As a Novel Tumor Suppressor, LHPP Promotes Apoptosis by Inhibiting the PI3K/AKT Signaling Pathway in Oral Squamous Cell Carcinoma

Abstract

Oral squamous cell carcinoma (OSCC) refers to the malignant tumor of the head and neck with a highest morbidity. It exhibits a poor prognosis and unsatisfactory treatment partially attributed to delayed diagnosis. As indicated from existing reports, the protein histidine phosphatase LHPP acts as a vital factor in tumorigenesis in liver, lung, bladder, breast and pancreatic tumor tissues. Thus far, the functional mechanism of LHPP in OSCC remains unclear. DGE analysis, OSCC cell lines and OSCC cases were found that LHPP was down-regulated in OSCC tissues and cells compared with that in normal oral mucosa tissues and cells, and was closely related to OSCC differentiation. Cell counting Kit 8 test, EdU proliferation test, scratches test, invasion test, monoclonal formation test, mouse xenograft tumor model, HE staining and immunohistochemistry showed that LHPP inhibited OSCC growth, proliferation and migration in vivo and in vitro. GO and KEGG enrichment analysis, LHPP transcription factor analysis and flow cytometry found that LHPP promotes the apoptosis of OSCC by decreasing the transcriptional activity of p-PI3K and p-Akt. Finally, our results suggested that LHPP inhibited the progression of OSCC through the PI3K/AKT signaling pathway, indicating that LHPP may be a new target for the treatment of OSCC.

Keywords: LHPP; Oral squamous cell carcinoma; PI3K/AKT pathway; apoptosis; proliferation.

Figure 1

Low LHPP expression in OSCC tissues and cells (A) The Volcano plot indicated the genes exhibiting the differential expression for normal and OSCC sample. (B) In accordance with the boxplot, LHPP in the OSCC samples is lower than normal sample. (C) Pairwise boxplot suggested LHPP with a relatively low expressing level within tumor sample. (D, E) The expression status of LHPP between different age and gender, revealing the relationships of LHPP with age and gender. (F) The survival analysis indicated that LHPP is a risk factor and is related to the survival and prognosis of patient. (G, H) The mRNA and protein levels in normal oral keratinocytes HOK cells and OSCC cells was assessed by RT-PCR and Western blotting. (I, J, K) Western blot of the LHPP expression in OSCC tissues and adjacent normal tissues (n= 3). *P < 0.05, ***P < 0.001, ****P < 0.0001.

Figure 2

LHPP expression is closely related to OSCC differentiation degree (A) HE staining and IHC staining of LHPP in normal oral mucosas and differentiated OSCC tissues. (B) MOD analysis of the LHPP expression in normal oral mucosas and differentiated OSCC tissues. ***P < 0.001, ****P < 0.0001.

Figure 3

Overexpressed LHPP cell lines were constructed (A, B, D, E) RT-PCR and WB assay for the LHPP expression in OE-LHPP group of SCC15 and SCC25 cell lines. (C, F) IFC staining of LHPP in NC, Vector and OE-LHPP group in SCC15 and SCC25 cell lines (200X). ***P < 0.001.

Figure 4

Over-expression of LHPP inhibited cell proliferation, migration and invasion in OSCC cells (A, B) Cell viability of SCC15 and SCC25 in NC, Vector, OE-LHPP groups cells for 24, 48 and 72 hours. (C) EdU assay of cell proliferation of SCC15 and SCC25 cells over-expressed LHPP or not for 24h. (D, E) The colony formation assay of SCC15 and SCC25 in NC, Vector, OE-LHPP groups for 14 days. (F, G) Transwell analysis for invasion of SCC15 for 24 h and SCC25 cells for 12 h. (H, I) RT-PCR analysis of snail, MMP2, N-cadherin and E-cadherin in the NC, Vector and OE-LHPP groups of SCC15 and SCC25. (J, K) The wound healing detection of SCC15 for 24, 48, and 72 hours and SCC25 cells for 12, 24, and 36 hours. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Overexpressed LHPP restricted the growth of xenograft tumors in vivo (A) Images of the xenograft tumors from BALB/c mice induced with NC, Vector, OE-LHPP groups cells. (B) Technical roadmap of tumor formation experimental procedures in nude mice. (C) Changes in tumor weight within 30 days after injection of cells in BALB/c mice. (D) The tumors peeled from BALB/c mice are presented. (E) Tumors volume after being inoculated with different groups of SCC15 cells for 30 days. (F, G, H) Representative images of HE and LHPP IHC staining of xenografts in NC, Vector and OE-LHPP groups. (I) Representative images of PCNA IHC staining of xenografts in NC, Vector and OE-LHPP groups. **p<0.01, ***P < 0.001.

Figure 6

Over-expression LHPP promoted cells apoptosis by suppressing PI3K/AKT signaling pathway (A) The network revealed the regulating relationship among LHPP, ATP4B and other related genes. (B) LHPP transcription factor prediction for LHPP by GCBI website. (C, D) RT-PCR analysis of the bcl-2 and bax expression level in NC, Vector and OE-LHPP group of SCC15 and SCC25 cell lines. (E, F, G) Flow cytometry to analyze apoptosis of OSCC cell. The apoptosis of OE-LHPP group was more significant than that of NC group and Vector groups. (H, I, J, K) WB assay for the bax, bcl-2 and cleaved-Caspase 3 expression in SCC15 and SCC25 cell lines. (L, M, N, O) The protein expression of AKT, p-AKT, PI3K, p-PI3K were estimated by performing the WB assay. *p<0.1, **p<0.01, ***P < 0.001.

Figure 7

PI3K/ AKT play a great role in the promotion mechanism of LHPP on oral squamous carcinoma cells apoptosis (A, B) The protein expressions of bax, bcl-2, cleaved-caspase 3, PI3K, p-PI3K, AKT and p-AKT were analyzed by performing the Western blotting assay. (C, D) The statistical investigation of the protein expression in SCC15 and SCC25 cells. (E) A predicted cellular signaling pathway model showing LHPP as tumor suppressor. *p<0.1, **p<0.01.