**5. Molecular mechanisms: The possible connection between malignancies and ABD**

ABD are characterized by autoantibodies against structural proteins of the skin, including molecules of dermal-epidermal junction and desmosomes. Cancer progression is a complex and multi-step process in which components of DEJ as well as a desmosomal molecules play a pivotal role in the development of metastasis. Probably, more than 90% of human cancers are of epithelial origin [159] and the autoimmunity against epidermal structures may play important role in those carcinogenesis. Thus, probably DEJ/desmosomes is the first barrier in tumor cells invasion. In light of this, understanding the molecular basis of pathological autoinflammation induction in autoimmune blistering dermatoses in relation to the mecha‐ nisms of malignant transformation is paramount for early detection and treatment of epithe‐ lial-derived cancers [160]. However, the precise molecular mechanisms underlying the association of malignancy with ABD still remain unknown. Nevertheless, understanding the link between the production of pathogenic autoantibodies in ABD and the development of the associated neoplasy should facilitate the development of more specific diagnostic tests and therapeutic strategies [160].

#### **5.1. Malignancy in relation to the autoimmunity in pemphigus group: The role of desmosomes components**

been put forward as a causative agent of ABD-type autoimmunity in PV, PF, BP and pemphi‐ gus vegetans. There has been multiple factors suspected of prostatic cancerogenesis, including viruses (BK, HSV2, HSV6, HSV11, HSV16, HSV18, HSV31, HSV33, HHV8, XMRV, CMV) and microbial agents (Chlamydia trachomatis, Mycoplasma hominis, Ureaplasma urealyticum, Neisseria gonorrhoeae and Treponema pallidum), yet data was inconclusive or supported no relation [144–151]. Surprisingly, Epstein-Barr virus infection has been found statistically associated with increased breast carcinoma risk [152]. It was speculated that the virus itself may play a role in ABD induction [153,154]. The data covering the issue of exogenous factors

Constant activation of immune system in ABD sustains chronic inflammation leading to tissue degeneration by proinflammatory molecules. As a result, the risk of the neoplasy increases [3]. However, that relation could be two-sided. Inflammation in tumor nest stimulates and modifies the immunity mechanisms. Furthermore, there is a hypothesis that the multiplicity of target antigens in ABD may result from intra- and intermolecular epitope spreading. The effect of epitope spreading in ABD is well-known fact and it seems to be a constitutional compound of autoimmunity. Structural similarities between autoantigen epitope and to-beautoantigen epitope may lead to production of autoaggresive cross-reacting immunoglobulins [9,155]. This phenomenon, which represents a broadening of the immune response from a single epitope to additional epitopes, is also described in cancer and recent findings suggests that epitope spreading may be a more significant predictor of effective immunity [156].

The initial anti-cancer immunity may ricochet to autoaggresion by epitope spreading and bystander effect alike [157]. Local inflammation in the course of malignant tumor or chemo‐ therapy/radiotherapy treatment may result in enhanced autoantigen presentation that causes T-cell priming, activation and expansion of additional specificity [158]. Therefore, in situations where immunosuppressive/anti-cancer treatment is absent or ceased, anti-cancer response may be responsible for development of autoimmunity to self-antigens characteristic for ABD.

**5. Molecular mechanisms: The possible connection between malignancies**

ABD are characterized by autoantibodies against structural proteins of the skin, including molecules of dermal-epidermal junction and desmosomes. Cancer progression is a complex and multi-step process in which components of DEJ as well as a desmosomal molecules play a pivotal role in the development of metastasis. Probably, more than 90% of human cancers are of epithelial origin [159] and the autoimmunity against epidermal structures may play important role in those carcinogenesis. Thus, probably DEJ/desmosomes is the first barrier in tumor cells invasion. In light of this, understanding the molecular basis of pathological autoinflammation induction in autoimmune blistering dermatoses in relation to the mecha‐ nisms of malignant transformation is paramount for early detection and treatment of epithe‐ lial-derived cancers [160]. However, the precise molecular mechanisms underlying the association of malignancy with ABD still remain unknown. Nevertheless, understanding the

**and ABD**

168 Highlights in Skin Cancer

contributing to both malignancy and ABD is yet generally inconclusive.

