Primary mucosal melanomas of the head and neck are characterised by overexpression of the DNA mutating enzyme APOBEC3B

Abstract

Aims Primary head/neck mucosal melanomas (MMs) are rare and exhibit aggressive biologic behaviour and elevated mutational loads. The molecular mechanisms responsible for high genomic instability observed in head/neck MMs remain elusive. The DNA cytosine deaminase APOBEC3B (A3B) constitutes a major endogenous source of mutation in human cancer. A3B‐related mutations are identified through C‐to‐T/−G base substitutions in 5′‐TCA/T motifs. Herein, we present immunohistochemical and genomic data supportive of a role for A3B in head/neck MMs. Methods and results A3B protein levels were assessed in oral ( n = 13) and sinonasal ( n = 13) melanomas, and oral melanocytic nevi ( n = 13) by immunohistochemistry using a custom rabbit α‐A3B mAb (5210‐87‐13). Heterogeneous, selective‐to‐diffuse, nuclear only, A3B immunopositivity was observed in 12 of 13 (92.3%) oral melanomas (H‐score range = 9–72, median = 40) and 8 of 13 (62%) sinonasal melanomas (H‐score range = 1–110, median = 24). Two cases negative for A3B showed prominent cytoplasmic staining consistent with A3G. A3B protein levels were significantly higher in oral and sinonasal MMs than intraoral melanocytic nevi ( P < 0.0001 and P = 0.0022, respectively), which were A3B‐negative (H‐score range = 1–8, median = 4). A3B levels, however, did not differ significantly between oral and sinonasal tumours ( P > 0.99). NGS performed in 10 sinonasal MMs revealed missense NRAS mutations in 50% of the studied cases and one each KIT and HRAS mutations. Publicly available whole‐genome sequencing (WGS) data disclosed that the number of C‐to‐T mutations and APOBEC3 enrichment score were markedly elevated in head/neck MMs ( n = 2). Conclusion The above data strongly indicate a possible role for the mutagenic enzyme A3B in head/neck melanomagenesis, but not benign melanocytic neoplasms.

Human APOBEC3B promotes tumor heterogeneityin vivoincluding signature mutations and metastases

“…IHC clearly shows a strong accumulation of human A3B in the nuclear compartment of cells in multiple murine tissues, consistent with prior reports for human A3B subcellular localization in human cell lines and tissues. [17][18][19][20] These observations demonstrate that higher levels of human A3B are tolerated in primary mouse tissues and, further, that its nuclear import mechanism is conserved, despite the fact that only distantly related polynucleotide deaminase family members are expressed in mice (i.e., Apobec3, Apobec1, and Aicda).…”

Aberrant APOBEC3B Expression in Breast Cancer Is Linked to Proliferation and Cell Cycle Phase

“…To obtain a global overview of the A3B expression pattern in breast cancer cells, paraffin-embedded sections of an exploratory panel of breast cancer cell lines were subjected to immunohistochemistry (IHC) using a validated antibody against A3B [ 26 , 27 , 28 ]. While this antibody recognizes A3A, A3B, and A3G, detection of A3B is easily distinguished from A3A and A3G during microscopy and immunoblotting due to A3B’s distinct nuclear localization and size, respectively ([ 26 , 27 , 28 ]; discussed in [ 12 ]). We initially chose to investigate MDA-MB-415 and BT474, two cell lines known to express A3B at relatively high levels [ 5 , 31 ].…”

“…Detection of A3B by conventional IHC on these cell lines was performed as described in [ 26 , 27 , 28 ]. For multiplex IHC, slides containing cell lines or primary breast cancer specimens were rehydrated by three xylene submersions of 10 min, followed by three 100% ethanol washes, one 70% ethanol wash, one 50% ethanol wash, and a final wash in ddH 2 O. Tissue material was then fixed to the glass slides by a 10-minute incubation in neutral buffered formalin, followed by a wash in ddH 2 O.…”

“…Earlier work by our group and others has pointed out that both A3B mRNA and protein levels associate with proliferation markers in multiple tumor types [ 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. Moreover, negative regulation of A3B transcription is facilitated through two distinct repressive E2F complexes, at least one of which associates with the cell cycle [ 24 , 25 , 29 ].…”

APOBEC3B coordinates R-loop to promote replication stress and sensitize cancer cells to ATR/Chk1 inhibitors

“…Genomic uracil lesions catalyzed by A3B are responsible for a large proportion of both dispersed and clustered mutations in breast, lung, cervix, head, neck and bladder cancers [ 4 – 6 ]. With defined roles in mutagenesis, A3B is reported to promote both initiation and progression of cancer, and overexpression of A3B is usually associated with poor clinical outcomes of cancer patients [ 7 – 9 ], including primary head/neck mucosal melanomas (MMs) [ 10 ]. Recently, emerging evidence has demonstrated that A3B activation was positively correlated with elevated levels of DNA RS, pointing to the additional function of A3B in promoting cancer [ 3 , 11 , 12 ].…”