Heme Sequestration Effectively Suppresses the Development and Progression of Both Lung Adenocarcinoma and Squamous Cell Carcinoma

Lung cancer is the leading cause of cancer-related death in the United States and worldwide (1). Lung cancer is a very heterogeneous disease at both cellular and histologic levels. Lung cancer can be divided into two major types: non–small cell lung cancer (NSCLC), which accounts for ∼85% of cases, and small cell lung cancer, which accounts for the remaining 15% of lung cancer cases (2). NSCLC is further subdivided into lung adenocarcinoma (ADC), lung squamous cell carcinoma (SCC), and large cell carcinoma. Lung cancer exhibits widespread intertumoral and intratumoral heterogeneity as well as incidences of subtype transdifferentiation (3–8). Such molecular heterogeneity and transdifferentiation afford multiple mechanisms for the development of drug resistance and pose great challenges for the treatment of lung cancer. Indeed, despite the advent of targeted therapies and immunotherapies, an effective treatment or cure for lung cancer remains an unlikely outcome for most patients. The five-year survival rate remains 10% to 20%, lower than many other cancers, including breast (90%) and prostate (99%) cancers (1). Thus, novel strategies are still needed for dramatic improvement in the overall survival rate of lung cancer patients.

Accumulating evidence increasingly demonstrates that mitochondrial metabolism and oxidative phosphorylation (OXPHOS) are crucial for tumorigenesis (9, 10). Increased oxidative metabolism does not conflict with the fact that most cancer cells exhibit enhanced glycolytic rates, as Warburg observed (11). Studies of NSCLC tumors in human patients showed that pyruvate and lactate from elevated glycolysis enter and intensify the TCA cycle in all tumors, although NSCLC tumors are metabolically heterogeneous (12, 13). Additionally, components of OXPHOS complexes and markers of mitochondrial biogenesis are found to be highly predictive of reduced overall survival in NSCLC patients (14). Likewise, the expression of OXPHOS genes is negatively correlated with the prognosis of lung ADC (15). Heme is essential for OXPHOS/mitochondrial respiration and drug metabolism (16). Multiple subunits in OXPHOS complexes II–IV contain heme. Heme coordinates the expression and assembly of OXPHOS complexes (17). It is also worth noting that heme can have an array of adverse effect to cells (18). Importantly, recent studies in our laboratory have shown that enhanced heme biosynthesis and uptake lead to elevated levels of mitochondrial heme, which cause intensified OXPHOS and ATP generation, thereby promoting tumorigenic functions in NSCLC cells (19). Thus, it would be interesting to examine if and how heme metabolism and function change as lung tumorigenesis initiates and progresses and if such changes differ in different subtypes of NSCLC.

Genetically engineered mouse models (GEMM) have provided valuable tools for understanding the development of the various lung cancer subtypes (20). Notably, KL mice offer a powerful GEMM for NSCLC. They harbor a conditional activating mutation (G12D) at the endogenous Kras locus, a conditional Lkb1 knockout, and conditional luciferase expression for monitoring tumorigenesis in live mice (21, 22). Lung tumors developed in KL mice include lung ADC and SCC. They exhibit broad tumor heterogeneity (21–23). KL tumors also express low levels of immune markers, including PD-L1, and are not responsive to immunotherapy (24). Thus, KL tumors represent those lung tumors that are more drug resistant and difficult to treat. Thus, KL mice provide a powerful tool to define the molecular features of resistant NSCLC tumors and to identify effective therapeutic strategy to overcome their resistance. In this report, using KL mice, we monitored the levels of markers of heme synthesis and uptake, along with markers of OXPHOS, tumor vasculature and oxygenation, and glucose metabolism, as lung tumorigenesis initiates and advances. We found that the levels of proteins and enzymes involved in heme synthesis and uptake strongly increased along with markers of glucose metabolism and tumor vasculature, as lung tumors developed in KL mice. Elevated levels of markers of heme synthesis and uptake are associated with both ADC and SCC. Further, using a heme-sequestering protein (HeSP2), we showed that heme sequestration can effectively suppress the progression of both ADC and SCC.