Pemphigus group is characterized by presence of autoantibodies against desmosomal cadherins. Research on human and animal models indicated that alternation in desmosomes components may lead to tumor progression and metastasis. However, little is known about the role of desmosome during cancer development [161]. It is clear, that the origin and maintenance of epidermis requires the coordinated regulation of proliferation, adhesion, migration and differentiation. Conceptually, desmosome complexes form when desmosomal cadherins – DCs (desmogleins – DSGs and desmocollins – DSCs) participate in heterotypic interactions that bring the plasma membranes of adjacent cells in close apposition [161]. The cytoplasmic tails of these cadherins interact with plakoglobin and plakophilins.

The data describing desmosome protein expression during human cancer progression are conflicting [161–163]. Molecular abnormalities of desmosomal proteins (DPs) are observed in ABD and epithelial malignancies. However, in malignancy, where cells may separate, detach and metastasize, it is possible that alterations in DPs expression may be the reason of carcino‐ genesis process. Several studies demonstrated that downregulation of DCs occurs during the progression of cancers and is often correlated with and predictive of tumor metastasis [161,162]. On the other hand, other studies reported overexpression of DCs during the cancer progression, and this pattern is associated with poor prognosis [163]. The regulatory mecha‐ nism controlling DCs expression are scanty explained. As known, gene expression may be modulated by genetic and/or epigenetic mechanisms and in this way may contribute to the development of pathologic autoimmunity or malignancies. Genetic changes as mutation, deletion, and gene rearrangement of DCs have been poorly found in cancer and ABD. Possible mechanism may also involve post-translational modification of protein, like phosphorylation, acetylation or methylation. In light of this, some data reported methylation of DCs, e.g. methylation of DSC3 in breast cancer. It was showed, that DSC3 is downregulated in colorectal cancer by DNA methylation [164]. Thus, further analysis of methylation status of DCs DNA may be useful to predict clinical outcomes in patients with malignancy.

Alternatively, the possible link between desmosomal components and malignancy may be the Perp protein. The Perp tetraspan membrane protein was originally identified as a transcrip‐ tional target of the p53 tumor suppressor upregulated during apoptosis [161,165]. However, Perp may have function as a target of the p53-related transcription factor (p63) involved in maintaining epithelial integrity by promoting desmosomal cell-cell adhesion. Electron microscopy and biochemical analyses showed that the blistering phenotype observed in the Perp-deficient epithelia is accounted for by both a reduction in desmosome number and compromised desmosome complex formation. It is suggested, that pemphigus autoantibodies may trigger internalization of Perp, which enhances adhesion defects [166]. Perp's position downstream of p63 and p53, as well as its essential role in normal desmosome function, suggest that it may be a target for mutation in human blistering diseases or cancer [167]. An interesting issue remains why a p53 target would play such a prominent role in adhesion in the skin. It was suggested [167], that this role relates to Perp being a key component of the transcriptional program for stratified epithelial development and maintenance specified by the p53 family member p63. Perp is a transcriptional target of p63 during development and in mature mouse skin [168]. Beyond a role in epithelial function, Perp's status as a p53 target involved in apoptosis may suggest a potential role as a tumor suppressor. Given that loss of both p53 and p63 cooperate in tumorigenesis, loss of Perp similarly may be expected to promote cancer development in some context. The future analysis of Perp will provide new insight into the role of desmosomes in epithelial homeostasis and cancer [167]. It was disputed, that Perpdeficiency promotes cancer by enhancing cell survival, desmosome loss and inflammation, and fundamental role for Perp and desmosomes in tumor suppression [161]. Interestingly, it seems that DSC3 is a p53 response gene and addition of wild-type p53 was found to be sufficient to induce expression of DSC3 in breast cancer [169]. Thus, it was of great interest to investigate whether this pathway is also active in tumor cells. In light of above, the induction of p53 may have impact on expression of DCs [164].

could play a contributory role in the initiation of early stages of cancer. In light of this, the evidence that altered expression of desmosomal proteins in various human malignancies has

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**5.2. Malignancy in relation to the autoimmunity in subepidermal blistering dermatoses:**

