Lung cancer is one of the leading causes of cancer-related deaths worldwide with a 5-year survival rate of less than 18%. Current treatment modalities include surgery, chemotherapy, radiation therapy, targeted therapy, and immunotherapy. Despite advances in therapeutic options, resistance to therapy remains a major obstacle to the effectiveness of long-term treatment, eventually leading to therapeutic insensitivity, poor progression-free survival, and disease relapse. Resistance mechanisms stem from genetic mutations and/or epigenetic changes, unregulated drug efflux, tumor hypoxia, alterations in the tumor microenvironment, and several other cellular and molecular alterations. A better understanding of these mechanisms is crucial for targeting factors involved in therapeutic resistance, establishing novel antitumor targets, and developing therapeutic strategies to resensitize cancer cells towards treatment. In this review, we summarize diverse mechanisms driving resistance to chemotherapy, radiotherapy, targeted therapy, and immunotherapy, and promising strategies to help overcome this therapeutic resistance.

Heme is an essential nutritional, metabolic, and signaling molecule in living organisms. Pathogenic microbes extract heme from hosts to obtain metallonutrient, while heme fuels mitochondrial respiration and ATP generation in lung tumor cells. Here, we generated small heme-sequestering proteins (HeSPs) based on bacterial hemophores. These HeSPs contain neutral mutations in the heme-binding pocket and hybrid sequences from hemophores of different bacteria. We showed that HeSPs bind to heme and effectively extracted heme from hemoglobin. They strongly inhibited heme uptake and cell proliferation and induced apoptosis in non–small cell lung cancer (NSCLC) cells, while their effects on nontumorigenic cell lines representing normal lung cells were not significant. HeSPs strongly suppressed the growth of human NSCLC tumor xenografts in mice. HeSPs decreased oxygen consumption rates and ATP levels in tumor cells isolated from treated mice, while they did not affect liver and blood cell functions. IHC, along with data from Western blotting and functional assays, revealed that HeSPs reduced the levels of key proteins involved in heme uptake, as well as the consumption of major fuels for tumor cells, glucose, and glutamine. Further, we found that HeSPs reduced the levels of angiogenic and vascular markers, as well as vessel density in tumor tissues. Together, these results demonstrate that HeSPs act via multiple mechanisms, including the inhibition of oxidative phosphorylation, to suppress tumor growth and progression. Evidently, heme sequestration can be a powerful strategy for suppressing lung tumors and likely drug-resistant tumors that rely on oxidative phosphorylation for survival.

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. KRAS mutations and LKB1 inactivation commonly occur in NSCLC and are associated with poor prognosis. Several studies enunciate the role of the immune system in lung cancer progression through cancer cells' ability to escape immune surveillance. Therefore, understanding NSCLC progression requires the examination of the molecular mechanisms behind this process. The importance of oxidative phosphorylation (OXPHOS) and mitochondrial metabolism in lung cancer has been continuously emphasized; OXPHOS and mitochondrial biogenesis markers predict low survival in NSCLC patients. Additionally, heme is an essential molecule in the OXPHOS pathway, and previous studies have shown that heme sequestration using heme sequestering protein 2 (HeSP2) arrests tumor progression. HeSP2 can potentially normalize the tumor immune microenvironment (TIME) via multiple mechanisms including the normalization of vessels which may promote immune cell infiltration, the reduction of hypoxia which may promote antitumor immune function, and the reduction of heme levels which potentially decreases the production of immunosuppressive enzymes requiring heme as a cofactor. In this study, control and HeSP2-treated tumors generated from a genetically engineered mouse model LSL-KrasG12D LKB1-/- (LSL-KRASG12D; LKBloxP/loxP; LSL-Luciferase (KLLuc) infected with AdenoCre virus and sacrificed at 10 months were used for immunohistochemical analysis. We examined immune markers under HeSP2 treatment to determine the immune system response to heme targeting and its effects on tumor progression. Results demonstrated that HeSP2 can modulate the tumor immune microenvironment increasing the levels of immune markers, which suggests that heme targeting can potentiate immunotherapy efficacy in lung cancer treatment. Citation Format: Maria del Carmen Chacon Castro, Eranda Berisha, Narges Salamat, Li Zhang. Heme sequestration modulates the tumor immune microenvironment in KRAS mutation/LKB1 inactivation non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1653.

