicated protein (row 3), and 10X images of lung tissue sections stained with DAPI (row 4) or antibodies against the indicated protein (row 5). The light green lines in DAPI montage images outline the tumors in the lung. The blue rectangles in H&E and white rectangles in DAPI montage images denote the regions shown in 10X. Scale bar, montage, 1mm; 4X, 200 µm; 10X, 100µm. D, Graph showing quantified levels of HCCS correlating with A. E, Graph showing quantified levels of SLC1A5 correlating with B. F, Graph showing quantified levels of GLS correlating with C. Protein levels were quantified, and data are plotted as mean {plus minus} SEM. G, Representative 40X IHC images of lung tissue sections showing the co-localization of ATP5F1A, COX7B, and CYCS with the mitochondrial marker TOMM20. Scale bar, 10 µm. H, OCR of NSCLC H226 cells cultured in normal medium, or heme depleted medium with 0.5 mM succinyl acetone (HD+SA). Heme (5 µM, 10 µM, and 20 µM, respectively) was added back to examine the effects of heme on OCRs. For statistical analysis, the levels in cells with heme added back were compared with the levels in cells without heme added back with a Welch two-sample t test. **, P<0.005.

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Cancer Prevention and Research Institute of Texas

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ARTICLE ABSTRACT

Lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC) are two most common subtypes of lung cancer. Here, to identify new, targetable molecular properties of both subtypes, we monitored changes in the levels of heme- and oxidative phosphorylation (OXPHOS)-related proteins during lung tumorigenesis. Heme is a central molecule for oxidative metabolism and ATP generation via OXPHOS. Notably, both lung ADC and SCC tumors can be induced in the genetically engineered KLLuc mouse model harboring the G12D Kras mutation and a conditional Lkb1 knockout. We found that the levels of the rate-limiting heme synthesis enzyme ALAS1 and uptake protein SLC48A1, along with OXPHOS complex subunits, progressively increased as lung tumorigenesis advanced. Our data demonstrated that elevated levels of heme- and OXPHOS-related proteins were associated with both ADC and SCC. Importantly, treatment of KLLuc mice with a heme-sequestering protein, HeSP2, that inhibits heme uptake in tumor cells effectively arrested lung tumor progression, and both ADC and SCC tumors were strongly suppressed. Additionally, HeSP2 effectively suppressed the growth of both SCC and ADC tumor xenografts in NOD/SCID mice. Further analyses indicated that HeSP2 effectively diminished OXPHOS in both ADC and SCC, reduced angiogenesis, alleviated tumor hypoxia, and suppressed cell proliferation. These results show that the advancing of lung tumorigenesis requires progressive increase in cellular heme synthesis and uptake, leading to intensified OXPHOS activity and ATP generation and promoting aggressive tumorigenic functions. Heme sequestration is an effective strategy for the suppression of both ADC and SCC tumor initiation and development.

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