Abstract
There is a critical need to identify new therapeutic vulnerabilities in pancreatic ductal adenocarcinoma (PDAC). Transcriptional co-regulators C-terminal binding proteins (CtBP) 1 and 2 are highly overexpressed in human PDAC, and CRISPR-based homozygous deletion of Ctbp2 in a mouse PDAC cell line (CKP) dramatically decreased tumor growth, reduced metastasis, and prolonged survival in orthotopic mouse allografts. Transcriptomic profiling of tumors derived from CKP vs. Ctbp2-deleted CKP cells (CKP/KO) revealed significant downregulation of the EGFR-superfamily receptor Erbb3, the heterodimeric signaling partner for both EGFR and ErbB2. Compared with CKP cells, CKP/KO cells also demonstrated reduced Erbb2 expression and did not activate downstream Akt signaling after stimulation of Erbb3 by its ligand neuregulin-1. ErbB3 expression in human PDAC cell lines was similarly dependent on CtBP2 and depletion of ErbB3 in a human PDAC cell line severely attenuated growth, demonstrating the critical role of ErbB3 signaling in maintaining PDAC cell growth. Sensitivity to the ErbB2-targeted tyrosine kinase inhibitor lapatinib, but not the EGFR-targeted agent erlotinib, varied in proportion to the level of ErbB3 expression in mouse and human PDAC cells, suggesting that an ErBb2 inhibitor can effectively leverage CtBP2-driven transcriptional activation of physiologic ErbB2/3 expression and signaling in PDAC cells for therapeutic benefit.
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Introduction
Pancreatic adenocarcinoma (PDAC) is among the most lethal of cancers, with an average 5-year survival from diagnosis of only 10% [1]. Only limited success is observed with existing therapies, and there is an immediate need to fully understand the underlying pathogenesis of PDAC to identify novel and effective therapies. We have previously demonstrated that the oncogenic transcription factor CtBP2 [2], which is frequently overexpressed in pancreatic cancer, as well as colon, breast, ovarian, prostate, and gastric cancers and associated with poor patient outcomes [3,4,5,6], is a critical dependency in PDAC, as Ctbp2 allelic loss led to dramatic decreases in tumor size and metastasis along with prolonged survival in PDAC-prone CKP (Pdx1-Cre; LSL-KrasG12D/+; Trp53fl/fl) mice [4]. Although the decreased tumor size and metastasis and prolonged survival seen in Ctbp2-deficient CKP mice were accompanied by reduced expression of cancer stem cell markers, the exact mechanism for dependency of PDAC tumor progression on Ctbp2 has remained unclear [4].
In this report, we have used a cell line derived from a CKP mouse PDAC tumor in which we homozygously deleted the Ctbp2 gene via CRISPR, to further interrogate the oncogenic mechanism of action of Ctbp2 in PDAC progression using an orthotopic allograft PDAC mouse model. Indeed, deletion of Ctbp2 significantly decreased PDAC tumor growth and metastasis and prolonged survival in mice orthotopically implanted with Ctbp2-deficient CKP cells, compared with mice implanted with parental CKP cells. Transcriptional profiling of CKP and Ctbp2-deficient CKP orthotopic PDAC tumors identified a novel connection between Ctbp2 and Erbb3 expression, also seen in human PDAC cell lines, and we further demonstrated Ctbp2-dependence for signaling by Erbb2/Erbb3 complexes after stimulation with the ErbB3 ligand neuregulin-1 (NRG-1). Depletion of ErbB3 in a human PDAC cell line strikingly curtailed growth, suggesting that ErbB3 and its physiologic signaling partner ErbB2, might serve as therapeutic targets in ErbB3-expressing PDAC. Indeed, mouse and human PDAC cells with higher levels of ErbB3 expression were significantly more sensitive to the ErbB2-targeted tyrosine kinase inhibitor (TKI) lapatinib. Taken together, our studies reveal novel regulation of ErbB3 by CtBP2 in PDAC, opening new avenues to target ErbB3-expressing PDAC tumors with inhibitors of EGFR superfamily signaling.
