Expression of Proton-Sensitive GPR31, GPR151, TASK1 and TASK3 in Common Skin Tumors

TWIK-related acid-sensitive potassium channels TASK1 and TASK3, as well as the G-protein-coupled receptors GPR31 and GPR151, are proton-sensitive membrane proteins. They can be activated or inhibited by low extracellular pH (pHe), which is a hallmark of the tumor microenvironment in solid tumors. However, the role of these channels in the development of skin tumors is still unclear. In this study, we investigated the expression profiles of TASK1, TASK3, GPR31 and GPR151 in squamous cell carcinomas (SCCs), basal cell carcinomas (BCCs), nevus cell nevi (NCN), and malignant melanomas (MMs). We performed immunohistochemistry using paraffin-embedded tissue samples from patients and found that most skin tumors express TASK1/3 and GPR31/151. The results show that BCCs are often negative for GPR31/151 as well as for TASK1/3, while nearly all SCCs express these markers. MMs and NCN show similar expression patterns. However, some tumors show a decreasing TASK1/3 expression in deeper dermal tumor tissue, while GPCRs were expressed more evenly. The lower frequency of GPR31/151 and TSAK1/3 expression in BCCs when compared to SCCs is a novel histological feature distinguishing these two entities. Moreover, BCCs also show lower expression of GPR31/151 and TASK1/3 as compared to NCN and MMs.


Introduction
The inverse pH gradient (extracellular pHe < intracellular pHi) is a hallmark of solid tumor cells [1]. Acidic metabolic waste products in the tumor microenvironment (TME) result from poor blood perfusion with subsequent hypoxia as well as from inflammation and high metabolic activity [1,2]. H+ ions and lactate accumulate in the TME due to metabolic changes in tumors as well as the altered activity/expression of membrane-bound transporters. The decrease in pHe activates proton-sensitive receptors, such as certain G-protein coupled receptors (GPCRs), transient receptor potential channels (TRPCs), acidsensing ion channels (ASICs) as well as TWIK-related acid-sensitive potassium channels (TASKs) [3]. Furthermore, tumor cells exhibit complex pH regulation via sodium hydrogen exchanger-1 (NHE1), monocarboxylate transporters (MCT1-4), bicarbonate transporters, vacuolar ATPases (VATPase) and carboanhydrases (CAII, CAIX, CAXII) [1,[4][5][6]. As a result, cancer cells exhibit a more alkaline pHi > 7.2 and correspondingly a lower pHe of 6.7-7 compared to normal cells [2]. It is remarkable that tumor cells regulate their intrato extracellular pH gradient in a very small range, meaning that they not only prevent

Rating
Dermatopathologists assessed the staining of the sections visually. Sections were labelled as ++ for strong positive reactions with >80% of cells being positive and/or when staining intensity was high, + for 20-80% of cells demonstrating a weak positive/partial positive reaction, and − for <20% of cells displaying weak staining (a negative reaction). The epidermis was used as a reference structure in scoring. Tumors with a decrease in expression towards deeper tissue layers were considered weak positive. The same rating was applied for inconsistent staining throughout the tumor.

Statistics
First, all rating results for all entities were compared using Kruskal-Wallis tests. For NCN and MMs, epidermal and dermal portions were separately used for testing. Pairwise comparisons were made via Bonferroni tests. Secondly, pairwise comparisons of BCCs vs. SCCs and of MMs vs. NCN were made for each protein using a Mann-Whitney U test, and the results are given as exact significance (shown as 2*(1-tailed significance), not corrected for ties, for BCCs vs. SCCs and epidermal portions of NCN/MMs) or asymptotic significance (2-tailed, for dermal portions of NCN/MMs).

Results
Initially, we selected 25 tumors for each entity. Unfortunately, there was not enough tissue left on some of the selected paraffin blocks to perform all four stainings. Furthermore, staining did not work properly (e.g., over-staining) on some of the samples or did not show tumor cell nests on the sliced tissue, and therefore could not be used for evaluation. In the end, we were left with varying numbers for each combination of tumor and staining.
The   (for each protein in  Supplementary Tables S1-S4, for each tumor in Supplementary Tables S5-S8). or did not show tumor cell nests on the sliced tissue, and therefore could not be used for evaluation. In the end, we were left with varying numbers for each combination of tumor and staining.
The mean age ± standard deviation was: SCCs: 85.43 ± 12.29 years (70% male and 30% female), BCCs: 81.19 ± 14.60 years (67% male and 33% female); NCN: 48.93 ± 16.86 years (53% male and 47% female); MMs: 66.48 ± 18.75 years (51% male and 49% female).  The scoring results for each tumor can be found in the  Supplementary Information (for each protein in Supplementary Tables S1-S4, for each  tumor in Supplementary Tables S5-S8).   or did not show tumor cell nests on the sliced tissue, and therefore could not be used for evaluation. In the end, we were left with varying numbers for each combination of tumor and staining.
Tumors with a decrease in expression towards deeper tissue layers were considered as being weakly positive. The same rating was applied for inconsistent staining throughout the tumor.   Overall, NCN, MMs and SCCs appeared to be predominantly weak or strongly positive for GPR31, GPR151, TASK1 and TASK3, whereas BCCs showed no expression of proton-sensing receptors or ion channels in 50% of cases. Furthermore, we observed a decrease in the expression of proton-sensitive proteins in deeper tissue sections of some NCN and MMs.
Tumors with a decrease in expression towards deeper tissue layers were considered as being weakly positive. The same rating was applied for inconsistent staining throughout the tumor.    Overall, NCN, MMs and SCCs appeared to be predominantly weak or strongly positive for GPR31, GPR151, TASK1 and TASK3, whereas BCCs showed no expression of proton-sensing receptors or ion channels in 50% of cases. Furthermore, we observed a decrease in the expression of proton-sensitive proteins in deeper tissue sections of some NCN and MMs.
Tumors with a decrease in expression towards deeper tissue layers were considered as being weakly positive. The same rating was applied for inconsistent staining throughout the tumor.

