2. Cancer Cell Secretome of Wild-Type P53 PDAC Cells Exhibits Suppressor Roles
Researchers previously published that gain-of-function mutant p53 isoforms exert their hyper-proliferative effects on cancer cells also through the alteration of their secretome
[16]. Here, they aimed to study whether WTp53 may exhibit suppressor roles through its influence on the secretome of PDAC cells, in accordance with the tumor suppressor role of WTp53. By using the previously published approach
[16], researchers induced the exogenous expression of WTp53 in p53-null AsPC-1 PDAC cells by using liposome-mediated transient transfection assay. In WTp53 PaCa3 cells, the endogenous p53 protein expression was transiently knocked down by transfection with siRNA. Forty-eight hours later, they checked the effective overexpression of p53 in AsPC-1 or knocking-down of WTp53 in PaCa3 cells by Western blotting and functionally analyzed their effect on cell growth induced by the presence or lack of wild-type p53, compared with their respective control (
Figure 1A). Subsequently, AsPC-1 or PaCa3 transfected cells were washed in PBS and then incubated in fresh culture medium for further 22 h to accumulate secreted proteins. The conditioned medium (CM) released by transfected AsPC-1 or PaCa3 cells was transferred to untransfected p53-null AsPC-1 cells, which were thus cultivated for 48 h with the secretome driven by the presence or absence of wild-type p53. This allowed us to study the functional effects of the secretome driven by wild-type p53 overexpression or by p53 knock-down, each one compared by their respective control. In accordance with the tumor suppressor role of wild-type p53,
Figure 1B,C show that p53-driven secretome is able to inhibit cell growth and promote apoptosis in AsPC-1 cells, as compared to its negative mock control. On the contrary, the conditioned medium of PaCa3 with silenced p53, is able to promote hyper-proliferative effects and to inhibit cell death of AsPC-1 cells, as compared to its negative control. The absence of extracellular p53 in WTp53-driven CM of AsPC-1 was previously proved by Western blotting
[16] and then further confirmed by mass spectrometry analysis in both AsPC-1 and PaCa3 cells. Furthermore, since p53 is able to transactivate autophagy-related genes and to induce autophagy flux
[18][19], researchers wondered whether even the conditioned medium driven by p53 can sustain autophagy.
Figure 1D shows that WTp53-driven secretome promotes autophagic vesicles. Specifically, the conditioned medium released by transfected AsPC-1 expressing WTp53 is able to promote autophagosome formation in untransfected AsPC-1 cells. Altogether, these data provide evidence that WTp53 influences the secretion of proteins or other molecules that can functionally contribute to the regulation of cell growth and cell death-related phenomena, such as apoptosis and autophagy responses.
Figure 1. Cancer cell secretome driven by wild-type p53 exhibits oncosuppressor roles. (A) Cell growth was measured by Cristal Violet assay in p53-null AsPC-1 cells transfected for overexpression of wtp53 and in PaCa3 after knocking-down of endogenous p53 to verify the transfection efficiency. Accompanying Western blotting of p53 and of GAPDH for control loading are reported. Statistical analysis * p < 0.05 wtp53 vs. Mock AsPC-1; sip53 vs. siCtrl PaCa3. (B) Cell growth was measured by Cristal Violet assay in untransfected p53-null AsPC-1 cells cultivated with WTp53-CM from AsPC1 or with sip53-CM from PaCa3, each one compared by their respective control. Statistical analysis * p < 0.05 CM-AsPC-1 WTp53 vs. CM-Mock; CM-PaCa3 sip53 vs. CM-Paca3 siCtrl. (C) Apoptosis was determined by the annexinV/FITC binding assay in AsPC-1 cultivated with wtp53-derived CM of AsPC-1 or with sip53-CM from PaCa3, each one compared by their respective control. Statistical analysis * p < 0.05 CM-AsPC-1 WTp53 vs. CM-Mock; CM-PaCa3 sip53 vs. CM-Paca3 siCtrl. (D) Autophagosome formation assay was determined by intracellular staining using the MDC probe in AsPC-1 cultivated with WTp53-derived CM of AsPC-1. Statistical analysis * p < 0.05 CM-AsPC-1wtp53 vs. CM-Mock.