Pathogenesis of blister formation in subepidermal blistering dermatoses is associated with destruction of dermal-epidermal junction and anchoring fibers. Recent studies shown inter‐ esting function of the hemidesmosome (HD) components in signal transduction, involving effect on cell behavior in tumor invasion [175]. Data indicated altered expression of DEJ proteins at different stages of carcinogenesis, what may suggest the association between tumor progression. Aberrant expression of DEJ proteins, which are associated with subepidermal blistering dermatoses (e.g. BP180, BP230, alfa6beta4 integrin, laminin-322) in epithelial cancers was demonstrated, what may indicate their role in tumor development and invasion [175]. Reduced expression of HD components may results in the detachment of cells from the basement membrane, facilitating piling or migration of cells [176]. On the other hand, carci‐ noma cells may upregulate the expression of HD molecules to enhance the attachment capacity of metastatic cells to the DEJ at the metastatic site in order to establish metastatic growth.

The aberrant expression of DEJ component may reflect dysfunction of HD, that occurs as an early event in multistep carcinogenesis of epithelium [177]. Downregulation of BP180 – one of the major autoantigen in bullous pemphigoid – was found in basal cells in mild dysplasias, upregulation of BP180 in suprabasal keratinocytes in moderate and severe dysplasis as well as in the central cells of squamous cell carcinomas (SCC; G2 and G3) using immunohisto‐ chemical (IHC) and in situ hybridization (ISH) methods [175]. Furthermore, this group of researchers indicated that overexpression of BP180 was found at the invasive front of the tumors. Authors suggested that reduced BP180 expression at the early step of carcinogenesis may reflect disturbed keratinocyte adhesion to the basement membrane. BP180 gene expres‐ sion is significantly induced by a tumor promoter PMA [175]. Previous findings obtained by the same authors revealed reduced BP180 expression in the peripheral cells of carcinoma islands in solid and keratotic basal cell carcinomas (BCC) and in the basal cells of invading buds in superficial BCCs [178]. The altered expression of DEJ proteins is likely to coincide with the disassembly of HD, which is an essential step in keratinocyte migration and carcinoma cell invasion [175]. Moreover, downreguation of BP180 and other hemidesmosome components was previously detected in vivo in prostate cancer and in the invasive cells of ductual mam‐ mary carcinoma [179,180]. On the other hand, upregulation of HD components has been reported in a variety of SCCs [177,181]. It was argued that BP180 upregulation in carcinoma cells at the invasive front of malignant tumors is important in the carcinoma cell migration [175]. Other study described upregulation and altered distribution of BP230 and alfa6beta4 integrin in the areas of invasive growth of head and neck SCCs [181]. PMA is a potent tumor promoter capable of inducing several genes that have a role in carcinogenesis and tumor invasion [182]. It is known, that laminin-332 gamma2 chain gene promoter is one of the targets of PMA activation, occurring via interaction with the activator protein 1 complexes [183]. The

been accumulating.

**The role of the DEJ proteins**

Malignancy may be also associated with pemphigus group via pemphigus-antibodies-induced signaling pathways. Thus, cadherins expression can function as a tumor and invasion suppressor due to its participation in processes such as morphological differentiation and contact inhibition of growth and motility. Several different molecular mechanisms for perturbation of cadherin function in epithelial tumors are reported: (i) transcriptional or genomic regulation of cadherin expression, (ii) mutations (e.g. deletion, insertion) of cadherin or catenin genes and (iii) regulation of adhesive function by signaling pathways. A signaling cascade initiated by pemphigus vulgaris antibodies may results in reduced availability of plakoglobin and abrogation of its function as transcriptional repressor of the proto-oncogene c-Myc. This in turn results in accumulation of c-Myc. Moreover, c-Myc expression is commonly upregulated in tumors. There is also evidence, that etiology of some skin cancer (e.g. BCC) may be dependent on several signaling pathways [170], which can be shared with pathological autoimmunity induction in pemphigus group. Other signaling pathways may engagement of Src, Wnt, hedgehog, epidermal growth factor receptor (EGFR) kinase (EGFRK), cAMP, protein kinases A and C (PKC), phospholipase C, mTOR, p38 MAPK, JNK [166]. Especially, it is postulated that DSG3 is a key player in Src signaling and overexpression of DSG3 elicited a phenotype similar to this with increased Src activity. Interestingly, this phenotype may be observed in some kind of cancers (e.g. SCC) [171]. Moreover, it is estimated that 35% of cancers show increased MAPK activity [172]. On the other hand, MAPK is involved in the process of acantholysis in pemphigus group.

