KLF7 promotes pancreatic cancer growth and metastasis by up-regulating ISG expression and maintaining Golgi complex integrity Romi Guptaa, Parmanand Malvia, Keshab Raj Parajulia, Radoslav Janostiakb, Suresh Bugidea, Guoping Caib, Lihua Julie Zhuc,d,e, Michael R. Greenc,1, and Narendra Wajapeyeea,1 aDepartment of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35233; bDepartment of Pathology, Yale University School of Medicine, New Haven, CT 06510; cDepartment of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA 01605; dProgram in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605; and eProgram in Bioinformatics and Integrative Biology, University of Massachusetts Medical School, Worcester, MA 01605 Contributed by Michael R. Green, April 3, 2020 (sent for review March 20, 2020; reviewed by Mu-Shui Dai and Zhenghe Wang) Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer Krüppel-like factor (KLF) proteins are transcriptional regu- with a dismal prognosis. Currently, there is no effective therapy for lators that are conserved among mammals from human to mouse PDAC, and a detailed molecular and functional evaluation of PDACs (16). Functionally, the KLF proteins can be classified into three is needed to identify and develop better therapeutic strategies. major groups. Group 1 (including KLFs 3, 8, and 12) and group 3 Here we show that the transcription factor Krüppel-like factor 7 (including KLFs 9, 10 11, 13, 14, 15, and 16) function largely as (KLF7) is overexpressed in PDACs, and that inhibition of KLF7 blocks transcriptional repressors (16). The group 2 proteins, which in- PDAC tumor growth and metastasis in cell culture and in mice. KLF7 clude KLF7 in addition to KLFs 1, 2, 4, 5, and 6, function pre- expression in PDACs can be up-regulated due to activation of a MAP dominantly as transcriptional activators (16). kinase pathway or inactivation of the tumor suppressor p53, two In this study, we show that KLF7 promotes PDAC growth and alterations that occur in a large majority of PDACs. ShRNA-mediated metastasis by up-regulating the expression of IFN-stimulated knockdown of KLF7 inhibits the expression of IFN-stimulated genes genes (ISGs) and by maintaining Golgi integrity. Our results (ISGs), which are necessary for KLF7-mediated PDAC tumor growth suggest that the PDAC-promoting, KLF7-regulated transcrip- MEDICAL SCIENCES and metastasis. KLF7 knockdown also results in the down- tional pathway is pharmacologically tractable for PDAC therapy. regulation of Discs Large MAGUK Scaffold Protein 3 (DLG3), result- ing in Golgi complex fragmentation, and reduced protein glycosyl- Results ation, leading to reduced secretion of cancer-promoting growth KLF7 Is Overexpressed in PDACs and Necessary for PDAC Tumor factors, such as chemokines. Genetic or pharmacologic activation Growth and Metastasis. Analysis of previously published mRNA of Golgi complex fragmentation blocks PDAC growth and metasta- expression data from patient-derived PDAC samples revealed sis similar to KLF7 inhibition. Our results demonstrate a therapeuti- that KLF7 mRNA expression was significantly overexpressed in cally amenable, KLF7-driven pathway that promotes PDAC growth and metastasis by activating ISGs and maintaining Golgi complex Significance integrity. KLF7 | pancreatic cancer | metastasis | IFN-stimulated genes | Golgi Pancreatic cancer remains a challenge for modern oncology complex practice because of its dismal prognosis, 5-y survival rate of <10%, and lack of effective therapies. Here we show that the transcription factor KLF7 promotes pancreatic cancer growth and ancreatic ductal adenocarcinoma (PDAC) is the fourth- metastasis by regulating IFN-stimulated genes (ISGs) and Golgi Pleading cause of cancer-related deaths in the United States complex integrity. Our results demonstrate a cell-intrinsic role and is expected to become the second-leading cause by 2020 (1, 2). for ISGs that is independent of innate immunity. We also dem- The five-year disease-free survival rate for patients with PDAC is < onstrate a previously unappreciated tumorigenic role of the extremely low and has remained 10% for several decades (2, 3). Golgi complex as a cellular apparatus that promotes the secre- Currently, there is no effective therapy for PDAC, and even im- tion of cancer-promoting growth factors. Our results have im- munotherapies that have worked effectively in patients with other plications for the understanding of pancreatic cancer as a disease cancer types have failed to provide meaningful clinical benefits in and for the development of new therapeutic interventions for patients with PDAC (1). Therefore, further molecular and func- pancreatic cancer. tional evaluation of PDAC is needed to identify and develop better therapeutic strategies. Author contributions: R.G., M.R.G., and N.W. designed research; R.G., P.M., K.R.P., R.J., Transcriptional deregulation is a major driver of cancer growth S.B., and G.C., performed research; R.G., S.B., M.R.G., and N.W. contributed new reagents/ and metastasis that shapes the cancer transcriptome and conse- analytic tools; R.G., P.M., K.R.P., R.J., S.B., G.C., L.J.Z., and N.W. analyzed data; and R.G., quently the cancer cell proteome (4–7). In fact, most cancers and P.M., M.R.G., and N.W. wrote the paper. their subtypes can be classified based on mRNA expression pat- Reviewers: M.