HeSP2 (previously known as HSP2) was purified using pET11a expression system. Dr. Mario Rivera (University of Kansas) kindly provided us the pET11a expression vector for Yersinia pestis HasA residues 1–193 (25). HeSP2 contains Q32H Y75M double mutations that were generated with the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies; ref. 19). Ad5-CMV-Cre was purchased from Baylor Vector Development Laboratory. The Opal 4 color IHC Kit was purchased from Akoya Biosciences. ATP Determination Kit was purchased from Molecular Probes.

All NSCLC cell lines, H1299 (ATCC cat. no. CRL-5803, RRID: CVCL_0060), A549 (ATCC cat. no. CRM-CCL-185, RRID: CVCL_0023), H460 (ATCC cat. no. HTB-177, RRID: CVCL_0459), HCC15 (DSMZ cat. no. ACC-496, RRID: CVCL_2057), H226 (ATCC cat. no. CRL-5826, RRID: CVCL_1544), H520 (ATCC cat. no. HTB-182, RRID: CVCL_1566), and SW900 (ATCC cat. no. HTB-59, RRID: CVCL_1731), were purchased from ATCC and maintained in RPMI medium. H1299, A549, and H460 were supplemented with 5% heat-inactivated FBS, whereas HCC15, H226, H520, and SW900 were supplemented with 10% heat-inactivated FBS and switched to 5% FBS, 24 hours before experiments. All experiments were conducted using cells between passages 3 to 5 from revival of the frozen stocks. Cell lines were authenticated by Genetica, LabCorp and were found to be >95% identical to the standard. Mycoplasma testing for cell lines was done using a MycoFluor Mycoplasma Detection Kit (Molecular Probes), and the results were negative. Some known mutations in these cell lines are as follows: HCC15 (homozygous TP53 mutation, c.776A>T; STK11 deletion; NRAS mutation, c.181 C>A; CDKN2A deletion, c.1_471del471), NCI-H226 (homozygous CDKN2A deletion, c.1_150del150), NCI-H520 (homozygous TP53 mutation, c.438G>A; homozygous CDKN2A deletion, c.134delG), SW900 (heterozygous KRAS mutation, c.35G>T; homozygous TP53 mutation, c.499C>T), H1299 (heterozygous NRAS mutation, c.181C>A; homozygous TP53 deletion), A549 (homozygous KRAS mutation, c.34G>A; homozygous STK11 mutation, c.109C>T; homozygous CDKN2A deletion, c.1_471del471), and H460 (homozygous KRAS mutation c.183A>T; homozygous CDKN2A deletion; c.1_471del471, homozygous KRAS mutation c.183A>T; homozygous STK11mutation, c.109C>T).

Measurement of heme synthesis and uptake in cells was carried out exactly as described previously (19, 26). Experiments were conducted in triplicates and were normalized with total cellular proteins.

LSL-Kras^G12D; Lkb1^flox/flox; LSL-Luciferase (KL_Luc) mice were generated, bred, and confirmed as described (27, 28). NOD/SCID mice (IMSR cat. no. CRL:394, RRID: IMSR_CRL:394) were purchased from Charles River Laboratories. In accordance with NIH guidelines, mice were bred and cared for in a University of Texas at Dallas (Richardson, TX)–specific pathogen-free animal facility. All animal procedures were performed under protocols approved by Institutional Animal Care and Use Committee at the University of Texas at Dallas. Animals were regularly examined for any signs of stress and euthanized according to preset criteria.