Results and discussion
Ctbp2 drives PDAC tumor growth and metastasis in an orthotopic allograft mouse model
To fully understand the role of CtBP2 in PDAC tumor biology, we developed a tractable orthotopic allograft model utilizing a mouse PDAC cell line derived from PDAC tumors of CKP mice [4] (Fig. S1A). The cell line (CKP) was validated as of ductal epithelial origin, as we observed higher mRNA expression of the epithelial/PDAC markers Krt19 (CK19) and Muc4 [7] in CKP cells compared to normal mouse pancreas tissue (Fig. S1B). Ctbp2 was then deleted homozygously in the CKP cell line using CRISPR/Cas9, with the Ctbp2-deleted CKP/KO cells demonstrating complete loss of Ctbp2 protein expression as confirmed by Ctbp2 immunoblot (Fig. 1A). To facilitate in situ imaging, the CKP and CKP/KO cell lines were next transduced with a luciferase cDNA, and luciferase expression in both cell lines (CKP-luc and CKP/KO-luc) was confirmed as equivalent by in vitro luciferase assay (Fig. S1C).
To better understand any differences between the phenotype of CKP and CKP/KO cells in vivo, we first determined whether Ctbp2 was required for growth in cell culture. Though both CKP-luc and CKP/KO-luc cell lines grew similarly over the initial 5 days of the assay, there was a modest but statistically significant 30% decrease in final live cell number achieved after 8 days of growth for the CKP/KO cells (Fig. S2A). To investigate the putative in vivo role of Ctbp2 in PDAC tumor growth and metastasis, CKP-luc and CKP/KO-luc cell lines were injected orthotopically into the pancreatic tail of 4-month-old immune-deficient non-obese diabetic (NOD)-SCID γ (NSG) mice, which were chosen so as to negate any impact of the host mouse immune system, allowing focus on the cell autonomous effects of Ctbp2. Mice were monitored for tumor growth over 3 weeks or until a humane endpoint was reached. Tumors in mice implanted with CKP/KO-luc cells grew significantly more slowly than CKP-luc tumors with final photon flux ~1/3 of that seen in CKP-luc tumors as determined by interval luciferase bioluminescent imaging (Fig. 1B, C). At necropsy, the average weight of pancreata from the CKP cohort was also 2-fold higher than the CKP/KO group, reflecting a higher overall tumor burden in the pancreas (Fig. 1D). Consistent with smaller tumor size, mice implanted with CKP/KO-luc cells survived significantly longer than mice implanted with CKP-luc cells, with calculated median survivals of 4.4 vs. 3 weeks, respectively (Fig. 1E).
We found a significant increase in the body weight of the mice carrying CKP tumors as compared to CKP/KO tumors (Fig. S2B), that was likely due to accumulation of ascitic fluid (a clear indicator of peritoneal metastasis in human PDAC [8]) which was present in all CKP mice, but in none of the CKP/KO mice (Fig. 1F). These findings therefore suggested that Ctbp2 might be crucial not only for PDAC growth, but also metastasis. Consistent with the universal finding of ascites in the CKP mice, mice implanted with CKP cells also demonstrated extensive peritoneal metastases (peritoneal surfaces of the liver, kidney, spleen, intestines), while only 1 of 5 CKP/KO injected mice exhibited peritoneal metastases, and only to the surfaces of the liver and spleen in the one mouse with metastasis (Fig. S2C and S2D; Table S1). This finding correlates with our previous observations in the CKP mouse model, where allelic loss of Ctbp2 eliminated metastasis to the peritoneum, the preferred site of metastasis in transgenic mice born with the CKP genotype [4].
We further investigated the underlying mechanism for the differential metastatic potential of CKP vs. CKP/KO tumors by studying the migratory potential of CKP vs. CKP/KO cells in vitro using a trans-well migration assay. Based on our previous data in colon cancer cells [9], we expected that CKP/KO cells would exhibit a deficiency in migratory ability and indeed, we observed a decrease in the ability of CKP/KO cells to migrate in this assay when compared with CKP cells (Fig. S2E). Thus, Ctbp2 directs both the growth and metastatic potential of PDAC tumor cells, with metastatic phenotypes in vivo correlating with activation of a migratory phenotype in vitro.
