S. G. Patel1, L. Li1, A. Nguyen1, P. Toste1, N. Wu1, C. Choi1, J. Smogorzewski1, T. Donahue1 1University Of California – Los Angeles,Surgery,Los Angeles, CA, USA
Introduction:
We have previously shown that exposure of pancreatic cancer (PDAC) tumor associated fibroblasts (TAFs) to cytotoxic chemotherapy unleashes a tumor supportive and pro-inflammatory phenotype, increasing tumor cell (TC) growth and invasion in vitro and in vivo. We hypothesize that Nfkb is the predominant transcription factor driving this TAF response to DNA damage, and therefore genetic knockout of the activator subunit, p65 in TAFs will attenuate the induction of these genes and tumor cell viability.
Methods:
Immortalized PDAC TAFs (Logsden Lab) were homozygously deleted for p65 using CRISPR:Cas9. RT-PCR and MTT assay were done using standard protocols. Conditioned media was collected for 24 hours from the TAFs preconditioned with 48 hours of 100 nM gemcitabine or basal media. PANC-1 TCs were cultured in this media for two days and MTT assay used to assess cell viability and proliferation. CHIP-Seq data and promoter motif enrichment was derived from NIH Epigenome roadmap public data and processed using GREAT.
Results:
Gemcitabine treatment of PDAC TAFs induced a pro-inflammatory gene expression program including statistically significant (p < 0.05) upregulation of a number of cytokines: IL-6 (9.2 fold), IL-8 (17.8 fold), IL-1a (62.1 fold), and Spp1 (2.7 fold) [Fig 1A]. The genes are part of a DNA damage response collectively known as the senescence associated secretory phenotype (SASP). Each of these genes have Nfkb binding sites identified from Chip-Seq for p65 or from binding site motif identification. The expression of these gemcitabine-induced SASP genes followed the cytosolic to nuclear translocation of the activating subunit of the Nfkb complex: p65 [Fig 1B]. Homozygous knockout of p65 in TAFs (TAF p65 -/-) resulted in loss of upregulation of multiple SASP mediators in gemcitabine treated cells as compared to wild-type TAFs with intact p65 (p<0.05). Additionally, two genes in our panel expressed at low levels under basal conditions in WT TAFs (IL8, IL-6) were silenced in the p65 KO TAFs. Conditioned media collected from naïve and 48 hour gemcitabine treated WT TAFs or non-target control (RelA NC) increased the proliferation of PDAC TCs (Panc-1, p<0.05); however, conditioned media from gemcitabine treated p65 KO (-/-) TAFs did not permit cancer cell growth in low serum conditions [Fig 1C].
Conclusion:
Gemcitabine treatment of PDAC TCs shows a cytotoxic effect in vitro, but is complicated by the emergence of resistance in vivo. TAFs influence on TCs is typically not appreciated in the in vitro setting. We show that culture media conditioned by TAFs is supportive of TC growth, in vitro, and inflammatory cytokines expressed subsequent to induction of DNA damage in TAFs are silenced in the absence of p65. We conclude that in the presence of gemcitabine, Nfkb signaling in stromal cells can promote cancer cell growth.