Lung tumors were induced by intranasal inhalation of Ad5-CMV-Cre virus into 6- to 11-week-old LSL-Kras^G12D; Lkb1^flox/flox; LSL-Luciferase (KL_Luc) mice at 3 × 10⁷ PFU per mouse, as described (28). Mice were anesthetized using intraperitoneal administration of Ketamine-Xylazine cocktail (ketamine, 100 mg/kg; xylazine, 12 mg/kg). Tumor growth was monitored using bioluminescence imaging (BLI) and histology. For studies on tumor progression, mice were sacrificed at 4, 6, 8, and 10 weeks after infection. For treatment experiments, mice were randomized into two groups (n ≥ 6 per group) that received saline (for control) or HeSP2 (25 mg/kg, i.v., every three days). Treatment was started postinfection when BLI signal from lung tumors reached above 5 × 10⁶ photons/sec, after 6 to 7 weeks postinfection. Body masses were recorded once every week. Mice were sacrificed after three weeks of treatment.

To generate subcutaneous xenograft models, 2 × 10⁶ HCC15 cells containing 50% Matrigel in serum-free medium were injected subcutaneously into the left flank region of 4- to 6-week-old female NOD/SCID mice (n  = 6 per group). Mice were randomized into treatment groups that received saline (for control) and HeSP2 (i.v. 25 mg/kg every three days), respectively. Body masses were recorded once every week. Treatments were started only after tumors were palpable to ensure successful implantation. Tumor growth was monitored with caliper measurements.

Oxygen consumption was measured, as described previously (26). Oxygen consumption rates (OCR) and ATP levels from freshly isolated tumors were measured as described (19). Both OCRs and ATP levels were normalized with protein amounts. For treatment with the inhibitor of heme synthesis, succinyl acetone, and heme-add back, cells were cultured in medium containing heme-depleted serum. Heme-depleted serum was prepared as described (29). Cells were grown in media containing 0.5 mmol/L succinyl acetone, then supplemented with 0, 5, 10, and 20 μmol/L heme, respectively, for six days.

Mice were sacrificed, and lungs were excised and prepared for histology. Lung tissues were fixed in 4% formalin. Paraffin embedding was performed at Histology core at University of Texas Southwestern Medical Center (Dallas, TX). The paraffin blocks were sectioned into 5 μm sections and stained with hematoxylin and eosin (H&E). Sections were scanned at 40× resolution with an Olympus VS120 slide scanner. Tumor lesion areas were measured using cellSens software from Olympus.

IHC was carried out exactly as described previously (30). Primary antibodies were diluted in 1× TBS/1% BSA/5% goat serum (16210-072, Gibco). The dilutions were 1:100 DeltaNp63 (Abcam cat. #ab172731, RRID:AB_2891015), 1:100 TTF-1 (Agilent cat. #M3575, RRID:AB_2877699), 1:200 Ki-67 (Cell Signaling Technology cat. #12202, RRID:AB_2620142), 1:200 cleaved caspase-3 (Cell Signaling Technology cat. #9661, RRID:AB_2341188), 1:100 Alas1 (Novus cat. #NB100-56415, RRID:AB_837550), 1:200 SLC48A1 (Novus cat. #NBP1-91563, RRID:AB_11009875), 1:50 HCCS (Abcam cat. #ab224321, RRID:AB_2891018), 1:200 cytochrome c (Novus cat. #NBP2-21569, RRID:AB_2891022), 1:100 COX5A (Abcam cat. #ab110262, RRID:AB_10861723), 1:500 COX5B (Abcam cat. #ab180136, RRID:AB_2891023), 1:500 COX7B (Abcam cat. #ab137094, RRID:AB_2891024), 1:1,000 ATP5A (Abcam cat. #ab14748, RRID:AB_301447), 1:200 SLC2A1 (Abcam cat. #ab40084, RRID:AB_2190927), 1:500 HK2 (Thermo Fisher Scientific cat. #PA5-29326, RRID:AB_2546802), 1:200 SLC1A5 (Abcam cat. #ab58690, RRID:AB_943478), 1:200 GLS (Abcam cat. #ab156876, RRID:AB_2721038), 1:35 VEGFA (Abcam cat. #9570, RRID: AB_308723), 1:50 VEGFR1 (Abcam cat. #ab2350, RRID:AB_303000), 1:200 HIF1α (Novus Biologicals cat. #NB100-449, RRID: AB_10001045), 1:200 CA9 (Novus biologicals cat. #NB100-417, RRID:AB_ 10003398), 1:200 CD34 (Abcam cat. #ab185732, RRID:AB_2811308), and CD31 (PECAM1; Cell Signaling Technology cat. #77699, RRID:AB_2722705).