-S.D., Oregon Health & Science University; and Z.W., Case Western Reserve University. terns alone (8, 9). Several key transcriptional regulators have been The authors declare no competing interest. shown to play important roles in PDAC tumor growth and me- tastasis (10–12). Key deregulated pathways in PDAC include on- Published under the PNAS license. cogenic mutations in the KRAS gene (found in >90% of PDACs) Data deposition: The RNA-seq data of this study have been submitted to the National TP53 Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO), https:// and mutations in the tumor-suppressor gene (found in up www.ncbi.nlm.nih.gov/geo/ (accession no. GSE107184). to two-thirds of PDACs), both of which mediate their cancer- 1To whom correspondence may be addressed. Email: [email protected] or Michael. promoting effects in part through their ability to regulate tran- [email protected]. scription (13–15). Overall, previous studies have shown that vari- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ ous transcription factors and transcriptional networks play major doi:10.1073/pnas.2005156117/-/DCSupplemental. roles in facilitating PDAC tumor growth and metastasis. First published May 19, 2020. www.pnas.org/cgi/doi/10.1073/pnas.2005156117 PNAS | June 2, 2020 | vol. 117 | no. 22 | 12341–12351 Downloaded by guest on September 27, 2021 PDAC samples compared with normal pancreas samples (Fig. 1 of p53, but not of LacZ, resulted in down-regulation of KLF7 at A and B and SI Appendix, Fig. S1A) (17–22). To investigate these the mRNA and protein levels (Fig. 3 C and D). To determine findings further, we analyzed KLF7 protein expression using whether p53 directly regulates KLF7 transcription, we first ana- immunohistochemistry and a tissue microarray of patient-derived lyzed the KLF7 promoter sequence using the in silico program PDAC samples (n = 50) and matched normal pancreas samples rVista2.0, which predicts potential DNA-binding sites for tran- (n = 50). We found that KLF7 protein was significantly overex- scription factors (28). The results identified a DNA-binding site pressed in a large majority of the PDAC samples compared with for p53 in the KLF7 promoter (SI Appendix, Fig. S3B). On the the matched normal pancreas samples (Fig. 1 C–E and SI Ap- basis of these findings, we performed chromatin immunopre- pendix,TableS1). Taken together, the results of these analyses cipitation (ChIP) to confirm the association of p53 with the en- show that KLF7 is overexpressed in PDAC. dogenous KLF7 promoter in AsPC1 cells expressing either Ad- Due to the fact that KLF7 is overexpressed in PDAC led us to p53 or Ad-LacZ. The ChIP results showed that p53 directly binds rationalize that KLF7 might be important for PDAC tumor to the KLF7 promoter in vivo (Fig. 3E). growth and metastasis. To determine this possibility, we knocked To further substantiate the role of p53 as a transcriptional down KLF7 expression in four different PDAC cell lines— repressor of KLF7, we knocked down p53 expression in hTERT- PANC1, AsPC1, MIAPaCa2, and SU.86.86—using two sequence- immortalized primary human pancreatic ductal epithelial (HPNE- independent short hairpin RNAs (shRNAs) (SI Appendix, Fig. hTERT) cells and analyzed KLF7 expression. We found that p53 S1 B and C). We then tested the outcome of the KLF7 knockdown knockdown increased the levels of KLF7 mRNA and protein in the on the ability of PDAC cells to form colonies in soft agar. We cells (Fig. 3 F and G). In addition, we confirmed that the p53 choose to perform the soft agar assay because measurement of knockdown resulted in reduced p53 recruitment to the KLF7 anchorage-independent growth in soft agar serves as a reliable promoter (Fig. 3H). Collectively, our results demonstrate that ac- surrogate assay for estimating in vivo tumorigenesis (23, 24). KLF7 tivation of the MAPK pathway or inactivation of p53 are sufficient knockdown in PDAC cells resulted in a significant reduction in for the transcriptional overexpression of KLF7 in PDAC cells. their ability to form colonies in soft agar (Fig. 2 A and B). We next asked whether KLF7 knockdown could inhibit PDAC Loss of KLF7 Results in the Repression of ISGs and Consequential tumor growth in vivo. To test this, we subcutaneously injected Inhibition of PDAC Growth and Metastasis. We next aimed to PANC1, AsPC1, and MIAPaCa2 PDAC cells, expressing either identify the mechanism by which KLF7 inhibition attenuates KLF7-specific shRNAs or a nonspecific (NS) control shRNA, PDAC tumor growth and metastasis.