Autophagy and RAS signaling: Clinical implications

Mon, 02/01/2021 - 12:21

Immunocytochemical staining of fixed and permeabilized HeLa cells treated with chloroquine, incubated with LC3B (green) and tubulin (red) antibodies, and counterstained with DAPI to visualized DNA.

By Christina Towers, PhD

The cellular recycling process known as autophagy is currently being targeted in over 60 clinical trials focused on treating different types of cancer1. To date, the only autophagy-targeted agents used in patients are late stage autophagy inhibitors that target the lysosome, including chloroquine (CQ) and hydroxychloroquine (HCQ). Over the last decade a host of studies performed in both cancer cell lines and mouse models have indicated that autophagy plays a tumor-promotional role in established tumors. Nonetheless, initial results from the first wave of trials have shown lackluster results when these drugs are used as single agents, however, there have been modest results seen when used in combination with other targeted agents. While there are a host of reasons these drugs might be underperforming in clinical trials, including a lack of specificity, mechanisms or resistance, and a lack of biomarkers – one clear shortcoming of the field is an inadequate understanding of autophagy dependence. It is still unclear which tumor types or sub-types may be most sensitive to autophagy-targeted agents.

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There is evidence, however, that tumors addicted to the RAS-RAF-MEK-ERK signaling pathway have extremely high levels of basal autophagy compared to other oncogene drivers. This may be especially true in pancreatic ductal adenocarcinoma (PDAC) where greater than 95% of PDAC tumors harbor a RAS activating mutation2,3. Moreover, genetically engineered mouse models of PDAC have shown exquisite sensitivity to genetic autophagy inhibition4. These results led to the hypothesis that RAS pathway activation may be activating autophagy as a pro-survival mechanism.

Western blot showing lysates from HeLa cell lines and LC3B knockout (KO) HeLa cell lines, both chloroquine treated and untreated, probed with rabbit monoclonal LC3B antibody and followed by HRP-conjugated secondary antibody. Immunohistochemical staining of formalin-fixed, paraffin-embedded breast cancer tissue with Beclin-1 antibody, followed by HRP-conjugated secondary antibody and DAB, and counterstained with hematoxylin.

Autophagy antibody pack (NB910-94159) for analysis of various significant autophagy targets includes sample sizes for antibodies targeting LC3 (NB100-2220SS), Beclin1 (NB110-87318SS), ATG5 (NB110-53818SS), ATG9A (NB110-56893SS), and ATG16L1 (NB110-60928SS). (Left) Lysates of HeLa parental cell line and LC3B knockout HeLa cell line (KO) untreated (-) or treated (+) with 50 uM Chloroquine for 18 hours. PVDF (Polyvinylidene difluoride) membrane was probed with 0.5 ug/mL of Rabbit Anti-LC3B Polyclonal Antibody (NB100-2220) followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody (HAF008). A specific band was detected for LC3B at a molecular weight of approximately 15 kDa (as indicated) in the parental HeLa cell line, but is not detectable in the knockout HeLa cell line. GAPDH is shown as a loading control. This experiment was conducted under reducing conditions. (Right) Analysis of a FFPE tissue section of human breast cancer using Beclin 1 antibody (NB110-87318) at 1:300 dilution, followed by a HRP labeled secondary antibody and DAB. Nuclei of the cells were counterstained with hematoxylin. The signal is strongest in a subset of stromal cells, likely the cancer associated fibroblasts.

It was therefore unexpected when Kinsey et al5 and Bryant et al6 published in the same issue of Nature Medicine in 2019 that inhibition of the RAS-RAF-MEK-ERK signaling pathway actually causes a robust increase in autophagic flux. Both groups utilized the mCherry-GFP-LC3 tandem construct to quantify autophagic flux across a panel or PDAC cell lines after either genetic or pharmacological inhibition of the pathway. These results indicate the relationship between the RAS-RAF-MEK-ERK signaling pathway and autophagy pathway may be more complex. The two groups go on to show that RAS pathway inhibition, at any stage in the pathway, synergizes with the clinically used autophagy inhibitors CQ and HCQ.  Importantly, Kinsley et al show these findings extend beyond PDAC to other RAS addicted tumors including NRAS-mutant melanoma and BRAF-mutant colon cancer.