Sections were incubated with primary antibodies overnight at 4°C and then with horseradish peroxidase–conjugated goat anti-rabbit IgG (Thermo Fisher Scientific cat. # 31460, RRID: AB_228341) or horseradish peroxidase–conjugated goat anti-mouse IgG (Thermo Fisher Scientific cat. #31430, RRID: AB_228307) at room temperature for 45 minutes. Sections were stained with tyramide signal amplification-conjugated fluorophores in 1× Plus Amplification Diluent (Akoya Biosciences cat. #FP1498).

IHC sections were scanned at a 10× resolution with an Olympus VS120 slide scanner and quantified using cellSens software from Olympus. For each lung tissue, protein expression in the tumor region is normalized to expression in the nontumor region. Microvessel density was calculated based on PECAM1 and CD34-positive objects as described previously (31). Experiments with all antibodies were repeated at least twice. Intensity or object counts from 10 ROIs were averaged for each tumor tissue. At least three sets of lung tissues per treatment were quantified, and the averages were calculated and plotted.

Data from different treatment groups of cells, mice, and tissues were compared, and statistical analysis was performed with a Welch two-sample t test.

The data generated in this study are available within the article and its supplementary data files.

Our previous experimental data have shown that the levels of heme synthesis and uptake, along with the levels of heme uptake protein and heme synthetic enzyme ALAS1, are elevated in NSCLC cell lines relative to nontumorigenic lung cell lines, leading to increased mitochondrial heme levels (19). Here, to monitor changes during the process of lung tumor development, we took advantage of the KL_Luc (LSL-Kras^G12D; Lkb1^flox/flox; LSL-Luciferase) mouse model (32). Previous studies have shown that these mice develop a full spectrum of NSCLC tumor types, including SCC, ADC, and mixed adenosquamous tumors (21–23). Consistent with previous studies, we detected small lung tumors starting at four weeks after tumor initiation by infection with the Ad-Cre virus (see Fig. 1A). Tumor burden/size progressively increased as lung tumorigenesis advanced to 10 weeks after tumor initiation (see Fig. 1A and E). We found that ADC appeared before SCC in KL_Luc mice, which is consistent with previous observations (21, 23). As shown in Fig. 1B, F, and G, ADC, revealed by the NKX2-1 (TTF-1) marker, was clearly detected starting at four weeks after tumor initiation, while SCC, revealed by the ΔNp63 (p40) marker, was detected at eight weeks after tumor initiation.

Next, we monitored the levels of the rate-limiting heme synthetic enzyme ALAS1 and the heme uptake protein SLC48A1. Increased levels of these proteins have been shown to strongly increase mitochondrial heme levels and OCRs, promoting ATP generation and tumorigenic functions in tumor cells (19). The levels of both ALAS1 (Fig. 1C and H) and SLC48A1 (Fig. 1D and I) were strongly and progressively increased as lung tumorigenesis advanced from 4 to 10 weeks after tumor initiation. These results are consistent with previous observation showing that elevated heme synthesis and uptake underpin tumorigenic functions in NSCLC cells (19). Further, these results indicate that heme synthesis and uptake are progressively increased to support tumorigenic functions in lung cells, as tumorigenesis advances.

