MAC is a rare skin adnexal tumor that is locally aggressive and has the potential for recurrence and metastasis. MAC is usually located on the head and neck, especially on the lip. Surgical resection using Mohs micrographic surgery or complete circumferential periphery is the standard treatment for MAC. Most MAC patients are white, with only sporadic cases in China. A systematic review reported 1968 MAC patients, the mean age was 61.8 years, and 54.1% were women [2]. In our study, all cases were located on the head and neck, and three of six were located on the lip. The mean age was 51.8 years, 66.6% (4/6) were women, and 25% (1/4) of patients suffered postoperative recurrence, based on the available follow-up information. The clinicopathologic features of MAC were first systemically discussed by Goldstein et al. in 1982 [1]. MAC usually has benign clinical characteristics and presents as asymptomatic, indurated flat plaques [12]. Microscopically, the tumor often shows both follicular [1] and sweat gland differentiation [13], occasionally dominated by one type. The cytopathologic features of MAC are mild and lack atypia and mitotic features, but it can infiltrate into the deep dermis, even skeletal muscle and peripheral nerves. Although MAC has dramatically malignant biological behavior, histologically, it may be confused with some benign skin adnexal tumors, such as desmoplastic trichoepithelioma [7, 14] and syringoma [6], especially when the biopsies are superficial, leading to delayed treatment and patients suffering larger wounds. Many studies have summarized the histological characteristics of MAC [1, 15]; however, few studies have identified the molecular genetic alterations in MAC as diagnostic and therapeutic markers to achieve early detection and treatment.

In recent decades, many studies have reported immunohistochemical studies of MAC. They revealed that carcinoembryonic antigen (CEA) [15] and CD23 [16] were positive in ductal lining cells and supported the sweat gland differentiation of MAC. CK [17, 18], CK5/6 [19], CK7 [18, 20], CK15 [21], CK19 [14], EMA [18, 22], ɑ-SMA [20], and Ln-ɤ 2 [23] were diffusely positive in most of the tumor cells; Bcl-2 [20] was focally positive; CK20 [20], c-erbB-2 [20], Ber-EP4 [14, 24], and CD34 [20] were negative, while CK20 and Ber-EP4 were positive in desmoplastic trichoepithelioma and desmoplastic basal cell carcinoma, respectively. p53 [20] was patchy and mottled, p63 [25] was positive in the periphery of tumor nests despite minimal staining within the center of the tumor islands, and adipophilin [26] was positive in the area of sebaceous differentiation, and Ki-67 usually stained less than 5%. Our study found that the expression of CK5/6, CK20, EMA, p63, p53, AR, PR, CD34, and Ki-67 in our six cases was consistent with the above results, and our results further validated the pathologic diagnosis of MAC in the six cases.

Recently, two studies proposed special molecular markers for MAC by systematically analyzing genetic changes through high-throughput sequencing at the DNA level. Chen et al. demonstrated that TP53 mutations and chromosomal deletion of CDKN2A and CDKN2B existed in a metastatic MAC of a 68-year-old man [9]. There has been no study on the protein expression of p16 (which is encoded by CDKN2A) in MAC. Our study showed that p16 was scattered, mottled positive at the protein level in 5/6 cases, but was negative in M5 (follow-up information is unavailable), which was consistent with the DNA alteration results of Chen et al. in his metastatic MAC case [9]. We noticed that all 2 MAC patients who showed evidence of p16 negativity in our study and Chen et al. were older patients (68 and 71 years old). While it is known that p16 expression was upregulated along with tissue aging and therefore was considered one of the most robust aging biomarkers characterized to date [27, 28]. Several studies indicated that, as a tumor suppressor, p16 is an intrinsic human clonal evolution regulator. Its forced overexpression or downregulation impairs the progression of in vitro clonal conversion [29, 30]. In several tissues, p16 governs the processes of stem cell self-renewal and its deregulation may result in tumor development [31]. Because the data is excessively limited, the relationship between MAC metastasis, age of patients, and p16 negative expression still needs further study. Chan et al. demonstrated that inactivated TP53 or the activated JAK/STAT signaling pathway plays important roles in MAC at the DNA level [10]. In our study, we analyzed RNA changes by next-generation transcriptome sequencing and found that the expressed genes between MAC patients and the normal population were different, and the differentially expressed genes could well distinguish the two populations. Pathway enrichment analysis found that the JAK/STAT signaling pathway (consistent with the DNA changes in Chan’s study), calcium signaling pathway, and cGMP-PKG signaling pathway have significant effects on MAC. However, we did not find mutated expression of p53 protein in all our MAC cases.

We focused our energy on calcium signaling because the JAK/STAT signaling pathway was revealed by Chan et al. The calcium signaling pathway is a cancer-associated pathway, and calcium ions (Ca2+) are important second messengers of varying cellular processes. Muscle contraction and hormone release as well as gene transcription are related to increasing cytosolic Ca2+ [32]. Ca2+ signaling is relevant to tumor progression, such as proliferation, migration, and apoptosis. Ca2+ signaling was significant in the hallmarks of cancer as described by Hanahan and Weinberg in 2000 [33] and 2011 [34]. Many proteins (channels, pumps, and exchangers) of the Ca2+ signaling pathway regulate cellular Ca2+ levels in compartments to precisely control different biological processes. The protein expression of Ca2+ signaling is altered in cancer, and specific cancer subtypes even manifest predominantly altered expression. It has been reported that a wide variety of proteins involved in Ca2+ signaling are highly expressed in malignancies, including breast, prostate, ovarian, thyroid, lung, and colon cancers [35,36,37]. In addition, specific Ca2+-permeable ion channels can cause patients to resist cancer therapies. Ca2+ signaling also plays a role in the tumor microenvironment. Inhibitors of Ca2+ signaling have undergone clinical trials and have been approved as orphan drugs for patients with solid cancers [38].

