KLHDC7B-DT aggravates pancreatic ductal adenocarcinoma development via inducing the crosstalk between cancer cells and macrophages.

Tumor microenvironment (TME) exerts key roles in pancreatic ductal adenocarcinoma (PDAC) development. However, the factors regulating the crosstalk between PDAC cells and TME are largely unknown. In this study, we identified a long noncoding RNA (lncRNA) KLHDC7B-DT, which was upregulated in PDAC and correlated with poor survival of PDAC patients. Functional assays demonstrated that KLHDC7B-DT enhanced PDAC cell proliferation, migration, and invasion. Mechanistically, KLHDC7B-DT was found to directly bind IL-6 promoter, induce open chromatin structure at IL-6 promoter region, activate IL-6 transcription, and upregulate IL-6 expression and secretion. The expression of KLHDC7B-DT was positively correlated with IL-6 in PDAC tissues. Via inducing IL-6 secretion, KLHDC7B-DT activated STAT3 signaling in PDAC cells in an autocrine manner. Furthermore, KLHDC7B-DT also activated STAT3 signaling in macrophages in a paracrine manner, which induced macrophage M2 polarization. KLHDC7B-DT overexpressed PDAC cells-primed macrophages promoted PDAC cell proliferation, migration, and invasion. Blocking IL-6/STAT3 signaling reversed the effects of KLHDC7B-DT on macrophage M2 polarization and PDAC cell proliferation, migration, and invasion. In conclusion, KLHDC7B-DT enhanced malignant behaviors of PDAC cells via IL-6-induced macrophage M2 polarization and IL-6-activated STAT3 signaling in PDAC cells. The crosstalk between PDAC cells and macrophages induced by KLHDC7B-DT represents potential therapeutic target for PDAC.


Indirect coculture of PDAC cells and macrophages
Macrophages (3 × 10 5 cells/well) were seeded into 24-well plates. PDAC cells (1 × 10 5 cells/well) with KLHDC7B-DT overexpression or silencing were seeded into the upper chambers of transwell inserts (Millipore, Bedford, MA, USA). The inserts were placed into the above described wells containing macrophages. After coculture for 72h, macrophages were collected to detect target genes expression. In addition, Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20201259/903697/cs-2020-1259.pdf by guest on 08 February 2021 after coculture for 72h, the educated macrophages were seeded into another 24-well plate. PDAC cells were seeded into the upper chambers of transwell inserts, which were placed into the wells containing educated macrophages. After coculture for another 24h, the migration and invasion of PDAC cells were detected as below described. Furthermore, the educated macrophages were seeded into the upper chambers of inserts which were placed into the wells containing PDAC cells. After coculture for another 24h, the proliferation of PDAC cells were detected as below described.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from frozen tissues and cultured cells using the TRIzol reagent (Invitrogen) following the manufacturer's manual. After quantification using

Cell proliferation assay
Cell proliferation was measured by Glo cell viability and 5-ethynyl-2'-deoxyuridine (EdU) incorporation assays as described before [35]. For Glo cell viability assay, 3,000 indicated PDAC cells were seeded per well in 96-well plates. At the indicated time, cell viabilities were measured using the CellTiter-Glo ® Luminescent Cell Viability Assay (Promega, Madison, WI, USA) according to the manufacturer's manual. EdU incorporation assays were conducted using the EdU Kit (Roche, Mannheim, Germany) according to the manufacturer's protocol. The percentages of EdU-positive cells were quantified using Zeiss fluorescence microscope based on at least five randomly fields. Cell migratory and invasive abilities were detected using transwell assays as described before [36]. For transwell migration assay, 20,000 indicated cells resuspended in FBS-free medium were seeded into the upper chambers of 8 µm pore inserts (Millipore). Complete medium added with or without macrophages was added to the lower chambers. After incubating or co-culturing with macrophages for 24 hours, the migrated cells were fixed, stained, and counted based on at least five randomly fields. For transwell invasion assay, the procedures were consistent with transwell migration assay except the pre-coating of Matrigel before the seeding of cells.

Confocal RNA fluorescence in situ hybridization
For in situ detection of KLHDC7B-DT in PDAC cells, the KLHDC7B-DT probes were designed and synthesized by Advanced Cell Diagnostics (ACD, Newark, CA, USA). The hybridization and fluorescence detection were performed using the RNAscope Fluorescent Multiplex Detection Kit (ACD). Confocal laser scanning microscopy (Leica, Wetzlar, Germany) was used to detect the localization of KLHDC7B-DT in PDAC cells.

