Investigating Molecular Mechanisms That Promote Survival or Death in ECM-detached Cancer Cells

In the United States, according to the American Cancer Society, 608,570 cancer deaths are projected to occur among men and women in 2021. Astonishingly, toward the end of the 20th century, the cancer death rate began continuously falling from its height in 1991 through 2018, for a total decline of 31%. This steady decline in cancer death is indubitably reflective of a multi-faceted effort in understanding the basic biology of cancer and developing effective therapeutic strategies. Therefore, to persist down the path of decreasing cancer death rates, investigations into one of the most prominent causes of cancer-related deaths must continue. By and large, metastasis, which is the spread of cancer cells from their original organ site, entrance into circulation, and re-establishment in distant, secondary organs, is responsible for 90% of fatalities caused by cancer. When considering obstacles to cancer cell viability, detaching from their extracellular matrix (ECM) and evading cell the associated death mechanisms are critical objectives cancer cells must achieve to successfully metastasis. Detachment from the ECM is known to induce a caspase-dependent cell death program, termed anoikis, that cancer cells can circumvent by multiple means. Nonetheless, overcoming caspase-dependent cell death alone is not sufficient to promote survival during metastasis, but our laboratory’s work has discovered that cancer cells must also address metabolic deficiencies that can lead to caspase-independent cell death programs. Throughout my graduate studies, I have had the honor of investigating two, separate projects for my thesis work that entails the following: 1) a survival mechanism to address detachment-induced metabolic challenges and 2) a caspase-independent cell death pathway driven by oxidative stress in ECM-detached cancer cells.

To begin, our laboratory’s research has demonstrated that a reduction of glucose uptake, accumulation of reactive oxygen species, and a subsequent decline in ATP production are each metabolic defects that occur upon ECM-detachment. Intriguingly, when ECM-detached cells were engineered to express the Erbb2 oncogene, a rectification of each metabolic deficiency was observed. Additionally, our previous work discovered that the serine/threonine serum and glucocorticoid kinase 1 (SGK1) is sufficient and required to promote ATP production in ECM-detached cancer cells and to facilitate anchorage-independent growth downstream of the Ras oncogene. However, what hadn’t been determined yet was the molecular mechanism that SGK1 utilizes to promote these functions and if disparate oncogenic insults relied on this mechanism. Therefore, utilizing four cancer cell lines with dissimilar oncogenes, we performed luciferase-based assays and soft agar experiments and found that SGK1 is not only sufficient and required to promote ATP generation in detachment and support anchorage-independent growth, but also that SGK1 preforms these functions with greater outcomes than AKT in detachment. Furthermore, through qRT-PCR and glucose uptake assays, we found that in ECM-detachment SGK1 upregulates the transcription of the glucose transporter GLUT1 and facilitates increased glucose flux into detached cells. Once glucose in inside the cells, we surprisingly found that SGK1 was not promoting ATP production through oxidative phosphorylation. Using glucose flux analysis, we discovered that SGK1 promotes ATP production and anchorage-independent growth via glucose metabolism in both glycolytic and pentose phosphate pathways (PPP). SGK1-mediated ATP production was found to be dependent upon the non-oxidate arm of the PPP due to the flux of glyceraldehyde-3-phosphate (G3P) into glycolysis. Taken together, our data published in Cell Reports reveals a novel molecular mechanism that serves as nexus between oncogenic signaling and metabolic reprograming in ECM-detached cells for their survival in an anchorage-independent conditions. With further studies, SGK1 could possibly present as a therapeutic target in various cancer types that have detached from their ECM.

Lastly, within the second project, our recent studies using in vitro and in vivo models have identified receptor interacting protein kinase 1 (RIPK1) as a critical mediator of ECM-detachment induced caspase-independent cell death via its capacity to promote mitophagy, which is defined as the degradation of mitochondria, and subsequent accumulation of mitochondrial reactive oxygen species (ROS) in cancer cells. Thus, our finding led us to the prominent question for my current project: How does RIPK1-mediated mitophagy lead to caspase-independent cell death in ECM-detached cancer cells. During mitophagy, mitochondria are encapsulated into vesicles known as autophagosomes, and these vesicles fuse with lysosomes to form autolysosomes that execute the degradation of mitochondria. Given that mitochondria contain iron-rich complexes and that lysosomes contain hydrogen peroxide, we posit that mitochondrial ROS is a result of the Fenton reaction (Fe²⁺+H₂O₂ = Fe³⁺ + H₂O +OH) occurring in autolysosomes during mitophagy in ECM-detachment. Additionally, because of its accumulation, mitochondrial ROS can lead to the loss of lysosomal membrane integrity, a process known as lysosomal membrane permeabilization (LMP), and cause the release of active cathepsins that have been observed to execute caspase-independent cell death once in the cytosol. To date, using a combination of subcellular fractionation, immunoblotting, immunofluorescence, and enzymatic activity assays, we have discovered that ECM-detachment induces LMP and that this LMP is dependent upon RIPK1-mediated mitophagy. Likewise, during detachment, not only have we observed the release of cathepsin B into the cytosol (a marker for LMP), but our data also show that detachment regulates the activity of cathepsin B in a RIPK1 and mitochondrial ROS-dependent fashion. With this discovery in addition to previous studies depicting its role in caspase-independent cell death, it is possible that cathepsin B is playing a prominent role in facilitating cell death that occurs as a result of RIPK1-mediated mitophagy. Therefore, our current focus entails investigating the requirement and mechanism of cathepsin B to promote caspase-independent cell death downstream of RIPK1 activity in ECM-detachment. In brief, elucidating a novel cell death pathway that could potentially reveal therapeutic targets for treatments, particularly in cancers that have acquired resistance to caspase-dependent cell death, truly highlights the significance of this project.