Breast cancer is the second leading cause of cancer-related deaths and comprises of approximately 30% of all cancer cases affecting women in the United States. There are three subtypes of breast cancer based on receptor profiles, with the third subtype being triple-negative breast cancer. TNBC accounts for about 10-15% of all breast cancers. The TNBC subtype lacks estrogen, progesterone, and HER2 receptors; it is associated with BRCA1 and BRCA2 mutations. This type of cancer has poor prognosis, tends to be more aggressive, and has limited treatment options compared to the other two subtypes. Heme is an essential molecule in the oxidative phosphorylation pathway and in the generation of reactive oxygen species (ROS). ROS generation causes oxidative stress and induces DNA damage in breast cancer. Therefore, targeting its activity potentially suppresses TNBC tumors. Heme-sequestering protein 2 (HeSP2) binds and sequesters heme. It has been shown that HeSP2 treatment of MDA-MB-231 cells inhibited cell proliferation and colony formation. Moreover, HeSP2 suppresses tumor growth in subcutaneously implanted TNBC MDA-MB-231 xenografts in NOD/SCID mice. Furthermore, in vitro studies using murine TNBC cell models 4T1-fluc Neo and EMT6-fluc Puro cells have also shown that cell proliferation and colony formation were inhibited after HeSP2 treatments of increasing concentrations. Thus, these results encourage the potential use of HeSP2 for breast tumor suppression. To further explore the effects of heme targeting and sequestration, we will determine heme flux in TNBC cell models by measuring the levels of heme uptake, export, synthesis, and degradation. Moreover, we will examine mitochondrial markers for OXPHOS complexes in HeSP2 treated TNBC xenografts using immunohistochemical analysis. Further studies are still underway to understand the role of heme in breast cancer, with preliminary results indicating that heme targeting, and sequestration effectively suppress triple-negative breast cancer and its progression. Citation Format: Eranda Berisha, Maria del Carmen Chacon Castro, Adnin Ashrafi, Narges Salamat, Parinaz Sadat Alemi, Li Zhang. Heme targeting and its mechanistic role in triple-negative breast cancer suppression. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4942.

Lung cancer is the leading cause of cancer-related death in the United States, with a 5-year survival rate of less than 25%. While most lung cancer patients receive radiation therapy, radioresistance severely impacts treatment outcomes. Thus, development of therapeutics to potentiate durable response to radiation therapy may be key to improving treatment outcomes. Research in our lab has previously implicated heme, a central biosynthetic molecule with important functions in diverse molecular and cellular processes, in lung cancer development and progression. To target heme, we designed heme-sequestering protein 2 (HeSP2) which displays potent anti-tumor activity in mouse tumor models. By decreasing tumor metabolic demand through heme sequestration, HeSP2 can significantly alleviate tumor hypoxia, a dominant radioresistance mechanism. Tumor hypoxia correlates with poor clinical outcomes by acting via multiple mechanisms such as inhibiting radiation-induced DNA damage and inducing the HIF-1 pathway that leads to antioxidant generation. Here, our preliminary data show that HeSP2 potentiates the antitumor efficacy of ionizing radiation. To see the effect of HeSP2 in combination with radiotherapy, in summary we subcutaneously implanted A549-luc, NSCLC cell lines with mutation in Kras and KLB1 in SCID mice. After 1-2 weeks mice were treated with saline (Control), HeSP2(25 mg/kg, i.v, twice a week), radiation (5 Gray, once per week at week 2 and 3), and HeSP2 combined with radiation (25 mg/kg, i.v, twice a week plus 5 Gray, once per week at week 2 and 3. Tumor tissues were harvested, processed and paraffin embedded for immunohistochemistry (IHC). Preliminary results indicate HeSP2 in combination with radiation reduce the levels of multiple angiogenic markers and vascular markers as well as microvessel density. These results indicate that heme sequestration in combination with radiation can be an effective strategy for suppressing lung tumors. Citation Format: Narges Salamat, Tianyuan Wang, Eranda Berisha, Maria Del Carmen Chacon Castro, Adnin Ashrafi, Debabrata Saha, Ralph P. Mason, Li Liu, Li Zhang. Evaluation of heme inhibitory therapy in combination with radiation of lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4979.