5. Identification of Secreted Proteins from Wild-Type P53-Driven Secretome
After the investigation of the functional involvement of WTp53-driven secretome in the inhibition of PDAC cell growth and chemosensitivity to GEM, researchers aimed to identify the secreted proteins by WTp53 in PDAC cells. Thus, they compared the protein composition of the CM released by PaCa3 cells expressing WTp53 as compared to that of PaCa3 cells after p53 knock-down. To avoid protein cross-contamination by serum, cells were washed to remove DNA–liposome complexes and cultured for further 22 h to accumulate secreted proteins in serum-free culture medium. This serum-free culture period was previously identified as the maximum time period without delay of cell growth or signals of cell death, thus avoiding indiscriminate cellular lysis
[16]. A peptide liquid chromatography separation followed by mass spectrometry analysis and database search with Protein Pilot and Mascot was then performed. SWATH-MS analyses were performed in triplicates for each analyzed sample, and they were imported in the Skyline software to perform the label-free quantification and the identification of hyper- and hyposecreted proteins. Researhers identified and quantified 210 secreted proteins in both p53 knock-down and WT PaCa3 cells. Among them, the quantitative proteomic analysis reported the modulation (fold change > 1.3 and
p-value < 0.05) of 47 secreted proteins, of which 30 were hyper secreted in p53 knock-down PaCa3 cells and 17 were hyposecreted (
Table 1) compared to WTp53. Among the most hypersecreted proteins after p53-knock-down in PaCa3 cells there are GTP-binding protein Di-Ras2 (FC = 36.4), BCL-6 corepressor-like protein 1 (FC = 24.3), centrosomal protein of 78 kDa (FC = 23.7), glial fibrillary acidic protein (FC = 5.5), importin-5 (FC = 4.7), transferrin receptor protein 1 (FC = 3.8), isocitrate dehydrogenase (FC = 3.0), ATP-citrate synthase (FC = 2.9) and cathepsin B (FC = 2.6). On the other hand, p53 silencing caused a particular decrease in farnesyl pyrophosphate synthase (FC = 0.3), protein S100-A4 (FC = 0.3), transgelin-2 (FC = 0.3), tubulin beta-3 chain (FC = 0.3) and putative heat shock protein HSP 90-alpha A4 (FC = 0.3) in PaCa3 secretome.
Table 1. Forty-seven modulated proteins in PDAC secretome of knock-down p53 Paca3 cells, as compared to wild-type p53 cells identified by SWATH-MS technology (p < 0.05). Fold change (FC) represents the ratio between CM protein abundance of p53 knock-down (KD) and p53 wild-type PaCa3 cells.
Uniprot ID |
Uniprot Accession Name |
Protein Name |
Gene Name |
FC (KD/wt p53) |
p-Value |
O00410 |
IPO5_HUMAN |
Importin-5 |
IPO5 |
4.7 |
4.31 × 10−4 |
O75874 |
IDHC_HUMAN |
Isocitrate dehydrogenase |
IDH1 |
3.0 |
3.56 × 10−4 |
P01008 |
ANT3_HUMAN |
Antithrombin-III |
SERPINC1 |
1.6 |
2.66 × 10−4 |
P02786 |
TFR1_HUMAN |
Transferrin receptor protein 1 |
TFRC |
3.8 |
8.59 × 10−4 |
P04792 |
HSPB1_HUMAN |
Heat shock protein beta-1 |
HSPB1 |
1.8 |
1.72 × 10−2 |
P04908 |
H2A1_HUMAN |
Histone H2A type 1-B/E |
HIST1 |
2.1 |
2.39 × 10−3 |
P07478 |
TRY2_HUMAN |
Trypsin-2 |
PRSS2 |
1.3 |
4.27 × 10−2 |
P07858 |
CATB_HUMAN |
Cathepsin B |
CTSB |
2.6 |
3.33 × 10−3 |
P14136 |
K2C8_HUMAN |
Glial fibrillary acidic protein |
GFAP |
5.5 |
1.49 × 10−2 |
P14555 |
PA2GA_HUMAN |