Furthermore, some data [173,174] demonstrated possibility that switching of DCs could play an important role in the development of some kind of cancer. Tumor development is in part characterized by the ability of cells to destruct of cell-cell adhesion and invade the surrounding tissue. Perhaps, the disturbances in desmosomal cadherin (as DSCs) expression could affect beta-catenin signaling [174]. It is known, that increased beta-catenin signaling is a common causative event in some kind of cancer (e.g. colorectal cancer), thus desmocollin switching could play a contributory role in the initiation of early stages of cancer. In light of this, the evidence that altered expression of desmosomal proteins in various human malignancies has been accumulating.

that it may be a target for mutation in human blistering diseases or cancer [167]. An interesting issue remains why a p53 target would play such a prominent role in adhesion in the skin. It was suggested [167], that this role relates to Perp being a key component of the transcriptional program for stratified epithelial development and maintenance specified by the p53 family member p63. Perp is a transcriptional target of p63 during development and in mature mouse skin [168]. Beyond a role in epithelial function, Perp's status as a p53 target involved in apoptosis may suggest a potential role as a tumor suppressor. Given that loss of both p53 and p63 cooperate in tumorigenesis, loss of Perp similarly may be expected to promote cancer development in some context. The future analysis of Perp will provide new insight into the role of desmosomes in epithelial homeostasis and cancer [167]. It was disputed, that Perpdeficiency promotes cancer by enhancing cell survival, desmosome loss and inflammation, and fundamental role for Perp and desmosomes in tumor suppression [161]. Interestingly, it seems that DSC3 is a p53 response gene and addition of wild-type p53 was found to be sufficient to induce expression of DSC3 in breast cancer [169]. Thus, it was of great interest to investigate whether this pathway is also active in tumor cells. In light of above, the induction

Malignancy may be also associated with pemphigus group via pemphigus-antibodies-induced signaling pathways. Thus, cadherins expression can function as a tumor and invasion suppressor due to its participation in processes such as morphological differentiation and contact inhibition of growth and motility. Several different molecular mechanisms for perturbation of cadherin function in epithelial tumors are reported: (i) transcriptional or genomic regulation of cadherin expression, (ii) mutations (e.g. deletion, insertion) of cadherin or catenin genes and (iii) regulation of adhesive function by signaling pathways. A signaling cascade initiated by pemphigus vulgaris antibodies may results in reduced availability of plakoglobin and abrogation of its function as transcriptional repressor of the proto-oncogene c-Myc. This in turn results in accumulation of c-Myc. Moreover, c-Myc expression is commonly upregulated in tumors. There is also evidence, that etiology of some skin cancer (e.g. BCC) may be dependent on several signaling pathways [170], which can be shared with pathological autoimmunity induction in pemphigus group. Other signaling pathways may engagement of Src, Wnt, hedgehog, epidermal growth factor receptor (EGFR) kinase (EGFRK), cAMP, protein kinases A and C (PKC), phospholipase C, mTOR, p38 MAPK, JNK [166]. Especially, it is postulated that DSG3 is a key player in Src signaling and overexpression of DSG3 elicited a phenotype similar to this with increased Src activity. Interestingly, this phenotype may be observed in some kind of cancers (e.g. SCC) [171]. Moreover, it is estimated that 35% of cancers show increased MAPK activity [172]. On the other hand, MAPK is involved in the process of

Furthermore, some data [173,174] demonstrated possibility that switching of DCs could play an important role in the development of some kind of cancer. Tumor development is in part characterized by the ability of cells to destruct of cell-cell adhesion and invade the surrounding tissue. Perhaps, the disturbances in desmosomal cadherin (as DSCs) expression could affect beta-catenin signaling [174]. It is known, that increased beta-catenin signaling is a common causative event in some kind of cancer (e.g. colorectal cancer), thus desmocollin switching

of p53 may have impact on expression of DCs [164].