CtBP2 regulates expression of ErbB3, which is critically required for PDAC cell growth
To better understand molecular pathways contributing to CtBP’s oncogenic activities in the orthotopic CKP PDAC model, we performed RNA sequencing (RNA-seq) analysis to compare differentially expressed genes between CKP and CKP/KO tumors (n = 5/group; false discovery rate = 0.01; Fig. 2A). A total of 780 protein coding genes were altered in response to deletion of Ctbp2, and of the top fifty genes that were differentially expressed between the two groups, we noted significant downregulation of the EGFR superfamily member Erbb3, which is a biochemical and functional partner to both the ErbB1/EGFR (EGFR hereafter) and ErbB2/Her2 (ErbB2 hereafter) proto-oncoproteins [10]. This finding was validated by qPCR analysis which demonstrated a striking 80% reduction in Erbb3 mRNA in CKP/KO compared with CKP tumors (Fig. 2B).
To investigate whether Erbb3 regulation by Ctbp2 was specifically occurring within CKP mouse tumor cells, as opposed to adjoining tumor stroma or normal tissue, we performed Ctbp2 and Erbb3 immunohistochemical (IHC) staining of pancreatic tumors from mice orthotopically implanted with CKP or CKP/KO cells, scoring mean staining intensity and the percentage of positively staining tumor cells for each marker across multiple tumors from each cell type (n = 5) (Fig. 2C, D). As expected, Ctbp2 and Erbb3 were both strongly expressed (mean intensityes = 3.0 for both) and present in nearly all (90 and 80% positive, respectively) CKP tumor cells, while the mean staining intensity of Ctbp2 and Erbb3 was minimal in tumors derived from CKP/KO tumor cells (0.8 and 0.4, respectively) with only a minority of tumor cells demonstrating any detectable expression (20 and 10% positive, respectively) (Fig. 2C, D). These data support direct and tumor cell-autonomous regulation of Erbb3 expression by Ctbp2 in CKP PDAC tumor cells.
To determine if Ctbp2 regulation of Erbb3 in mouse tumors was generalizable to human PDAC, we examined a panel of 9 human PDAC cell lines for CtBP1/2 and ErbB3 protein expression, and while only 5 of the 9 lines expressed detectable ErbB3, all cell lines expressed both CtBP1 and 2 equivalently (Fig. S3). To validate CtBP2 control of ErbB3 expression in human PDAC cells as seen in mouse CKP cells, we expressed control or CtBP2 shRNA in the ErbB3-positive human PDAC cell line HPAC, and as observed in CKP/KO cells, there was a drastic reduction in ErbB3 mRNA and protein levels after CtBP2 depletion (Fig. 3A, B).
Active ErbB2/ErbB3/Akt/MAPK signaling in PDAC cells requires CtBP2
Having shown CtBP2-dependent ErbB3 regulation in PDAC cells and tumors, we next explored the potential functional significance of this pathway, including the signaling partners of ErbB3 and their dependence on CtBP activity. ErbB3 signals only when heterodimerized with other EGFR superfamily members that encode active tyrosine kinase domains, such as EGFR, ErbB2, or ErbB4 [11]. Hence, we examined EGFR, Erbb2, and Erbb4 expression in CKP and CKP/KO cells and surprisingly found a significant decrease in Erbb2, but not EGFR levels, in CKP/KO cells relative to CKP cells (Fig. 3A), while Erbb4 was not detectably expressed (data not shown). We found similar results after stable knockdown of CtBP2 in HPAC cells, with a drastic reduction in ErbB2/3 mRNA and protein levels in shCtBP2-expressing vs. shCtrl-expressing HPAC cells (Fig. 3B). These results align with human tumor data where ErbB2 and ErbB3 are expressed in synchronous fashion in PDAC [12] and in other cancers, such as breast cancer [13]. In terms of the potential clinical significance of these findings in PDAC patients, interrogation of the TCGA database revealed significantly higher levels of both ErbB2 and ErbB3 mRNA in PDAC tumors compared to adjacent normal pancreatic tissue (Fig. S4A and S4B), and importantly, survival after PDAC diagnosis is inversely correlated with ErbB3 mRNA expression level [14]. Indeed, shRNA knockdown of ErbB3 in HPAC cells severely attenuated the rate of cell growth in culture as compared to HPAC cells expressing a control shRNA (Fig. 3C).