Subcellular Fractionation
Subcellular fractionation was performed as described before [37]. The RNAs in biochemically fractionated components were measured using qRT-PCR as described above.

Western blot
Total proteins were extracted from indicated cells were with the RIPA Lysis Buffer

Enzyme-linked immunosorbent assay (ELISA)
Indicated PDAC cells with 80% confluence were washed three times and cultured in FBS-free medium for another 48 hours. Then, the cell supernatant was collected and the concentration of IL-6 in the supernatant was detected using Human IL-6 ELISA Kit (ab178013, Abcam).

Statistical analysis
Statistical analyses were conducted using GraphPad Prism 6.0 Software. . The normality of the data was tested using the Shapiro-Wilk normality test. Student's t-test was used to analyze the differences between two groups. One-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test or Tukey's multiple comparisons test was used to analyze the differences between more than two groups as indicated in figure legends. Wilcoxon matched-pairs signed rank test, Mann-Whitney test, log-rank test, Spearman correlation analysis, and Pearson's chi-square test were performed to analyze the clinical data as indicated in figure and table legends. P < 0.05 was considered as statistically significant.

KLHDC7B-DT was upregulated in PDAC and correlated with poor survival of PDAC patients
To search the lncRNAs correlated with survival of pancreatic adenocarcinoma (PAAD) patients, we analyzed the cancer genome atlas (TCGA) data using the online tool GEPIA (http://gepia.cancer-pku.cn/). The most significant survival-correlated genes in PAAD were shown in Supplementary Table 1. Among these genes, we noted CTA-384D8.35, which was also named as KLHDC7B-DT. Through analyzing TCGA data using GEPIA, we found that increased expression of KLHDC7B-DT was correlated with both poor overall survival and disease free survival of PAAD patients (Supplementary Fig. 1A-B). In addition, KLHDC7B-DT was found to be significantly increased in PAAD tissues compared with that in normal pancreatic tissues ( Supplementary Fig. 1C). To further investigate the expression and clinical relevance of KLHDC7B-DT in PDAC, we collected 64 pairs of PDAC tissues and adjacent noncancerous pancreatic tissues. The expression of KLHDC7B-DT in these tissues was measured and the results showed that KLHDC7B-DT was markedly increased in PDAC tissues compared with noncancerous pancreatic tissues (Fig. 1A). Correlation  (Table 1) attenuated by IL-6 neutralizing antibody (Fig. 5A). EdU incorporation assays showed that the increased EdU positive cells of MIA PaCa-2 caused by KLHDC7B-DT overexpression were also attenuated by IL-6 neutralizing antibody (Fig. 5B).
Transwell migration assays showed that the increased cell numbers of migration of MIA PaCa-2 cells caused by KLHDC7B-DT overexpression were attenuated by IL-6 neutralizing antibody (Fig. 5C). Transwell invasion assays showed that the increased cell numbers of invasion of MIA PaCa-2 cells caused by KLHDC7B-DT overexpression were attenuated by IL-6 neutralizing antibody (Fig. 5D). In addition,