Phospholipase A2 |
PLA2G2A |
2.4 |
1.86 × 10−4 |
P19338 |
NUCL_HUMAN |
Nucleolin |
NCL |
1.4 |
9.66 × 10−4 |
P22692 |
IBP4_HUMAN |
Insulin-like growth factor-binding protein 4 |
IGFBP4 |
1.9 |
9.48 × 10−3 |
P25398 |
RS12_HUMAN |
40S ribosomal protein S12 |
RPS12 |
1.6 |
2.92 × 10−2 |
P37837 |
TALDO_HUMAN |
Transaldolase |
TALDO1 |
1.4 |
4.35 × 10−3 |
P40925 |
MDHC_HUMAN |
Malate dehydrogenase |
MDH1 |
1.8 |
9.26 × 10−3 |
P53396 |
ACLY_HUMAN |
ATP-citrate synthase |
ACLY |
2.9 |
6.18 × 10−4 |
P54652 |
HSP7C_HUMAN |
Heat shock-related 70 kDa protein 2 |
HSPA2 |
1.7 |
6.79 × 10−3 |
P62158 |
CALM_HUMAN |
Calmodulin |
CALM1 |
1.5 |
1.31 × 10−2 |
P80188 |
NGAL_HUMAN |
Neutrophil gelatinase-associated lipocalin |
LCN2 |
2.0 |
7.20 × 10−5 |
Q01105 |
SET_HUMAN |
Protein SET |
SET |
1.6 |
1.55 × 10−2 |
Q04828 |
AK1C1_HUMAN |
Aldo-keto reductase family 1 member C1 |
AKR1C1 |
1.5 |
8.20 × 10−3 |
Q15582 |
BGH3_HUMAN |
Transforming growth factor-beta-induced protein ig-h3 |
TGFBI |
1.4 |
5.93 × 10−3 |
Q32P51 |
RA1L2_HUMAN |
Heterogeneous nuclear ribonucleoprotein A1-like 2 |
HNRNPA1L2 |
1.3 |
7.76 × 10−3 |
Q5H9F3 |
BCORL_HUMAN |
BCL-6 corepressor-like protein 1 |
BCORL1 |
24.3 |
5.74 × 10−5 |
Q6UWE0 |
LRSM1_HUMAN |
E3 ubiquitin-protein ligase LRSAM1 |
LRSAM1 |
2.2 |
6.48 × 10−3 |
Q86TI0 |
TBCD1_HUMAN |
TBC1 domain family member 1 |
TBC1D1 |
2.5 |
8.13 × 10−5 |
Q92598 |
HS105_HUMAN |
Heat shock protein 105 kDa |
HSPH1 |
1.9 |
1.65 × 10−2 |
Q96HU8 |
DIRA2_HUMAN |
GTP-binding protein Di-Ras2 |
DIRAS2 |
36.4 |
4.02 × 10−4 |
Q96QV6 |
H2A1A_HUMAN |
Histone H2A type 1-A |
HIST1H2AA |
1.3 |
1.29 × 10−3 |
A8MST6 |
CEP78_HUMAN |
Centrosomal protein of 78 kDa |
CEP78 |
23.7 |
2.99 × 10−3 |
B2RPK0 |
HGB1A_HUMAN |
Putative high mobility group protein B1-like 1 |
HMGB1P1 |
0.6 |
8.43 × 10−3 |
O14556 |
G3P_HUMAN |
Glyceraldehyde-3-phosphate dehydrogenase |
GAPDHS |
0.5 |
2.66 × 10−2 |
P01034 |
CYTC_HUMAN |
Cystatin-C |
CST3 |
0.6 |
4.58 × 10−3 |
P02538 |
K2C6B_HUMAN |
Keratin, type II cytoskeletal 6A |
KRT6A |
0.5 |
1.53 × 10−3 |
P02768 |
ALBU_HUMAN |
Serum albumin |
ALB |
0.6 |
4.06 × 10−4 |
P07737 |
PROF1_HUMAN |
Profilin-1 |
PFN1 |
0.5 |
9.28 × 10−6 |
P08238 |
HS90B_HUMAN |
Heat shock protein HSP 90-beta |
HSP90AB1 |
0.6 |
1.14 × 10−2 |
P14324 |
FPPS_HUMAN |
Farnesyl pyrophosphate synthase |
FDPS |
0.3 |
9.93 × 10−4 |
P23284 |
PPIB_HUMAN |
Peptidyl-prolyl cis-trans isomerase B |
PPIB |
0.7 |
1.40 × 10−2 |
P26447 |
S10A4_HUMAN |
Protein S100-A4 |
S100A4 |
0.3 |
1.72 × 10−4 |
P37802 |
TAGL2_HUMAN |
Transgelin-2 |
TAGLN2 |
0.3 |
2.18 × 10−3 |
P60709 |
ACTB_HUMAN |
Actin, cytoplasmic 1 |
ACTB |
0.6 |
5.29 × 10−5 |
Q04760 |
LGUL_HUMAN |
Lactoylglutathione lyase |
GLO1 |
0.5 |
5.79 × 10−4 |
Q13885 |
TBB5_HUMAN |
Tubulin beta-3 chain |
TUBB3 |
0.3 |
2.70 × 10−3 |
Q58FG1 |
HS90A_HUMAN |
Putative heat shock protein HSP 90-alpha A4 |
HSP90AA4P |
0.3 |
2.16 × 10−3 |
Q92688 |
AN32B_HUMAN |
Acidic leucine-rich nuclear phosphoprotein 32 family member B |
ANP32B |
0.4 |
5.73 × 10−3 |
Q9Y536 |
PPIA_HUMAN |
Peptidyl-prolyl cis-trans isomerase A-like 4A |
PPIAL4A |
0.4 |
3.52 × 10−3 |
Interestingly, these secreted protein from p53 knock-down and WTp53 PaCa3 cells were further compared with the secreted proteins of AsPC-1 cells GOF R175H and R273H mutp53 isoforms and their respective controls that researchers previously identified using the same method