Next, we interrogated the functional significance of CtBP2 regulation of ErbB2/3 for oncogenic downstream signal transduction, as ErbB2/ErbB3 heterodimers actively signal through both the PI3K/Akt and MAP Kinase (MAPK) pathways [10]. Serum-starved CKP and CKP/KO cells were treated with the ErbB3-specific ligand NRG-1 [15] at increasing doses (0, 0.25, 0.5, 1 ng/ml) for 15 min and cell lysates analyzed for Akt/MAPK signaling by immunoblotting for total and activated phosphorylated (p-) forms of Erbb2, Erbb3, Akt, and Erk1/2. In CKP cells, NRG-1 stimulation activated Erbb2/Erbb3/Akt/MAPK signaling as evidenced by dose-dependent induction of phosphorylated species of Erbb2, Erbb3, Akt, and Erk1/2 (Fig. 3D). However, CKP/KO cells showed little or no induction of Erbb2 or Erbb3 phosphorylation upon treatment with NRG-1 due to low total Erbb2/3 protein levels, and consistent with decreased Erbb2/3 signaling, downstream Akt and Erk1/2 phosphorylation was also absent. These results support Ctbp2 as a critical regulator of Erbb2/Erbb3/Akt/MAPK signaling in PDAC cells.
Sensitivity to inhibitors of EGFR-family tyrosine kinases is regulated by the CtBP2/ErbB3 axis
To determine if physiologic EGFR-family signaling in PDAC cells that express ErbB3 could be pharmacologically targeted for therapeutic benefit, we explored the sensitivity of CKP and CKP/KO cells to inhibitors that target active heterodimeric EGFR/ErbB3 and ErbB2/ErbB3 signaling complexes. For this purpose, we exposed CKP and CKP/KO cells to the EGFR/ErbB2 dually specific tyrosine kinase inhibitor (TKI) lapatinib or the EGFR-specific TKI erlotinib. Of note, erlotinib exhibits little or no cross-inhibition of ErbB2 kinase, while lapatinib inhibits both ErbB2 and EGFR/ErbB1 kinases [16, 17]. We found that CKP cells were ~3-fold more sensitive to lapatinib than CKP/KO cells (IC50s 0.4 µM and 1.4 µM, respectively; p < 0.05), which could be explained by the decreased Erbb2/3 expression and signaling in CKP/KO cells when compared to CKP cells (Fig. 4A, upper and lower panels). We further tested lapatinib sensitivity across additional human PDAC cell lines with differential expression of ErbB3 (Fig. S3), and consistent with results in CKP vs. CKP/KO cells, cells with low or undetectable ErbB3 expression showed significantly higher lapatinib IC50 values compared to cells with high ErbB3 expression (Figs. 4B and S3). To determine if inhibition of EGFR/ErbB3 complexes by lapatinib might be contributing to its PDAC cell inhibitory activity, we observed that, unlike lapatinib, the IC50 of the EGFR-specific TKI erlotinib was not significantly different between CKP and CKP/KO cells, (IC50s = 5.6 and 2.6, respectively; p = NS; Fig. 4A, middle and lower panels).
In this study, we have developed a novel orthotopic allograft PDAC mouse model to show that loss of the oncogenic transcription factor Ctbp2 [2] decreases PDAC tumor growth and metastasis. Indeed, ~100% of mice in the CKP cohort demonstrated peritoneal metastases along with malignant ascites, while only ~20% of mice in the CKP/KO cohort demonstrated peritoneal metastases, and none developed ascites. Demonstrating the faithfulness of this model, the phenotype mirrors that observed of allelic loss of Ctbp2 in CKP transgenic mice, which abrogated the development of ascites and peritoneal metastases that is universal to the CKP model [4]. In contrast to the costly and time-consuming transgenic CKP model, the CKP orthotopic allograft model serves as a tractable PDAC mouse model that will aid study of tumor progression and metastasis mechanisms, as well as potential therapeutic strategies for PDAC. Though our data was generated in immunodeficient mice to focus on the cell autonomous effects of Ctbp2, the model also has the significant advantage of allowing study in immunocompetent syngeneic (C57B/6) mice that would facilitate studies of mechanisms of immune escape as well as immunotherapeutic strategies, the lack of which are an enormous unmet need in human PDAC.