KLHDC7B-DT induced macrophages M2 polarization in an IL-6-dependent paracrine manner
PTEN-PI3K-AKT signaling and STAT3 signaling have been revealed to be involved in the macrophages M2 polarization [38][39][40]. IL-6 is also frequently reported to activate AKT and STAT3 [41,42]. Therefore, we further investigated whether KLHDC7B-DT modulated macrophages M2 polarization using the in vitro indirect coculture system (Fig. 6A). We first examined the effects of KLHDC7B-DT on AKT and STAT3 in THP-1 differentiated macrophages. As shown in Fig. 6B, phosphorylation levels of AKT and STAT3 in macrophages were increased after coculture with KLHDC7B-DT overexpressed MIA PaCa-2 cells compared with those in macrophages coculture with control MIA PaCa-2 cells. Conversely, phosphorylation levels of AKT and STAT3 in macrophages were reduced after coculture with KLHDC7B-DT silenced Capan-1 cells compared with those in macrophages coculture with control Capan-1 cells (Fig. 6C). M1 macrophage markers including IL-12, TNF-α, and iNOS were decreased in macrophages after coculture with KLHDC7B-DT overexpressed MIA PaCa-2 cells compared with those in macrophages coculture with control MIA PaCa-2 cells (Fig. 6D). The decreases of M1 macrophage markers were largely reversed by an IL-6 neutralizing antibody (Fig. 6D).
M2 macrophage markers including IL-10, CD163, and ARG1 were increased in macrophages after coculture with KLHDC7B-DT overexpressed MIA PaCa-2 cells compared with those in macrophages coculture with control MIA PaCa-2 cells (Fig.   6E). The increases of M2 macrophage markers were also largely reversed by an IL-6 neutralizing antibody (Fig. 6E). Conversely, the M1 macrophage markers were increased in macrophages after coculture with KLHDC7B-DT silenced Capan-1 cells compared with those in macrophages coculture with control Capan-1 cells (Fig. 6F).
The M2 macrophage markers were reduced in macrophages after coculture with KLHDC7B-DT silenced Capan-1 cells compared with those in macrophages coculture with control Capan-1 cells (Fig. 6G). Due to IL-4 is well-known to induce macrophages M2 polarization and IFN-γ is well-known to induce macrophages M1 polarization, we next investigated the potential effects of KLHDC7B-DT on IL-4 and IFN-γ. As shown in Supplementary Fig. 4A

M2 macrophages induced by KLHDC7B-DT facilitated PDAC cell proliferation, migration, and invasion
We further investigated the potential roles of M2 macrophages induced by KLHDC7B-DT on PDAC cell proliferation, migration, and invasion using the in vitro indirect coculture system (Fig. 7A). Glo cell viability assays revealed that the cell viabilities of MIA PaCa-2 were increased after coculture with macrophages educated  (Fig. 7G). Transwell migration assays revealed that the cell numbers of migration of Capan-1 were decreased after coculture with macrophages educated by KLHDC7B-DT silenced Capan-1 cells compared with those after coculture with macrophages educated by control Capan-1 cells (Fig. 7H). Transwell invasion assays revealed that the cell numbers of invasion of Capan-1 were decreased after coculture with macrophages educated by KLHDC7B-DT silenced Capan-1 cells compared with those after coculture with macrophages educated by control Capan-1 cells (Fig. 7I).
Thus, these data suggested that M2 macrophages induced by KLHDC7B-DT facilitated PDAC cell proliferation, migration, and invasion.