Heme serves as a prosthetic or cofactor for multiple subunits of mitochondrial OXPHOS complexes and coordinates the generation of OXPHOS complexes. Elevated heme synthesis and uptake should promote OXPHOS complex synthesis. We therefore detected the levels of OXPHOS complex subunits at various time points of lung tumorigenesis. We found that the levels of OXPHOS complex subunits, like heme synthetic enzyme and transporter, progressively increased as lung tumorigenesis advanced from 4 to 10 weeks after tumor initiation. These include cytochrome c (CYCS; Fig. 2A and F), cytochrome c oxidase subunit COX7B (Fig. 2B and G), and ATP synthase subunit alpha ATP5F1A (Fig. 2C and H). Likewise, the levels of cytochrome c-type heme lyase HCCS also increased strongly as lung tumorigenesis advanced (Supplementary Fig. S1A and S1D). We have also confirmed the specificity of the antibodies by showing that ATP5F1A, COX7B, and CYCS all colocalized with the mitochondrial localization marker TOMM20 (see Supplementary Fig. S1G). Furthermore, Supplementary Fig. S1H shows that NSCLC cells supplemented with higher levels of exogenous heme exhibited significantly higher OCRs. This result indicates that increased heme availability is linked to elevated OXPHOS activity.

Next, we examined the levels of proteins involved in the utilization of two key fuels promoting tumorigenic functions, glucose, and glutamine. We found that the levels of the glucose transporter SLC2A1 (Fig. 2D and I) and the key glycolysis enzyme hexokinase HK2 (Fig. 2E and J) progressively and strongly increased as lung tumorigenesis advanced from 4 to 10 weeks after tumor initiation. Note that the increase of SLC2A1 was somewhat delayed compared with HK2, likely due to its low expression associated with ADC (see below). The levels of the glutamine transporter SLC1A5 (Supplementary Fig. S1B and S1E) and the key enzyme of glutamine consumption, glutaminase GLS1 (Supplementary Fig. S1C and S1F), also strongly increased in lung tumors compared with normal tissues. These results show that enzymes of key bioenergetic pathways, including OXPHOS, glucose, and glutamine consumptions, along with key heme synthetic enzyme and transporter, are upregulated progressively as lung tumorigenesis advances, to support increased needs for cellular energy.

As tumorigenesis advances, it is expected that angiogenesis will be induced. Indeed, we found that the levels of the angiogenic factor VEGFA (Fig. 3A and F) and its receptor VEGFR1 (Fig. 3B and G) increased progressively as lung tumorigenesis advanced from 4 to 10 weeks after tumor initiation. Likewise, the levels of the vascular marker CD34 (Fig. 3C and H), as well as microvessel density (Fig. 3I), progressively increased along with lung tumorigenesis. Further, we examined tumor hypoxia by detecting the levels of hypoxia-inducible factor HIF1A and the long-term hypoxia marker carbonic anhydrase 9 (CA9). The levels of HIF1A increased in all 4- to 10-week tumors (Fig. 3D and J), and the levels of CA9 increased progressively (Fig. 3E and K), indicating tumor hypoxia progressively intensified in 4- to 10-week tumors.

ADC and SCC have previously been shown to exhibit different metabolic properties (27, 33). One particularly notable example is the selectively high expression of GLUT1/SLC2A1 in SCC. Indeed, we detected high levels of GLUT1/SLC2A1 expression associated with SCC, whereas low levels of SLC2A1 expression were associated with ADC in the KL_Luc tumors (Fig. 4A and F). Notably, elevated levels of the glutamine transporter SLC1A5 were associated with both ADC and SCC (Fig. 4B and G). Next, we examined the levels of the rate-limiting heme synthetic enzyme ALAS1 and heme transporter SLC48A1 in ADC and SCC. We found that high levels of ALAS1 (Fig. 4C and H) and SLC48A1 (Fig. 4D and I) were associated with both ADC and SCC. Furthermore, elevated levels of OXPHOS complex subunits COX5A (Fig. 4E and J), COX5B (Supplementary Fig. S2A and S2D), COX7B (Supplementary Fig. S2B and S2E), and CYCS (Supplementary Fig. S2C and S2F) were associated with both ADC and SCC. These results suggest that OXPHOS activities are elevated in both ADC and SCC to support tumorigenic functions of these cancer cells.