GPR31
All of the SCCs showed a weakly positive expression of GPR31 (Figure 1b

GPR31
All of the SCCs showed a weakly positive expression of GPR31 (Figure 1b Figure S16). TASK3 expression was significantly lower in BCCs as compared to SCCs (p < 0.01 Kruskal-Wallis and post hoc Bonferroni tests, p = 0.011 in Mann-Whitney U tests, details in Supplementary Tables S9 and S10). No significant differences in expression were found between NCN and MMs.

Discussion
We examined the expression profiles of GPR31, GPR151, TASK1 and TASK3 in the most common types of skin cancer. We found mixed results and inhomogeneous expression profiles of the different receptors even in the same tumor type, which might be explained by different patients with diverse genetics, unequal growth patterns/tumor thickness and opposing functions of receptors in the human body. There is also a high probability of channel/receptor interplay between pH-sensitive GPCRs and TASKs

GPR151
In SCCs, GPR151 expression was weakly positive in 10/17, strongly positive in 6/17 and negative in only 1/17 of the cases (Figure 1c, Supplementary Figure S5 (Figure 4c, Supplementary Figure S8). Overall, melanocytic tumors appear to lose this marker with increasing depth. GPR151 expression was significantly lower in BCCs as compared to SCCs (p < 0.01 in Kruskal-Wallis and post hoc Bonferroni tests, p = 0.009 for BCCs vs. SCCs in Mann-Whitney U tests, details in Supplementary Tables S9 and S10). Furthermore, SCC showed more frequent positive expression than MM (p < 0.01 Kruskal-Wallis and post hoc Bonferroni, details in Supplementary Tables S9 and S10, both epidermal and dermal). No significant differences in expression were found between NCN and MM.

TASK1
Only 1/16 SCCs showed a negative staining, while 13/16 showed a weakly positive and 2/16 showed a strong expression of TASK1 (Figure 1d, Supplementary Figure S9). Eight out of 20 of the BCC tissue samples showed no expression of TASK1 and 12/20 appeared to be weakly positive (Figure 2d, Supplementary Figure S10 Figure S12). Overall, TASK1 seems to be more expressed in deeper sections of the tissue. TASK1 expression was significantly lower in BCCs as compared to SCCs (p < 0.01 Kruskal-Wallis and post hoc Bonferroni tests, p = 0.029 in Mann-Whitney U tests, details in Supplementary Tables S9 and S10). No significant differences in expression were found between NCN and MM.

TASK3
SCCs showed strong expression in 6/17 and weak expression in 10/17 of tissue samples. Only 1/17 was negative for TASK3 (Figure 1e, Supplementary Figure S13). Ten out of 18 of BCC showed a weak expression of TASK3, whereas 7/18 showed no expression and the remaining 1/18 showed a strong expression (Figure 2e, Supplementary Figure S14 Figure S16). TASK3 expression was significantly lower in BCCs as compared to SCCs (p < 0.01 Kruskal-Wallis and post hoc Bonferroni tests, p = 0.011 in Mann-Whitney U tests, details in Supplementary Tables S9 and S10). No significant differences in expression were found between NCN and MMs.

Discussion
We examined the expression profiles of GPR31, GPR151, TASK1 and TASK3 in the most common types of skin cancer. We found mixed results and inhomogeneous expression profiles of the different receptors even in the same tumor type, which might be explained by different patients with diverse genetics, unequal growth patterns/tumor thickness and opposing functions of receptors in the human body. There is also a high probability of channel/receptor interplay between pH-sensitive GPCRs and TASKs [30,31]. One of the most striking results found was the lower frequency of GPR31/151 and TASK1/3 expression in BCC when compared to SCC.
There is no difference in the RNA expression levels of the investigated proteins according to studies analyzed at NCBI Geo (Supplementary Table S11). However, protein expression was obviously altered between BCCs and SCCs, as seen in our immunohistochemistry stainings. BCCs seem to lose the pH-sensitive proteins investigated in this study. Studies on cBioportal were analyzed for mutation frequencies of GPR31, GPR151, KCNK3, and KCNK9 in the investigated tumor entities. For non-melanoma skin cancer, SCCs showed a markedly higher mutation frequency than BCCs (Supplementary Figure S21). However, protein expression was found to be reduced in BCCs as compared to SCCs, which could be due to post-transcriptional processes. For MMs, up to 8% mutation frequency was found for GPR31/151 and KCNK3. However, more than 30% mutations were found in one study on KCNK9 (Supplementary Figure S22). Interestingly, a large portion of these were classified as amplification. Further studies are needed to address the role of KCNK9/TASK3 in MMs' progression from nevi.