Upon mechanistic exploration of how Ctbp2 might be regulating PDAC tumor progression, an unbiased transcriptomic profiling approach revealed that loss of Ctbp2 significantly downregulated expression of the Erbb3 growth factor receptor, a member of the EGFR family of growth factor receptors, which is expressed in a subset of human PDAC tumors [12, 18] and has been previously linked to adverse clinical outcomes in both PDAC and breast cancer [14, 19]. Validating these RNA-seq results, directed qPCR of orthotopic CKP vs. CKP/KO tumor mRNA revealed striking decreases in mRNA and protein levels of Erbb3, as well as its signaling partner Erbb2, in tumors lacking Ctbp2. Moreover, Ctbp2 loss abrogated downstream signaling to the growth-promoting Akt/MAPK pathways after engagement of Erbb3 by its native ligand, NRG-1. Consistent with a critical role for ErbB3 signaling in maintaining the PDAC growth phenotype, depletion of ErbB3 dramatically attenuated the growth of human HPAC PDAC cells. Thus, our findings suggest that CtBP2 regulates PDAC growth and metastasis via concerted transcriptional regulation of ErbB2 and ErbB3, and that ErbB3 is critically required to maintain growth of PDAC cells that maintain its expression via active signaling by at least, the Akt/MAPK pathways (Fig. 4C). Notably, this work is the first to address whether native expression of ErbB3 in PDAC cells is actually required to maintain growth capacity, as a prior report suggesting ErbB3 plays an important role in PDAC tumor growth and progression relied on exogenous overexpression of ErbB3 [18].
Our data strongly supports targeting of ErbB2/3 signaling in ErbB3-expressing PDAC, and our work also supports an emerging paradigm in the field of EGFR-family targeted therapeutics, based on recent data showing: (1) Responsiveness of ErbB2(low) tumors to anti-ErbB2 agents [20] as almost all PDAC would be classified as ErbB2(low) [21]; (2) the critical nature of ErbB2/ErbB3 complexes in oncogenic signaling evidenced by the success of pertuzumab that targets ErbB2/3 heterodimerization in breast cancer refractory to trastuzumab [22], and; (3) the improvement in survival seen with dual targeting of ErbB2/3 with trastuzumab and the ErbB2-specific TKI tucatinib [23]. Our data support further investigation of a similar dual targeting strategy in ErbB2(low)/ErbB3(high) PDAC tumors using extracellular domain directed antibodies against ErbB2/3 interaction, such as pertuzumab, or against ErbB3 itself [24, 25], combined with TKI inhibition of the ErbB2 kinase, with lapatinib as we report here, or with the next generation TKI tucatinib, as has shown success in breast cancer [23].
Data availability
The datasets generated during the current study are available in the GEO repository.
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Acknowledgements
The authors would like to thank B. Hu (VCU) for assistance with generating the orthotopic model. We would like to thank A. Ivanov (Cleveland Clinic) for gifting the HPAF-II cell line and T. Donahue (University of California, Los Angeles) for gifting the PaTu8988T and Suit2 cell lines. Services and products in support of the research project were generated by the Virginia Commonwealth University Cancer Mouse Models Core Laboratory, supported, in part, with funding from NIH-NCI Cancer Center Support Grant P30 CA016059.
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K.K.C. and S.R.G. designed experiments. K.K.C., H.P., P.K.D., T.M. and B.C performed the experiments. K.K.C., M.M.D., B.S., M.G.D., M.I. and S.R.G. analyzed the data. K.K.C. drafted the paper; S.R.G. edited the paper.
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Chougoni, K.K., Park, H., Damle, P.K. et al. Coordinate transcriptional regulation of ErbB2/3 by C-terminal binding protein 2 signals sensitivity to ErbB2 inhibition in pancreatic adenocarcinoma. Oncogenesis 12, 53 (2023). https://doi.org/10.1038/s41389-023-00498-8
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DOI: https://doi.org/10.1038/s41389-023-00498-8