To further evaluate and compare heme metabolism and OXPHOS activities in ADC versus SCC cells, we examined four representative SCC cell lines: HCC15, NCI-H226, NCI-H520, and SW 900. In comparison, we examined three previously characterized ADC cell lines—H1299, A549, and H460—that have been shown to exhibit elevated heme synthesis, uptake, and OXPHOS activities (19). We found that the levels of heme synthesis (Supplementary Fig. S3A), heme uptake (Supplementary Fig. S3B), and OXPHOS activity (Supplementary Fig. S3C) in SCC cell lines were generally comparable with the levels in ADC cell lines, although the levels are heterogeneous in both SCC and ADC cell lines. Together, these results indicate that heme synthesis, uptake, and OXPHOS are intensified in both SDC and SCC cells to support their tumorigenic functions.

To test the possibility of targeting heme availability for the suppression of lung tumorigenesis and progression, we took advantage of a heme-sequestering protein 2 (HeSP2, previously named HSP2), which has been shown to inhibit heme uptake, OXPHOS activity, ATP generation, and tumorigenic functions in NSCLC cell lines (19). We carried out the treatment of endogenously developed lung tumors in KL_Luc mice with HeSP2. We found that HeSP2 strongly suppressed the growth and progression of endogenously developed lung tumors (Fig. 5A). Using markers for ADC (NKX2-1) and SCC (ΔNp63), we showed that the growth and progression of both ADC and SCC were suppressed by HeSP2 (Fig. 5B). Quantification of tumor lesion areas showed that the effects of HSP2 on both ADC and SCC progression were strong and statistically significant (Fig. 5C).

Our previous studies have shown that HeSP2 strongly suppresses the growth of ADC tumor xenografts, such as H1299 and A549 tumors (19). Here, to further confirm the effect of HeSP2 on suppressing SCC tumors, we examined the effect of HeSP2 on the growth of HCC15 SCC tumor xenografts in NOD/SCID mice. Figure 6A–C shows that HeSP2 strongly suppressed the growth of HCC15 tumors. The treatments of HeSP2 did not cause significant changes in body masses of mice (Fig. 6D). The measurements of OCRs (Fig. 6E) and ATP levels (Fig. 6F) in isolated tumor cells from mice showed that HeSP2 substantially reduced OCR and ATP generation in tumors.

Additionally, we assessed the effect of HeSP2 treatment on OXPHOS in endogenously developed lung tumors in KL_Luc mice by detecting the levels of OXPHOS complex subunits. We found that HeSP2 reduced the levels of OXPHOS complex subunits, including COX5A (Fig. 7A and G), COX5B (Supplementary Fig. S4A and S4E), COX7B (Supplementary Fig. S4B and S4F), ATP5F1A (Supplementary Fig. S4C and S4G), and CYCS (Supplementary Fig. S4D and S4H) in KL_Luc tumors. These results indicate that HeSP2 treatment should reduce OXPHOS activity and are consistent with the effect of HeSP2 on OCR in SCC tumor xenografts.

To further ascertain the molecular mechanisms underlying the suppression of tumor growth, we examined the effects of HeSP2 on markers of tumor angiogenesis and vasculature in KL_Luc mice. HeSP2 significantly reduced the levels of angiogenic factor VEGFA (Fig. 7B and H) and receptor VEGFR1 (Fig. 7C and I), as well as the endothelial cell markers PECAM1 (Fig. 7D and J) and CD34 (Fig. 7E and L). Consequently, HeSP2 strongly decreased microvessel density in treated tumors (Fig. 7K and M) calculated based on both IHC images of PECAM1 and CD34. HeSP2 also diminished tumor hypoxia, as shown by reduced levels of hypoxia markers HIF1A (Supplementary Fig. S5A and S5D) and CA9 (Supplementary Fig. S5B and S5E). These results show that HeSP2 treatment normalized tumor vasculature and alleviated tumor vasculature in KL_Luc mice.