GPR31/GPR151
GPR31 is evenly expressed in SCCs, MMs and in NCN in both epidermal and dermal portions, but NCN showed more negative staining results than MMs. In contrast to SCCs (100% +), BCCs were negative in~50% of the samples, which is an interesting immunohistochemical feature to distinguish both entities. Even though there is little information on the expression of GPR31 in melanomas and NMSC, the role in cancer progression has been proven in other tissues. GPR31 expression is significantly upregulated in colorectal cancer tissues [16]. High GPR31 expression indicates poor prognosis of colorectal cancer. Furthermore, the expression of 12-HETER1 positively correlates with the grades of prostate cancer, and knockdown of GPR31 in cancer cells inhibited HCC recurrence in NAFLD [15,32,33]. Taking these considerations and our results into account together, GPR31 might serve as a potential diagnostic target, but more functional studies are required to fully understand the role of GPR31 in skin tumor progression.
Among all investigated tumors, BCC showed the lowest expression rates of GPR151. Contrarily, nearly all SCCs showed positive expression of GPR151. In MMs and NCN, we found inhomogeneous results concerning the expression of GPR151 in epidermal and dermal tissue portions.
Another interesting observation was the weak or strong positive staining of the lymphocytes. As Morita et al. found, in CX3CR1+ immune cells, GPR31 is activated in the intestinal lumen, leading to their dendrite protrusion and enhancing intestinal immune responses [34], while Lämmermann et al. summarized GPCRs and especially the protonsensitive GPR31 and GPR151, which are commonly found in immune cells [35].

TASK1/TASK3
All investigated tumors, except for BCCs, showed frequent expression of TASK3. As mentioned before, TASK3 is responsible for maintaining the resting membrane potential of the cell. Besides TASK3, numerous K + channels have been reported to be differentially expressed in human cancer and regulate different aspects of tumorigenicity [28,36]. The mechanism responsible for this connection is the hyperpolarization caused by the opening of TASK3 due to acidification, which then promotes cell cycle progression through G 1 -S. We can say that TASK1/3 are widely expressed in the more aggressive common skin tumors SCC and MM, but are rarely found to be highly expressed in BCC. As Sun et al. found that the inhibition of TASK3 via Y4 reduced the proliferative index and decreased the tumor burden in vivo, TASK3 could also be a promising therapeutic target in cancer [37]. TASK1 shows quite similar expression patterns to TASK3, except for MMs, where it is expressed less frequently. In MMs, we found mixed results for TASK1 expression, and in the dermal portion of NCN, no/low expression of TASK1 was predominant. Antigny et al. found that TASK1 inhibition promoted increased proliferation, vasoconstriction and inflammation [38]. Based on the clearly positive staining of lymphocytes for TASK1/3, we can confirm that these K + channels are expressed in lymphocytes and therefore play a significant role in the human immune response, which makes them a potential drug target for T cell-mediated autoimmune diseases such as multiple sclerosis [39].

Conclusions
Taking everything into account, our findings need to be verified by a larger sample size and by the study of different patient collectives (based on the hypothesis that the expression of GPCRs/TASKs varies between patients, type of cancer and micro-and macroenvironmental circumstances) to investigate the different roles of GPCRs/TASKs more precisely.
As this is the first study on the expression of these pH-sensitive proteins (GPR31/151, TASK1/3) in skin tumors in the literature, our approach is descriptive. However, one of the most striking results found was the lower frequency of GPR31/151 and TASK1/3 expression in BCC when compared to SCC and melanocytic tumors (NCN/MM). This makes the study of these pH-sensitive proteins a novel histological/diagnostic tool to distinguish these two entities (BCC/SCC). GPR31 is expressed in all SCC but only in half of the BCC.
In terms of methodology, automated evaluation of immunohistochemistry could be a potential approach for future studies [40]. As a further step, it would then be important to conduct clinical studies on whether these receptors/channels have a significant effect on the overall survival of MM patients. In a similar context, studies should focus on the expression of GPR31, GPR151, and TASK1/3 in metastases of SCCs and MMs. Further studies could focus on how different growth patterns or tumor stages correlate with the expression of the investigated proteins.  Table S1: Scoring for GPR31, Table S2: Scoring for GPR151, Table S3: Scoring for TASK1, Table S4: Scoring for TASK3, Tables S5-S8: Scoring results for each tumor entity, Tables S9 and S10: Statistical analysis test results, Table S11: NCBI Geo mRNA expression comparison results. Funding: This study was funded by the German Research Foundation (DFG), grant SCHR 1288/6-1.

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki. The investigated tissue samples were from biopsies older than 10 years, and hence free to use according to German legislation.