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Selective inhibition of stemness through EGFR/FOXA2/SOX9 axis reduces pancreatic cancer metastasis

Oncogene volume 40, pages 848–862 (2021)Cite this article

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Abstract

Pancreatic cancer (PC) is difficult to defeat due to mechanism (s) driving metastasis and drug resistance. Cancer stemness is a major challenging phenomenon associated with PC metastasis and limiting therapy efficacy. In this study, we evaluated the pre-clinical and clinical significance of eradicating pancreatic cancer stem cells (PCSC) and its components using a pan-EGFR inhibitor afatinib in combination with gemcitabine. Afatinib in combination with gemcitabine significantly reduced KrasG12D/+; Pdx-1 Cre (KC) (P < 0.01) and KrasG12D/+; p53R172H/+; Pdx-1 Cre (KPC) (P < 0.05) derived mouse tumoroids and KPC-derived murine syngeneic cell line growth compared to gemcitabine/afatinib alone treatment. The drug combination also reduced PC xenograft tumor burden (P < 0.05) and the incidence of metastasis by affecting key stemness markers, as confirmed by co-localization studies. Moreover, the drug combination significantly decreases the growth of various PC patient-derived organoids (P < 0.001). We found that SOX9 is significantly overexpressed in high-grade PC tumors (P < 0.05) and in chemotherapy-treated patients compared to chemo-naïve patients (P < 0.05). These results were further validated using publicly available datasets. Moreover, afatinib alone or in combination with gemcitabine decreased stemness and tumorspheres by reducing phosphorylation of EGFR family proteins, ERK, FAK, and CSC markers. Mechanistically, afatinib treatment decreased CSC markers by downregulating SOX9 via FOXA2. Indeed, EGFR and FOXA2 depletion reduced SOX9 expression in PCSCs. Taken together, pan-EGFR inhibition by afatinib impedes PCSCs growth and metastasis via the EGFR/ERK/FOXA2/SOX9 axis. This novel mechanism of pan-EGFR inhibitor and its ability to eradicate CSC may serve as a tailor-made approach to enhance chemotherapeutic benefits in other cancer types.

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Fig. 1: Effects of afatinib and gemcitabine on mouse pancreatic tumoroids and xenograft models.
Fig. 2: Afatinib and gemcitabine combination treatment inhibited human pancreatic cancer patients-derived tumoroids.
Fig. 3: Afatinib inhibited human and mouse pancreatic cancer stem cells and stemness.
Fig. 4: Cancer stem cell-associated self-renewal markers SOX9, CD44, EpCAM, and CD133 expression in normal pancreas, grade 2 and grade 3 PDAC, chemo-treated and untreated pancreatic cancer tissues.
Fig. 5: Afatinib inhibited CSC stemness by downregulating multiple CSC markers in vitro and in vivo xenograft PC models.
Fig. 6: Afatinib decreased the CSC population via EGFR/ERK/FOXA2/SOX9 signaling in PCSCs.

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Funding

This research paper is supported, in parts, by grants from the National Institutes of Health (This work was supported, in parts, by the National Institutes of Health (P01 CA217798, R01 CA183459, R01 CA195586, R01 CA206444, R01 CA210637, R01 CA228524, R01 CA247471, U01 CA200466, and U01 CA210240).

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Author notes

  1. These authors contributed equally: Garima Kaushik, Parthasarathy Seshacharyulu

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE, USA

    Garima Kaushik, Parthasarathy Seshacharyulu, Sanchita Rauth, Palanisamy Nallasamy, Satyanarayana Rachagani, Rama Krishna Nimmakayala, Raghupathy Vengoji, Kavita Mallya, Ramakanth Chirravuri-Venkata, Amar B. Singh, Maneesh Jain, Moorthy P. Ponnusamy & Surinder K. Batra

  2. Division of Surgical Oncology, Fred & Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA

    Jason M. Foster & Quan P. Ly

  3. Department of Biostatistics, UNMC, Omaha, NE, USA

    Lynette M. Smith

  4. Department of Pathology and Microbiology, UNMC, Omaha, NE, USA

    Subodh M. Lele & Surinder K. Batra

  5. Department of Gastrointestinal Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA

    Mokenge P. Malafa

  6. Fred and Pamela Buffett Cancer Center, Eppley Institute for Research in Cancer and Allied Disease, University of Nebraska Medical Center, Omaha, NE, USA

    Maneesh Jain, Moorthy P. Ponnusamy & Surinder K. Batra

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  1. Garima Kaushik
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Contributions

SKB, MPP, GK, and PS designed the experiments, and SKB and MPP offered directions. GK, PS, SR, PN, SR, RKN, RV, and KM performed the experiments and analyzed the results. RCV performed bioinformatic analysis and analyzed the Data. Pathological analysis conducted by SML. Statistical analysis was performed by LMS. GK and PS drafted the paper, and SKB, MPP, MJ, ABS, MPM, JMF, and QPL reviewed the paper and suggested significant changes to the drafts. All authors read and approved the final paper.

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Correspondence to Moorthy P. Ponnusamy or Surinder K. Batra.

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SKB is one of the co-founders of Sanguine Diagnostics and Therapeutics, Inc. The other authors disclosed no potential conflicts of interest.

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Kaushik, G., Seshacharyulu, P., Rauth, S. et al. Selective inhibition of stemness through EGFR/FOXA2/SOX9 axis reduces pancreatic cancer metastasis. Oncogene 40, 848–862 (2021). https://doi.org/10.1038/s41388-020-01564-w

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