Furthermore, we detected the levels of proliferation marker MKI67 and apoptosis marker cleaved caspase-3 (CASP3). Figure 7F and N shows that HeSP2 strongly reduced the levels of Ki67. HeSP2 increased the levels of cleaved CASP3 in treated tumors (Supplementary Fig. S5C and S5F). These results show that decreased proliferation and increased apoptosis contribute to tumor suppression by HeSP2. Together, these results show that HeSP2 effectively decreased OXPHOS activity, normalized tumor vasculature, alleviated tumor hypoxia, inhibited tumor cell proliferation, and induced apoptosis, thereby leading to the suppression of ADC and SCC tumor growth and progression.

Despite improved early detection and the use of targeted therapies and immunotherapies, the lung cancer five-year survival rate remains 10% to 20% in most countries. Targeted therapies are limited by the lack of targetable genetic mutations in many patients and the development of drug resistance (34, 35). Recently, immunotherapies, including nivolumab, pembrolizumab, and atezolizumab, have been used for treating NSCLC (36). Immunotherapies have extended the median overall survival by several months in both the first-line and second-line settings in most clinical trials (35, 37, 38). Furthermore, only a fraction of patients can benefit from immunotherapy (39). For example, patients with LKB1 mutations often do not respond to immune therapy (24, 40). Clearly, identifying new molecular features of NSCLC that enable novel therapeutic strategies is of great biological and clinical importance.

Heme is a central metabolic and signaling molecule that regulates diverse processes, including transcription and cell signaling (41–43). Heme also serves as a prosthetic group in proteins and enzymes involved in oxygen utilization and metabolism (16). One crucial function of mitochondria is to carry out mitochondrial respiration/OXPHOS for ATP generation. Heme function and mitochondrial respiration are tightly linked. Multiple subunits in OXPHOS complexes II–IV contain heme. Heme also coordinates the expression and assembly of OXPHOS complexes (17). Clearly, heme possesses unique signaling and structural properties that enable it to coordinate elevated OXPHOS in cancer cells (9). Here, using the KL_Luc mouse model harboring a conditional activating mutation (G12D) at the endogenous Kras locus and a conditional Lkb1 knockout, we are able to monitor the changes in the levels of enzymes and transporters involved in heme synthesis and uptake, OXPHOS, as well as those involved in the uptake and utilization of glucose and glutamine (Figs. 1 and 2; Supplementary Fig. S1). Our data showed that the levels of transporters and enzymes involved in heme acquisition and ATP generation via oxidative metabolism of glucose and glutamine all progressively increased along with lung tumorigenesis. Previously, work in the authors' lab has shown that elevated heme flux and function underlie enhanced OXPHOS and tumorigenicity of NSCLC cells (19). The data shown here indicate that elevated levels of heme and OXPHOS complex enzymes correlate with the stages of lung tumor development. Evidently, as lung tumorigenesis advances, more heme is synthesized and taken up to support OXPHOS, which leads to increased ATP generation, thereby further promoting tumor progression. Additionally, our data showed that as with intensified heme and OXPHOS function, tumor angiogenesis and hypoxia increased with lung tumorigenesis (Fig. 3).

Notably, consistent with previous studies (27), IHC showed that elevated levels of the glucose transporter GLUT1/SLC2A1 are preferentially associated with only SCC (Fig. 4A and F). However, elevated levels of heme and OXPHOS complex enzymes are associated with both ADC and SCC (Fig. 4C–E and H–J; Supplementary Fig. S2), indicating that elevated heme function and OXPHOS are important for the development of both ADC and SCC. This is further supported by data from ADC and SCC cell lines. Measurements in cell lines showed that the levels of heme synthesis, uptake, and oxygen consumption fall in the comparable range in both ADC and SCC cell lines (Supplementary Fig. S3). These results together strongly support the conclusion that elevated heme and OXPHOS function are a common feature of ADC and SCC.