IL-6 blocking attenuated the roles of KLHDC7B-DT educated macrophages in PDAC cell proliferation, migration, and invasion
Given that our above results had shown that KLHDC7B-DT induced macrophages M2 polarization in an IL-6-dependent paracrine manner, we further investigated whether the roles of KLHDC7B-DT educated macrophages in PDAC cell proliferation, migration, and invasion were dependent on IL-6 induced M2 polarization. An IL-6 neutralizing antibody was added to the in vitro coculture system of KLHDC7B-DT overexpressed MIA PaCa-2 cells and macrophages. Then, the educated macrophages were further co-cultured with MIA PaCa-2 cells (Fig. 8A), and the effects of the educated macrophages on MIA PaCa-2 cell proliferation, migration, and invasion were detected. Glo cell viability assays showed that the increased cell were attenuated by IL-6 neutralizing antibody (Fig. 8B). EdU incorporation assays showed that the increased EdU positive cells of MIA PaCa-2 caused by KLHDC7B-DT educated macrophages were also attenuated by IL-6 neutralizing antibody (Fig. 8C). Transwell migration assays showed that the increased cell numbers of migration of MIA PaCa-2 cells caused by KLHDC7B-DT educated macrophages were attenuated by IL-6 neutralizing antibody (Fig. 8D). Transwell invasion assays showed that the increased cell numbers of invasion of MIA PaCa-2 cells caused by KLHDC7B-DT educated macrophages were attenuated by IL-6 neutralizing antibody (Fig. 8E). Collectively, these findings showed that IL-6 blocking attenuated the roles of KLHDC7B-DT educated macrophages in PDAC cell proliferation, migration, and invasion. Combining with Fig. 6 data, these data also suggested that the roles of KLHDC7B-DT educated macrophages in PDAC cell proliferation, migration, and invasion were dependent on IL-6 induced M2 polarization.
KLHDC7B-DT is located in chromosome 22q13.33, and has only one exon.
KLHDC7B-DT has 637 nucleotides in length. Previous reports showed that KLHDC7B-DT was increased in cervical cancer, clear cell renal cell carcinoma, and lung adenocarcinoma [47][48][49]. But the roles and mechanisms of action of KLHDC7B-DT in cancers have not been studied. In this study, TCGA data and our own cohort both showed that KLHDC7B-DT was increased in PDAC tissues.
Moreover, TCGA data and our own cohort both found that increased expression of KLHDC7B-DT was correlated with worse overall survival and disease free survival.
Furthermore, the binding of lncRNAs may modulate DNA methylation levels of their binding regions [52]. These different epigenetic modifications further induced the activation or inhibition of their bound targets. The epigenetic modification complexes, such as histone methyltransferase, histone deacetylase, and also others, which are involved in the regulation of IL-6 transcription by KLHDC7B-DT need further investigation.
Intriguingly, we also found that IL-6 secreted by PDAC cells and induced by KLHDC7B-DT could also activate STAT3 and AKT signaling in macrophages in a paracrine manner. The activation of STAT3 and AKT signaling has been frequently revealed to induce macrophages M2 polarization [38][39][40]. Consistently, in this study, we found that KLHDC7B-DT induced macrophages M2 polarization through Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20201259/903697/cs-2020-1259.pdf by guest on 08 February 2021 promoting IL-6 secretion in the in vitro coculture system of PDAC cells and macrophages. Macrophages may be induced to be differentiated into M1 type or M2 type. The M1 macrophages are generally revealed to have tumor suppressive roles, and while the M2 macrophages are reported to have oncogenic roles [55]. The roles of IL-6 in macrophages polarization are complex and various. In some particular conditions, IL-6 was regarded as pro-inflammatory M1 signature marker [56][57][58].
However, in other conditions IL-6 was expressed by M2 macrophages, such as the increased production of IL-6 by M2 macrophages which were induced by heparanase enzyme highly expressed pancreatic carcinoma cells [59]. Moreover, IL-6 was also reported to induce M2 polarization. IL-6 was reported to stimulate adipose tissue macrophages M2 polarization [60], promote mouse macrophages and human monocytes M2 polarization during Trypanosoma cruzi infection [61]. IL-6 augmented IL-4-induced macrophages M2 polarization [60,62,63]. IL-6 promoted M2 polarization and IL-6 receptor antibodies antagonized M2 polarization in triple-negative breast cancer [39]. IL-6 induced M2 macrophages alternative activation in glioblastoma [64]. IL-6 neutralizing antibodies reduced alternatively activated M2 macrophages population, but did not affect classically activated M1 macrophages population in glioblastoma [64]. IL-6 produced by prostate epithelial cells induced macrophages M2 polarization in prostate cancer [40]. Here, we also showed that IL-6 induced macrophages M2 polarization in PDAC. These reports showed that the roles and mechanisms of IL-6 in macrophage polarization are largely tissue-specific, which need further detailed investigations. Moreover, we further In conclusion, this study identified KLHDC7B-DT as an overexpressed, prognosis-related, and oncogenic lncRNA in PDAC. KLHDC7B-DT directly bound to IL-6 promoter, activated IL-6 transcription, upregulated IL-6 expression and secretion.
The secreted IL-6 activated STAT3 signaling in PDAC cells in an autocrine manner.
The activation of IL-6/STAT3 signaling in PDAC cells promoted PDAC malignancy.
The secreted IL-6 also activated STAT3 signaling in macrophages in a paracrine manner. The activation of IL-6/STAT3 signaling in macrophages induced Downloaded from http://portlandpress.com/clinsci/article-pdf/doi/10.1042/CS20201259/903697/cs-2020-1259.pdf by guest on 08 February 2021 macrophages M2 polarization, which also promoted PDAC malignancy (Fig. 9). The crosstalk between PDAC cells and macrophages induced by KLHDC7B-DT represents potential therapeutic target for PDAC.       Scale bars, 100µm. Data are represented as mean ± SD based on three independent experiments. *P < 0.05, **P < 0.01, ns, not significant, by one-way ANOVA followed by Tukey's multiple comparisons test.  Data are represented as mean ± SD based on three independent experiments. *P < 0.05, **P < 0.01, ns, not significant, by one-way ANOVA followed by Tukey's multiple comparisons test.