Proper execution of cellular function, maintenance of cellular homeostasis and cell survival depend on functional integration of cellular processes and correct orchestration of cellular responses to stresses. Cancer transformation is a common negative consequence of mismanagement of coordinated response by the cell. In this scenario, by maintaining the balance among synthesis, degradation, and recycling of cytosolic components including proteins, lipids, and organelles the process of autophagy plays a central role. Several environmental stresses activate autophagy, among those hypoxia, DNA damage, inflammation, and metabolic challenges such as starvation. In addition to these chemical challenges, there is a requirement for cells to cope with mechanical stresses stemming from their microenvironment. Cells accomplish this task by activating an intrinsic mechanical response mediated by cytoskeleton active processes and through mechanosensitive protein complexes which interface the cells with their mechano-environment. Despite autophagy and cell mechanics being known to play crucial transforming roles during oncogenesis and malignant progression their interplay is largely overlooked. In this review, we highlight the role of physical forces in autophagy regulation and their potential implications in both physiological as well as pathological conditions. By taking a mechanical perspective, we wish to stimulate novel questions to further the investigation of the mechanical requirements of autophagy and appreciate the extent to which mechanical signals affect this process.

Proper execution of cellular function, maintenance of cellular homeostasis and cell survival depend on functional integration of cellular processes and correct orchestration of cellular responses to stresses. Cancer transformation is a common negative consequence of mismanagement of coordinated response by the cell. In this scenario, by maintaining the balance among synthesis, degradation, and recycling of cytosolic components including proteins, lipids, and organelles the process of autophagy plays a central role. Several environmental stresses activate autophagy, among those hypoxia, DNA damage, inflammation, and metabolic challenges such as starvation. In addition to these chemical challenges, there is a requirement for cells to cope with mechanical stresses stemming from their microenvironment. Cells accomplish this task by activating an intrinsic mechanical response mediated by cytoskeleton active processes and through mechanosensitive protein complexes which interface the cells with their mechano-environment. Despite autophagy and cell mechanics being known to play crucial transforming roles during oncogenesis and malignant progression their interplay is largely overlooked. In this review, we highlight the role of physical forces in autophagy regulation and their potential implications in both physiological as well as pathological conditions. By taking a mechanical perspective, we wish to stimulate novel questions to further the investigation of the mechanical requirements of autophagy and appreciate the extent to which mechanical signals affect this process.

Introduction: Vasculogenic mimicry (VM) describes a process by which cancer cells establish an alternative perfusion pathway in an endothelial cell-free manner. Despite the strong correlation with reduced patient survival, the mechanisms by which a tumor can create this self-generated irrigation system are still not fully understood. The process of VM in vitro can occur in laminin-111-containing Matrigel and requires the PI3K pathway. However, the membrane protein component and signaling pathways involved in this process are unknown. Methods: In an established in vitro model of VM of ovarian and breast cancer cells (HEY and MDA-MB-231, respectively) on Matrigel coating, we utilized gene silencing and blocking antibodies to elucidate the signaling pathways involved in this process. RNASeq was used to identify novel transcripts and siRNA was utilized to verify the requirement of candidate RNA/protein in tubular formation. Results: siRNA and antibody blocking of integrin β1, but not β3, prevented VM formation in vitro. Individual silencing of cortactin, TKS5, MMP-2, MMP-9 and MMP-14 affected tubular formation. RNAseq analysis suggested that VM has minimal dependence on de novo transcriptional activity yet reported a strong upregulation of small Integral Membrane Protein 11 (SMIM11A). This result was verified by qPCR and siRNA silencing of SMIM11A prevented VM formation. Discussion: Laminin 111 may interact with integrin β1, and the consequent activation of the PI3K pathway could potentially remodel cytoskeletal proteins and promote the activity of MMPs. We report for the first time a biological role for SMIM11a. This gene is regulated at the RNA level and is required for the formation of tubular structure in vitro. As VM is strongly associated with poor patient survival, understanding the formation of this alternative irrigation system may deliver new druggable targets. Citation Format: Gabriel Mingo, Javiera Pradenas, Nicole Babbitt, Pamela González, Cristina Bertocchi, Gareth Owen. SMIM11A, a novel protein involved in the mechanism of vasculogenic mimicry formation in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3841.

Introduction: Vasculogenic mimicry (VM) describes a process by which cancer cells establish an alternative perfusion pathway in an endothelial cell-free manner. Despite the strong correlation with reduced patient survival, the mechanisms by which a tumor can create this self-generated irrigation system are still not fully understood. The process of VM in vitro can only occur in Matrigel; however, the protein component and signaling pathways involved in this process are unknown. Methods: Using an established in vitro model of VM, of ovarian and breast cancer cells (HEY and MDA-MB-231, respectively) on Matrigel coating, we utilized pharmacological inhibitors, gene silencing and blocking antibodies to elucidate the signaling pathways involved in the process of VM. Immunofluorescence and sirius red staining were used to determine the glycoprotein-rich component lining the lumen of the tubular structures. Results: Differently to what observed in the presence of Matrigel, VM did not occur when cancer cells were cultivated on plastic, glass or heat denatured Matrigel (10 mins at 65°C). Using exclusively Collagen I or Laminin 111 to mimic the extracellular matrix we observed than only in the presence of Laminin 111 could VM formation occur. Laminin is secreted and deposited by HEY cells and constitutes a part of the luminal lining. Silencing of integrin β1, but not β3, by siRNA and antibody blocking prevents this process. Chemical inhibition of PI3K pathway and metalloproteases (MMP) activation demonstrate that these pathways are also essential. RNAseq analysis suggests that this process has minimal dependence on de novo transcriptional activity. Discussion and conclusion: We have shown that VM only occurs when cells are seeded on Matrigel but not on plastic, glass or heat denatured Matrigel, suggesting that this phenomenon is susceptible to substrate/matrix rigidity. Furthermore, we identified Laminin as the essential matrix protein secreted and deposited by cancer cells to allow for VM assembly. Its interaction with integrin β1, and the consequent regulation of the activity of MMP leads to the remodeling of the ECM to favor the connection of the VM channels to the microcirculation system. This pathway is not heavily dependent on transcription but requires the PI3K pathway. As VM is strongly associated with poor patient survival, understanding the formation of this alternative irrigation system may deliver new druggable targets. Citation Format: Gabriel Mingo, Andres Valdivia, Varina Aldana, Javiera Pradenas, Nicole Babbitt, Pamela Gonzalez, Francisco Nualart, Jorge Díaz, Lisette Leyton, Cristina Bertocchi, Gareth Owen. A characterization of cancer vasculogenic mimicry: Extracellular matrix induced cellular signaling to lumen formation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3150.