Immunocytochemical staining of immersion-fixed chloroquine treated control and LC3B-knockout HeLa cells probed with rabbit polyclonal LC3B-antibody and anti-rabbit secondary antibody (red) and counterstained with DAPI (blue).LC3B was detected in immersion fixed Chloroquine treated HeLa cells (left) but was not detected in LC3B knockout HeLa cells (right) using rabbit anti-human LC3B polyclonal antibody (NB100-2220) at 0.3 ug/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated anti-Rabbit IgG Secondary Antibody (NL004) (red) and counterstained with DAPI (blue). Specific staining was localized to cytoplasm

These results could have important implications clinically. Indeed, Kinsley et al presents a case study with a PDAC patient who was refractory to all standard-of-care therapy.  Four months of treatment with escalating doses of hydroxychloroquine and the MEK inhibitor, trametinib, led to a dose dependent decrease in the PDAC blood born cancer agent, CA19-9, by 95% and a corresponding 50% reduction in tumor burden. Previously, similar case studies in BRAF-mutant brain cancer patients have also indicated synergistic affects after treatment with autophagy inhibitors in combination with Ras pathway inhibition7. Together, these studies indicate that co-targeting with RAS pathway inhibitors and autophagy inhibitors may improve overall patient outcomes, particularly in tumors addicted to the RAS-RAF-MEK-ERK signaling pathway.

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Christina TowersChristina Towers, PhD   
University of Colorado (AMC)
Dr. Towers studies the roles of autophagy, apoptosis and cell death in cancer.


  1. Levy, J., Towers, C. G., & Thorburn, A. (2017).Targeting autophagy in cancer. Nature reviews. Cancer.
  2. Yang, A., Rajeshkumar, N. V., Wang, X., Yabuuchi, S., Alexander, B. M., Chu, G. C., Von Hoff, D. D., Maitra, A., & Kimmelman, A. C. (2014). Autophagy is critical for pancreatic tumor growth and progression in tumors with p53 alterationsCancer discovery.
  3. Perera, R. M., Stoykova, S., Nicolay, B. N., Ross, K. N., Fitamant, J., Boukhali, M., Lengrand, J., Deshpande, V., Selig, M. K., Ferrone, C. R., Settleman, J., Stephanopoulos, G., Dyson, N. J., Zoncu, R., Ramaswamy, S., Haas, W., & Bardeesy, N. (2015). Transcriptional control of autophagy-lysosome function drives pancreatic cancer metabolismNature.
  4. Yang, S., Wang, X., Contino, G., Liesa, M., Sahin, E., Ying, H., Bause, A., Li, Y., Stommel, J. M., Dell'antonio, G., Mautner, J., Tonon, G., Haigis, M., Shirihai, O. S., Doglioni, C., Bardeesy, N., & Kimmelman, A. C. (2011). Pancreatic cancers require autophagy for tumor growthGenes & development.
  5. Kinsey, C. G., Camolotto, S. A., Boespflug, A. M., Guillen, K. P., Foth, M., Truong, A., Schuman, S. S., Shea, J. E., Seipp, M. T., Yap, J. T., Burrell, L. D., Lum, D. H., Whisenant, J. R., Gilcrease, G. W., 3rd, Cavalieri, C. C., Rehbein, K. M., Cutler, S. L., Affolter, K. E., Welm, A. L., Welm, B. E., … McMahon, M. (2019). Protective autophagy elicited by RAF→MEK→ERK inhibition suggests a treatment strategy for RAS-driven cancersNature medicine.
  6. Bryant, K. L., Stalnecker, C. A., Zeitouni, D., Klomp, J. E., Peng, S., Tikunov, A. P., Gunda, V., Pierobon, M., Waters, A. M., George, S. D., Tomar, G., Papke, B., Hobbs, G. A., Yan, L., Hayes, T. K., Diehl, J. N., Goode, G. D., Chaika, N. V., Wang, Y., Zhang, G. F., … Der, C. J. (2019). Combination of ERK and autophagy inhibition as a treatment approach for pancreatic cancerNature medicine.
  7. Mulcahy Levy, J. M., Zahedi, S., Griesinger, A. M., Morin, A., Davies, K. D., Aisner, D. L., Kleinschmidt-DeMasters, B. K., Fitzwalter, B. E., Goodall, M. L., Thorburn, J., Amani, V., Donson, A. M., Birks, D. K., Mirsky, D. M., Hankinson, T. C., Handler, M. H., Green, A. L., Vibhakar, R., Foreman, N. K., & Thorburn, A. (2017). Autophagy inhibition overcomes multiple mechanisms of resistance to BRAF inhibition in brain tumorseLife.

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