AF1q Expression Associates with CD44 and STAT3 and Impairs Overall Survival in Adenoid Cystic Carcinoma of the Head and Neck

Malignancies originating from salivary gland tissue are rare and have a reported incidence rate of 5–10 per 1,000,000. [1] In addition, salivary gland malignancies appear as a heterogeneous group with several different histologic subtypes. Currently, the WHO differentiates between 22 malignant epithelial tumors of the salivary glands. [2] Both their scarcity and heterogeneity make it particularly difficult to study and understand all histologic subtypes.

Adenoid cystic carcinoma (ACC) confronts patients and clinicians with several serious issues and aggressive features that were first described by Dockerty and Mayo in 1942. [3] This kind of salivary gland carcinoma is characterized by distant metastases, high rate of recurrences and perineural invasion. Overall survival rates are reported to approximate 80% after 5 years and 60% after 10 years. [4] Nevertheless, over 40% of all ACC patients are confronted with distant metastases during the course of their disease. [5] Particularly patients diagnosed with the solid form of ACC are at a high risk of increased mortality and of developing distant metastasis. [6]

Park et al. have recently identified AF1q as a factor that promotes distant metastasis in breast cancer. [7] AF1q, a MLL fusion partner, was first described by Tse and colleagues as a fusion gene (1; 11) (q21; q23) that is specifically expressed in leukemic and hematologic precursor cells. [8] Although the chromosomal abnormality that was detected then is not observed in solid tumors, AF1q expression has been found in various other malignancies including solid tumors. [9, 10] The oncogenic potential of AF1q may be a result of its capability to activate the Wnt pathway. [7] AF1q and its cofactors TCF1, beta-catenin and LEF1 form a transcriptional complex and activate the expression of CD44, a downstream target of the Wnt pathway. Aberrant Wnt signaling is associated with invasive characteristics of ACC in-vitro. [11] Activation of the Wnt pathway through beta catenin is associated with decreased survival rates in patients with major and minor salivary gland malignancies. [12, 13] Additionally, expression of AF1q enhances the oncogenic STAT3 signaling pathway via upregulation of PDGF-B and consecutive phosphorylation of the PDGF receptor beta. [14] Inhibition of STAT3 may be therefore a promising strategy in the treatment of ACC. Specifically, Bu and colleagues have demonstrated that selective inhibition of STAT3 by the small molecule inhibitor S3I-201 results in decreased cellular proliferation, migration and invasion of ACC cell lines. [15]

In this context, we hypothesized that AF1q might be expressed in ACC of the head and neck. Moreover, we aimed to investigate its effects on patient survival and potential associations with the Wnt and STAT3 signaling pathways.

In our study expression of AF1q was immunohistochemically detectable in almost two thirds of the patients, and more than half of our cohort expressed AF1q at a high or moderate level. Expression of AF1q was found in a number of other solid malignancies such as breast cancer, ovarian cancer, and colorectal cancer. [7, 19, 20] Subsequently, we compared survival of patients with AF1q^high and AF1q^low tumors in our study. One of the major findings was that patients with AF1q^high tumors showed a significant decrease in OS. Other studies reported similar findings. Hu and colleagues were able to demonstrate via The Cancer Genome Atlas dataset of colorectal cancer patients that AF1q expression resulted in reduced OS and DFS rates. Park and coworkers reported similar results in breast cancer patients. [7] In addition, their study group was able to identify AF1q as a prognosticator for brain metastases. Interestingly, we did not find any difference in the occurrence of distant metastases at the time of initial diagnosis or in the course of disease. Nevertheless, lymph node metastases were detected >3 times more often in patients with AF1q^high tumors. However the level of significance was not reached. The fact of AF1q not being associated with metastatic disease might be contributed to the low number of patients eligible for this analysis. In a study investigating 96 colorectal cancer patients, AF1q upregulation was significantly associated with lymph node metastases [19] Moreover, expression of AF1q was predominately found in patients with solid components of ACC. A solid growth pattern in ACCs is generally associated with worse clinical outcome. Strictly solid ACCs were only found in AF1q^high patients.

In addition, we examined the expression of CD44, as a downstream target of the Wnt pathway, and of STAT3 in our cohort. Our data suggests that AF1q expression may lead to an activation of both pathways since CD44 and STAT3 are expressed at a higher intensity in AF1q^high patients. The promoting role of AF1q for the oncogenic STAT3 pathway was described by Park et al. [14] Furthermore, AF1q has been shown to induce expression of PDGF-B/PDGFBR-B, which results in a markedly enhanced STAT3 activity. Additionally, AF1q results in the transcriptional activation of CD44 downstream of Wnt. [7] Activated Wnt signaling has recently been associated with AF1q activation and the enhancement of cancer stem cell population in breast cancer. [21]

In conclusion, we have been able to link the expression of AF1q in patients with ACC to a significant decrease in OS. Our results could not confirm higher rates of distant metastases in ACC patients compared to other data reported in the literature. However, lymph node metastases were more often found in patients AF1q^high tumors. Additionaly, we linked AF1q expression to higher levels of STAT3 and CD44. As a matter of fact, association with the STAT3 as well as the Wnt signaling pathway might open new therapeutic perspectives in fighting ACC. Taken together, our results argue in favor of further investigation of AF1q in ACC of the salivary glands in order to confirm our results and to examine AF1q as a potential therapeutic target.