170 Highlights in Skin Cancer

acantholysis in pemphigus group.

#### **5.2. Malignancy in relation to the autoimmunity in subepidermal blistering dermatoses: The role of the DEJ proteins**

Pathogenesis of blister formation in subepidermal blistering dermatoses is associated with destruction of dermal-epidermal junction and anchoring fibers. Recent studies shown inter‐ esting function of the hemidesmosome (HD) components in signal transduction, involving effect on cell behavior in tumor invasion [175]. Data indicated altered expression of DEJ proteins at different stages of carcinogenesis, what may suggest the association between tumor progression. Aberrant expression of DEJ proteins, which are associated with subepidermal blistering dermatoses (e.g. BP180, BP230, alfa6beta4 integrin, laminin-322) in epithelial cancers was demonstrated, what may indicate their role in tumor development and invasion [175]. Reduced expression of HD components may results in the detachment of cells from the basement membrane, facilitating piling or migration of cells [176]. On the other hand, carci‐ noma cells may upregulate the expression of HD molecules to enhance the attachment capacity of metastatic cells to the DEJ at the metastatic site in order to establish metastatic growth.

The aberrant expression of DEJ component may reflect dysfunction of HD, that occurs as an early event in multistep carcinogenesis of epithelium [177]. Downregulation of BP180 – one of the major autoantigen in bullous pemphigoid – was found in basal cells in mild dysplasias, upregulation of BP180 in suprabasal keratinocytes in moderate and severe dysplasis as well as in the central cells of squamous cell carcinomas (SCC; G2 and G3) using immunohisto‐ chemical (IHC) and in situ hybridization (ISH) methods [175]. Furthermore, this group of researchers indicated that overexpression of BP180 was found at the invasive front of the tumors. Authors suggested that reduced BP180 expression at the early step of carcinogenesis may reflect disturbed keratinocyte adhesion to the basement membrane. BP180 gene expres‐ sion is significantly induced by a tumor promoter PMA [175]. Previous findings obtained by the same authors revealed reduced BP180 expression in the peripheral cells of carcinoma islands in solid and keratotic basal cell carcinomas (BCC) and in the basal cells of invading buds in superficial BCCs [178]. The altered expression of DEJ proteins is likely to coincide with the disassembly of HD, which is an essential step in keratinocyte migration and carcinoma cell invasion [175]. Moreover, downreguation of BP180 and other hemidesmosome components was previously detected in vivo in prostate cancer and in the invasive cells of ductual mam‐ mary carcinoma [179,180]. On the other hand, upregulation of HD components has been reported in a variety of SCCs [177,181]. It was argued that BP180 upregulation in carcinoma cells at the invasive front of malignant tumors is important in the carcinoma cell migration [175]. Other study described upregulation and altered distribution of BP230 and alfa6beta4 integrin in the areas of invasive growth of head and neck SCCs [181]. PMA is a potent tumor promoter capable of inducing several genes that have a role in carcinogenesis and tumor invasion [182]. It is known, that laminin-332 gamma2 chain gene promoter is one of the targets of PMA activation, occurring via interaction with the activator protein 1 complexes [183]. The relationship of BP180 to malignancy is discussed. BP180 has possible phosphorylation sites and may by phosphorylated in SCC [177]. Enhanced expression and abnormal distribution of BP180 in various precancerous and cancerous tissues was revealed, including e.g. SCCs and Bowen's disease [177].

**6.1. Anti-desmosomal autoimmunity circle / pemphigus group cases**

An elderly female with painful oral erosions, flat white-speckled infiltrations, mucosal edema and enlarged submandibular lymph nodes was admitted to oncology center outpatients. Histopathological examination of the retromolar mucosa of the left alveolar process of maxilla showed paraepidermoid epithelium with focal high-grade dysplasia and