Importantly, we showed that heme sequestration with HeSP2 is effective at suppressing the progression of both ADC and SCC in KL_Luc mice (Fig. 5). This is in complete agreement with previous studies showing that HeSP2 (HSP2) effectively suppresses the growth of NSCLC H1299 and A549 tumor xenografts in mice (19, 44). Additionally, we demonstrated that HeSP2 effectively suppresses HCC15 SCC tumor xenografts in mice (Fig. 6). Together, these results show that HeSP2 strongly suppresses the growth and progression of endogenously developed lung ADC and SCC tumors, as well as ADC and SCC tumor xenografts. Thus, targeting heme and OXPHOS via heme sequestration is a novel, effective strategy for the suppression of both ADC and SCC tumors.

To sum up, our data presented in this report provides the following new insights about NSCLC: (i) The levels of enzymes and proteins relating to heme synthesis and uptake increase progressively as lung tumorigenesis advances. (ii) The levels of enzymes relating to OXPHOS, glucose metabolism, and glutamine metabolism increase progressively during lung tumorigenesis. (iii) Angiogenesis and tumor hypoxia increase progressively during lung tumorigenesis. (iv) The increase in heme- and OXPHOS-related proteins and enzymes accompanies both ADC and SCC, and heme sequestration effectively suppresses the growth and progression of both ADC and SCC tumors. Notably, our results indicate that HeSP2 alleviates tumor hypoxia and normalizes vasculature (Fig. 7B–E and H–M, Supplementary Fig. S5A and S5B, S5D and S5E). Tumor hypoxia strongly affects cancer progression. It promotes several processes that are crucial for migration, invasion, metastasis, immune surveillance, and drug resistance (45–47). Tumor hypoxia generally indicates poor prognosis in human cancer. Even early-stage NSCLC exhibits substantial tumor hypoxia (48). Thus, alleviating tumor hypoxia presents a high potential to improve the outcome of NSCLC treatment (49). Given the effectiveness of HeSP2 in alleviating tumor hypoxia, it is highly likely that HeSP2 or agents with similar properties can be used in combination with chemotherapy, radiotherapy, and immunotherapy, to dramatically improve the treatment of lung ADC and SCC. The application of the strategy of heme sequestration can fundamentally advance cancer therapy.

L. Zhang reports a patent for US 2020/0361999 A1 pending; and L. Zhang is the president of HemePro Therapeutics, LLC. UT Dallas is actively managing the potential conflict of interest. No disclosures were reported by the other authors.

S. Dey: Conceptualization, data curation, formal analysis, validation, investigation, visualization, methodology, writing–original draft, writing–review and editing. A. Ashrafi: Conceptualization, data curation, formal analysis, validation, investigation, methodology, writing–original draft, writing–review and editing. C. Vidal: Data curation, formal analysis, investigation, methodology, writing–review and editing. N. Jain: Data curation, investigation, methodology, writing–review and editing. S.P. Kalainayakan: Conceptualization, data curation, validation, investigation, methodology, writing–review and editing. P. Ghosh: Conceptualization, data curation, validation, investigation, methodology, writing–review and editing. P.S. Alemi: Data curation, investigation, visualization, writing–review and editing. N. Salamat: Data curation, investigation, methodology, writing–review and editing. P.C. Konduri: Data curation, investigation, methodology, writing–review and editing. J.-w. Kim: Conceptualization, supervision, methodology, writing–review and editing. L. Zhang: Conceptualization, resources, formal analysis, supervision, funding acquisition, validation, methodology, writing–original draft, project administration, writing–review and editing.

This work was supported by Cancer Prevention and Research Institute of Texas grant RP200021.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.