Genomic Analysis of Waterpipe Smoke-Induced Lung Tumor Autophagy and Plasticity.

The role of autophagy in lung cancer cells exposed to waterpipe smoke (WPS) is not known. Because of the important role of autophagy in tumor resistance and progression, we investigated its relationship with WP smoking. We first showed that WPS activated autophagy, as reflected by LC3 processing, in lung cancer cell lines. The autophagy response in smokers with lung adenocarcinoma, as compared to non-smokers with lung adenocarcinoma, was investigated further using the TCGA lung adenocarcinoma bulk RNA-seq dataset with the available patient metadata on smoking status. The results, based on a machine learning classification model using Random Forest, indicate that smokers have an increase in autophagy-activating genes. Comparative analysis of lung adenocarcinoma molecular signatures in affected patients with a long-term active exposure to smoke compared to non-smoker patients indicates a higher tumor mutational burden, a higher CD8+ T-cell level and a lower dysfunction level in smokers. While the expression of the checkpoint genes tested-PD-1, PD-L1, PD-L2 and CTLA-4-remains unchanged between smokers and non-smokers, B7-1, B7-2, IDO1 and CD200R1 were found to be higher in non-smokers than smokers. Because multiple factors in the tumor microenvironment dictate the success of immunotherapy, in addition to the expression of immune checkpoint genes, our analysis explains why patients who are smokers with lung adenocarcinoma respond better to immunotherapy, even though there are no relative differences in immune checkpoint genes in the two groups. Therefore, targeting autophagy in lung adenocarcinoma patients, in combination with checkpoint inhibitor-targeted therapies or chemotherapy, should be considered in smoker patients with lung adenocarcinoma.

Hypoxia increases mutational load of breast cancer cells through frameshift mutations.

Tumor hypoxia-induced downregulation of DNA repair pathways and enhanced replication stress are potential sources of genomic instability. A plethora of genetic changes such as point mutations, large deletions and duplications, microsatellite and chromosomal instability have been discovered in cells under hypoxic stress. However, the influence of hypoxia on the mutational burden of the genome is not fully understood. Here, we attempted to elucidate the DNA damage response and repair patterns under different types of hypoxic stress. In addition, we examined the pattern of mutations exclusively induced under chronic and intermittent hypoxic conditions in two breast cancer cell lines using exome sequencing. Our data indicated that hypoxic stress resulted in transcriptional downregulation of DNA repair genes which can impact the DNA repair induced during anoxic as well as reoxygenated conditions. In addition, our findings demonstrate that hypoxic conditions increased the mutational burden, characterized by an increase in frameshift insertions and deletions. The somatic mutations were random and non-recurring, as huge variations within the technical duplicates were recognized. Hypoxia also resulted in an increase in the formation of potential neoantigens in both cell lines. More importantly, these data indicate that hypoxic stress mitigates DNA damage repair pathways and causes an increase in the mutational burden of tumor cells, thereby interfering with hypoxic cancer cell immunogenicity.

Role of Hypoxic Stress in Regulating Tumor Immunogenicity, Resistance and Plasticity.

Hypoxia, or gradients of hypoxia, occurs in most growing solid tumors and may result in pleotropic effects contributing significantly to tumor aggressiveness and therapy resistance. Indeed, the generated hypoxic stress has a strong impact on tumor cell biology. For example, it may contribute to increasing tumor heterogeneity, help cells gain new functional properties and/or select certain cell subpopulations, facilitating the emergence of therapeutic resistant cancer clones, including cancer stem cells coincident with tumor relapse and progression. It controls tumor immunogenicity, immune plasticity, and promotes the differentiation and expansion of immune-suppressive stromal cells. In this context, manipulation of the hypoxic microenvironment may be considered for preventing or reverting the malignant transformation. Here, we review the current knowledge on how hypoxic stress in tumor microenvironments impacts on tumor heterogeneity, plasticity and resistance, with a special interest in the impact on immune resistance and tumor immunogenicity.