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Due to improper biopsy technique full examination of the material was impossible. Therefore, the patient was readmitted to the oncology ward, with tentative diagnosis of carcinoma planoepitheliale with bilateral metastases to neck lymph nodes, for bilateral selective lymphadenectomy and probational excision of the lesions at right buccal region and left soft palate. Histopathological examination on probational biopsy showed mu‐ cous membrane fragments with features of basement membrane disruption and acantholy‐ sis. There was abundant infiltration consisting of lymphoid cells with plasmocyte prevalence and presence of granulocytes. The overall image did not support the diagno‐ sis of carcinoma planoepitheliale, yet suggested pemphigus. Chest x-ray showed leftsided pleural effusion with costodiaphragmatic recess filling. At the posterolateral side of the lung, thin layer of liquid reaching 7th rib in the posterior axillary line was observed. Ultrasonography of the neck showed non-enlarged thyroid gland (10 ml of volume) with hypoechogenic node (11 mm x 8 mm) and 3 lesser hypoechogenic nodes of diameters up to 4 mm in the left lobe. Enlarged lymph nodes, with narrow lymph sinuses (up to 15 mm x 8 mm) and unclear character, were seen bilaterally in the upper part of the neck vessels

The patient was directed to dermatological ward for further diagnostics. Laboratory tests showed increased sedimentation rate. DIF of perilesional skin of the vulva revealed IgG(+), IgG1(++) and C3(+) deposits in the intercellular spaces of the spinous layer of epidermis. hDIF on pulled-out hair revealed IgG(+), IgG1(+) and IgG4(+) deposits in the intercellular spaces of the outer root sheath. DIF of larynx mucosa showed fragmented specimen, mainly without epithelium. There was fragmented epithelium consisting of several cells at the specimens margin (evident acantholysis) with surrounding noticeable IgG(+/-) and C3(+/-) deposits. IIF study on monkey esophagus revealed circulating IgG class of pemphigus-type autoantibodies against desmosomal proteins of keratinocytes – titer above 1/80. On the basis of abovemen‐

After amelioration of the lesions, due to pulse steroid treatment, she was dismissed from the ward. Two months later, she suffered from aggravation of mucosal erosions, haematopnoe and dyspnoe. The woman was admitted to oncology center, where chest computed tomogra‐ phy (CT) scan visualized solid mass in the left lung (Fig. 1A). Via bronchofiberoscopy, carcinoma planoepitheliale of the left lung (G4) with hepatic metastases was diagnosed. Palliative radiotherapy (20 Gy/T) of the mediastinal/left pulmonary region led to aggravation

*6.1.1. Case 1 — Mucocutaneous pemphigus vulgaris / lung cancer*

tioned tests the diagnosis of mucocutaneous PV was reached.

few neoplastic cells.

beyond mandibular angles.

of mucocutaneous PV (Fig. 1B).

As it was demonstrated that expression of BP180 was decreased or absent in cutaneous neoplasms [184], some authors speculated that some type of carcinomas itself might expose BP180 antigenic epitope, which would normally be hidden, thus inducing the production of autoantibodies that lead to the onset of BP [185]. It was proposed that BP180 neoexpression could be associated with early/malignant transformation of keratinocytes as widely expression of BP180 was demonstrated in SCC in contrast to normal skin, where this protein is restricted to basal keratinocytes [186].

Laminin-322 (previously named laminin-5) is the main autoantigen in anti-epiligrin cicatricial pemphigoid, mucouse membrane pemphigoid. Laminin-332 and alfa6beta4integrin may play an important role in tumor progression via activation of phosphatidylinositol-3 kinase signaling (PI3K) [187]. There was shown, that it is highly expressed in several types of squamous and other epithelial tumours [188]. Moreover, in these tumors, laminin-322 shows tendency to accumulate at the interface of the tumor with the surrounding stroma, and expression of this proteins correlates with tumor invasiveness [188]. Interestingly, keratino‐ cytes from patients with junctional epidermolysis bullosa, that did not express laminin-322 or beta4integrin, showed a lack of tumorigenecity in immunodeficient mice after transformation [188]. Perhaps, the binding of collagen VII to laminin-322 may be essential for tumorigenesis [188]. Furthermore, the laminin-322-derived signaling is an important component of tumori‐ genesis. As mentioned above, constitutive activation of the PI3K pathway leading to RAC1 GTPase activation may be the triggering factor inducing tumor invasion.
