Several functions of TPD52 have been studied in various cancer cells [1–21]. However, the role of TPD52 in hypoxia has never been reported, to the best of our knowledge. The central tumor region is exposed to hypoxia, whereby the cancer cells obtain the ability to resist radiation and drug therapies . Our previous study reported that TPD52 is strongly expressed at the center of OSCC. Similarly, a histological specimen of a tongue OSCC tissue (Supplemental Fig. S1) shows the expression of TPD52 at a hypoxic region in the tumor. Then, we examined whether hypoxic conditions induce the expression of the TPD family (Fig. 1). Only TPD52 was increased by hypoxia in OSCC cells at both the mRNA and protein levels. However, this was not observed in NHEK cells. Moreover, TPD52 mRNA and protein levels increased most prominently in SAS cells. These results suggest that TPD52 may be involved in the survival and proliferation of OSCC cells.
HIF is known to be an important factor for the survival of various cancer cells under hypoxic conditions  and is involved in infiltration (reviewed in ), metastasis , metabolic reprogramming, and resistance to chemo and radiation therapies . Further, anticancer drugs targeting HIF are currently being developed (reviewed in ). We investigated whether increased expression of TPD52 under hypoxia was regulated by HIF (Fig. 2). Contrary to our expectation, knockdown of HIF-1/2α did not increase TPD52 mRNA and protein under hypoxia. In addition, the promoter assay for TPD52 showed not only that TPD52 promoter activity was decreased by hypoxia, but also that knockdown of HIF-1/2α showed little effect. These results indicate that the increase in TPD52 under hypoxia is independent of transcription. HIF-1α is always degraded by the action of PHDs under normoxia, and the activity of PHDs is decreased by hypoxia due to lack of O2 . Cobalt is a well-known PHD inhibitor, which induces the expression of HIF-1α via the phosphoinositide 3-kinase (PI3K) pathway, resulting in the activation of HIF-1α even under normoxia . Therefore, we carried out the next experiment, where HIF-1α was induced by the addition of CoCl2 under normoxia. However, this did not increase TPD52 mRNA, nor did it increase TPD52 promoter activity. These results reinforced the observation that TPD52 is HIF-independent and transcription-independent.
We also reported  that the expression of TPD52 mRNA is post-transcriptionally regulated through the stability of TIA-1 and TIAR, major components of SGs . Stress granules are structures that are temporarily formed in the cytoplasm by stress stimuli, such as hypoxia , endoplasmic reticulum stress , heat shock , and viral infection . We examined whether the increase in TPD52 mRNA resulted from an increase in mRNA stability, and, if so, whether TIA-1 and TIAR were involved. Therefore, we carried out RNA degradation and RIP assays. As a result, hypoxia increased the stability of TPD52 mRNA by approximately 2-fold. The downregulation of TIA-1 and TIAR decreased stability under normoxia, in agreement with our previous study . Interestingly, under hypoxia, the knockdown of these genes showed drastic effects, whereby mRNA increased by hypoxia stability was abolished more than that observed in normoxia. The RIP assay showed that hypoxia reduced the binding abilities of TIA-1 and TIAR to TPD52 mRNA. Therefore, it was suggested that the increase in TPD52 mRNA under hypoxia may be due to increased binding of TIA-1/TIAR, which was triggered by hypoxic stress. TIA-1 and TIAR are important components of SGs . Translation efficiency, mRNA turnover, and mRNA stability are regulated by SG formation . Supplemental Fig. S2 shows the aggregation of TIA-1 and TIAR in the cytosol of the cells under hypoxia, indicating the formation of SGs. Thus, the involvement of SG formation in the increase of TPD52 mRNA might be shown in the present study, although further investigation is required.
In order to investigate the proliferation, survival, and apoptosis of TPD52 on OSCC cells exposed to hypoxic conditions, we investigated the combined effects of TPD52 knockdown and inhibition of HIF (Fig. 4). PX-478, a chemical inhibitor of HIF, inhibits the translation of HIF-1α . TPD52 knockdown or PX-478 alone led to reduced MTT activity and increased caspase 3/7 activity under hypoxia. However, the combined use of these drastically increased the effects. Since the repressing effects of TPD52 knockdown and inhibition of HIF are thought to be independent of each other, as shown in the previous subsection, the result may be due to the interception of two (or possibly more) pathways. Next, the combined effects of TPD52 overexpression and the addition of PX-478 were examined. Overexpression of TPD52 alone increased MTT activity and decreased caspase 3/7 activity, showing the opposite effects of knockdown. However, the effects of TPD52 overexpression were almost entirely abolished. This may be triggered by HIF inhibition, thereby resulting in the loss of cell viability maintenance. Therefore, we hypothesized that the combined effects can reduce xenografted tumor and moved on to in vivo studies.
TPD52 knockdown with the addition of PX-478 in tumor-xenograft mice demonstrated decreased tumor volume, as observed in the in vitro study (Fig. 5). These results suggest the possibility of a clinical application for cancer therapeutics. In immunohistochemistry, knockdown of TPD52 and addition of PX-478 each induced cell death at the center of the tumor, where the environment was most hypoxic. This also increased the expression of p62 and Akt at the tumor periphery. p62 is an autophagy-related protein that is reported to accumulate in the cytosol during autophagy failure . Akt is related to macro-autophagy, and the activation of Akt initiates autophagy, followed by the consumption of p62 and formation of autophagosomes . Conversely, Shang et al. reported  that suppression of TPD52 induces autophagy-induced cell death during irradiation, resulting from the consumption of p62, while Zhao et al.  reported that knockdown of TPD52 decreased Akt. Therefore, the results of the present study suggest that TPD52 may inhibit autophagy signaling by modulating the Akt signaling pathway. However, inhibition of HIF with knockdown of TPD52 might induce cell death in cancer cells under hypoxia due to cellular starvation through aberrant acceleration of autophagy.
HIF is reportedly involved in the growing malignancy of various cancer cells through upregulation of survivability under hypoxia  and is thought to be a molecular target for cancer therapeutics . However, several groups, including ours, have studied the role of TPD52 in the proliferation and survival of various cancer cells [1–19]. In the present study, we focused on the roles of TPD52 in OSCC cells under hypoxia. As a result, we first revealed that TPD52 is increased under hypoxia in a HIF-independent manner, and that the combination of TPD52 knockdown and HIF inhibition reduced cell viability and induced cell death, including apoptosis. These results may lead to novel cancer therapeutics by controlling the expression of TPD52 in cancer tissue. However, details regarding TPD52 in cancer cells under hypoxia are still to be investigated, and the following studies are now ongoing.