[16]. They identified several secreted proteins in common between CM-PaCa3 wild-p53 and CM-AsPC-1 GOF R175H and R273H. Specifically, the secreted heat shock protein beta-1 inhibited by WTp53 was hypersecreted by R175H-mutp53 AsPC-1
[16]; Cathepsin B and Transforming growth factor-beta-induced protein ig-h3 hyposecreted by WTp53 were hypersecreted in AsPc-1 cells GOF R273H; the heat shock protein HSP90-beta hypersecreted in WTp53was hyposecreted in AsPC-1 cells GOF R273H
[16].
In order to validate some differentially secreted proteins they performed Western bot analysis extending the evaluation also to other PDAC cell lines and to normal human pancreatic duct epithelial (HPDE1) as control (Figure 4). The results obtained confirm that p53 is able to determine hypersecretion of GAPDH and beta-actin proteins, as revealed in p53-null AsPC-1 cells in which p53 was overexpressed. On the other side, p53 knock-down reduced GAPDH and beta-actin secretion in Hs 766T and PaCa3 PDAC cell lines. It is noteworthy that p53 silencing in HPDE1 did not determine any significant change in protein secretion, suggesting that the deep modulation of protein secretome in PDAC cells by p53 may be a cancer-associated phenomenon.
Figure 4. Immunoblot validation of p53-dependent hyposecreted proteins. p53-null AsPC-1 cells were transiently transfected with WTp53 or mock control plasmids, Hs 776T and PaCa3 WTp53-PDAC cell lines and HPDE1 human pancreas non-tumor cells were transiently transfected with siP53 to downregulate TP53; a scramble siRNA (siCTRL) was used as a control. A total of 48 h after transfection, cells were washed and further incubated for 22 h in serum-deprived media. Secreted proteins were precipitated overnight and p53-dependent GAPDH and ß-Actin protein expression was confirmed by immunoblot. Amido black staining was used as loading control.
6. Dysregulated Pathways, Protein Interaction Networks and Upstream Regulators Related to Wtp53-Driven Secreted Proteins
Researchers analyzed modulated proteins by bioinformatics tools to obtain a global overview of the interaction network by which WTp53 can control tumor development through secreted proteins in PDAC. Ingenuity Pathway Analysis (IPA) was employed to identify the main pathways and biological processes associated with the oncosuppressor role of p53. The canonical pathways linked to the silencing of p53 are mainly related to aldosterone signaling, gluconeogenesis, protein ubiquitination, ferroptosis signaling, acetyl-CoA biosynthesis, glucocorticoid receptor signaling, clathrin-mediated endocytosis, SPINK1 pancreatic cancer pathway, methylglyoxal degradation and remodeling of epithelial adherens junctions (Figure 5A). The analysis of the molecular and cellular functions revealed an involvement of proteins associated with cell death and survival, protein synthesis and cellular movement functions (Figure 5B).
Figure 5. p53-driven canonical pathways and molecular and cellular functions. Bar-plot of canonical pathway significance (−log(p-value)) of altered secreted proteins in response to the silencing of p53 in PDAC (A). Activation/inhibition (z-score) was not predicted. Molecular and cellular functions with significance (p-value) and number of associated proteins (B).