The four candidate genes (CACNA1S, ATP2A1, RYR1, and MYLK3) of Ca2+ signaling have special functions in tumor progression. CACNA1S (also named Cav1.1) encodes one of the five subunits of L-type voltage-gated Ca2+ channels, which are located in the cellular membrane and are associated with Ca2+ influx. Grasset et al. reported that high expression of Cav1.1 promotes the collective migration of squamous cell carcinoma cells by increasing intracellular Ca2+, while Cav1.1 gene silencing by using blockers (diltiazem and verapamil) of L-type Ca2+ channels decreases the invasive properties of tumor cells both in vitro and in vivo [39]. RYR1, which is located in the endoplasmic reticulum (ER) membrane, participates in Ca2+ release from the ER and has a significant influence on autophagy and the activity of Ca2+ release-activated Ca2+ channels (CRACs), thus participating in the biological processes of tumors [40]. ATP2A1, also named sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 1 (SERCA1), is related to Ca2+ influx and ER refilling to assist ER Ca2+ levels [41] and plays a major role in muscular excitation and contraction. Chemaly et al. reported that cell apoptosis and survival were controlled by SERCA1 through ER stress and increased Ca2+ levels in the cytoplasm [42], which may be a potential therapeutic target in tumors. Thapsigargin, an inhibitor of SERCA, has been described by Ball et al. and can effectively inhibit the function of SERCA [43]. MYLK3, a kinase, phosphorylates cardiac myosin heavy (MYH7B) and light (MYL2) chains, potentiating the force and rate of cross-bridge recruitment in myocytes [44]. MYLK3 also is a member of the MAPK signaling pathway, another cancer-related signaling pathway. It was revealed that patients with MYLK3 methylation showed prolonged overall survival in ovarian cancer treated with surgery [45]. In general, the membrane channel protein CACNA1S should locate upstream of the whole system. It works together with the ER channel proteins, ATP2A1 and RYR1; regulates the intracellular calcium concentration, then affects the concentration and function of MLYK3 downstream through the calcium/calmodulin pathway, and finally affects the formation and biological behavior of tumor cells.

To our knowledge, there have been no reports on the protein expression of CACNA1S and RYR1 in solid tumors by now. What is more, although patients with MYLK3 methylation showed prolonged overall survival in ovarian cancer, there was no statistically significant association between expression of the gene and overall survival in these patients [45], while it is exciting that ATP2A1 mRNA and protein are overexpressed in ovarian cancer tissues compared to normal ovarian surface epithelial cell [46, 47]. Furthermore, inhibition of ATP2A1 activity by curcumin disrupts the Ca2+ homeostasis and hence promotes apoptosis in ovarian cancer cells [47]. Our transcriptomic analysis showed that CACNA1S, MYLK3, RYR1, and ATP2A1 are upregulated in MAC at the mRNA level. Then, we verified protein levels of these four genes of the calcium signaling pathway by IHC because biofunction was directly occupied by proteins. Meanwhile, we also aimed to verify the value of these four candidate genes in pathologic differential diagnosis between MAC and its histological mimics. We demonstrated that the CACNA1S, MYLK3, RYR1, and ATP2A1 proteins were more highly expressed in MAC tumor cells than in normal sweat glands and syringoma, while were generally negative in tumor cells of trichoepithelioma and basal cell carcinoma, infundibulocystic type. Thus, these four candidate genes were upregulated at both the RNA and protein levels in MAC. Our findings indicated that the calcium signaling pathway may have a special influence on biological behavior in MAC, and the four genes (CACNA1S, MYLK3, RYR1, and ATP2A1) may be new diagnostic molecular markers and therapeutic targets for MAC.

The cGMP/PKG signaling pathway was also one of the enriched pathways in our study. It had revealed that activation of the cGMP/PKG pathway plays an anticancer effect in melanoma [48], head and neck squamous cell carcinoma [49], and breast [50] and colon cancer [51]. However, some studies reported opposite conclusions, and they demonstrated that activation of the cGMP/PKG signaling pathway enhances the protumorigenic effect [52, 53]. Moreover, there were complex interactions between the cGMP-PKG signaling pathway and the calcium signaling pathway. Interestingly, our results showed that the differently expressed genes were overlapped between the cGMP/PKG signaling pathway and the calcium signaling pathway in MAC, except MYH7. While MYH7 was also bio-functional correlated with WLYK3, one of the four genes we focused on. We put emphasis on the calcium signaling pathway other than the cGMP/PKG pathway, because the number of alternative genes was more and its Q value was lower than the cGMP/PKG pathway.

To our knowledge, this is the first report of transcriptional analysis of MAC worldwide. Our data entirely illustrated changes in MAC at the RNA level, but proteomic studies are still needed to confirm our results. A limitation of our study was that only five MAC cases were used for high-throughput sequencing because of the low morbidity of MAC in China. Transcriptome studies with more cases are also needed in the future.

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