To predict the upstream molecules (genes, transcription factors, microRNA, etc.) that may play a role in the observed secretome modulation and, thus, in response to a p53 silencing in PDAC, they performed the upstream regulator analysis through IPA software. IPA analysis suggested that TP53, IPMK, MYC and MTOR are among the most significant upstream regulators predicted as inhibited or activated (
Figure 6). Interestingly, the most significant upstream regulator, with a
p-value of 2.57
−10, was TP53. TP53 was predicted inhibited (z-score of −1.289) and the 40.4% of the total modulated proteins resulted under its regulation. Among these, eight proteins that are inhibited by TP53 were detected as hypersecreted (ACLY, TGFB1, TFRC, TALDO1, LCN2, IGFBP4, HSPH1, CTSB) after p53 knock-down, whereas five proteins that are activated by TP53 resulted to be hyposecreted (ACTB, TAGLN2, S100A4, PFN1, FDPS) after the p53 knock-down in PaCa3 cells. Moreover, the upstream regulators MYC and MTOR were predicted as activated (z-score of 2.157 and 2.984, respectively) with a
p-value of 2.75
−7 and 1.17
−6, respectively. Instead, the upstream regulator IPMK was predicted as inhibited (z-score of −2.00) with a
p-value of 9.87
−8. MYC and MTOR are well-known oncogenes usually activated in cancer, whereas IPMK can act as a co-activator of TP53
[23][24].
Figure 6. Upstream gene regulator analysis. In p53 knocking-down conditions, TP53 and IPMK are the most significant inhibited upstream regulators, while MYC and MTOR resulted the most significant activated regulators.
Researchers then employed STRING software in order to investigate protein–protein interactions and to explore the functional enriched pathways. They manually added p53 to the list of regulated proteins and found that it directly interacts with nine hypersecreted (Figure 7A) and eight hyposecreted proteins (Figure 7B). Concerning the hypersecreted proteins after p53 knock-down, the network analysis reported a cluster of interaction between p53 and SET, HSPB1, K2C8, NUCL, CATB, TFR1, MDHC, HSP7C, ACLY; while among the hyposecreted proteins after p53 knock-down there was a cluster of interaction involving S10A4, ALBU, PPIB, HS90B, HS90A, ACTB, G3P, TBB5. The functional enrichment analysis also showed that most of the hypersecreted proteins belong to extracellular space and exosomes, supporting their role in cell–cell communications.
Figure 7. STRING analysis. Protein–protein interactions among modulated proteins. p53 was manually added to identify potentially related connections. The network of hypersecreted proteins is reported in (A) while the network of hyposecreted proteins is reported in (B).
Finally, the diseases and functions analysis performed through the IPA software highlighted the involvement of proteins associated with leukocytes movement (HSPB1, PPIB, ACTB, HS90B, ALB and S10A4), carcinoma (CATB, ALB, CYTC and SAA4) and advanced malignant tumor (CATB, BGH3, ACTB, CYTC and SAA4) disease classes (Table 2).
Table 2. Diseases and functions analysis. Leukocytes movement, carcinoma and advanced malignant tumor resulted the most significant diseases and functions classes associated with modulated proteins. The proteins with their function and regulation after p53 knock-down are reported in the table.
Diseases and Functions |
Leukocytes movement |
Protein |
Regulation |
Function |
HSPB1 |
Up-regulated |
Decrease movement of leucocytes |
PPIB |
Down-regulated |
Increase movement of leucocytes |
ACTB |
Down-regulated |
Increase movement of leucocytes |
HS90B |
Down-regulated |
Increase movement of leucocytes |
ALB |
Down-regulated |
Increase movement of leucocytes |
S10A4 |
Down-regulated |
Increase movement of leucocytes |
Carcinoma |
Protein |
Regulation |
Function |
CATB |
Up-regulated |
Increase carcinoma |
ALB |
Down-regulated |
Decrease carcinoma |
CYTC |
Down-regulated |
Decrease carcinoma |
SAA4 |
Down-regulated |
Decrease carcinoma |
Advanced malignant tumour |
Protein |
Regulation |
Function |
CATB |
Up-regulated |
Increase advanced malignant tumour |
BGH3 |
Up-regulated |
Increase advanced malignant tumour |
ACTB |
Down-regulated |
Decrease advanced malignant tumour |
CYTC |
Down-regulated |
Decrease advanced malignant tumour |
SAA4 |
Down-regulated |
Increase advanced malignant tumour |