Although AP-1 has been demonstrated to play a crucial role in HPV - induced carcinogenesis and oncogenic HR-HPVs have been detected in a sizable number of ESCC, there is no study that describes the role of AP-1 in ESCCs. In the present study, we demonstrate for the first time, constitutive activation of AP-1 in ESCCs and change in binding partners that form active AP-1complex in ESCCs. ESCC with HPV infections constituted a significant proportion (19%; 14/75) and interestingly all these cases harbored the most prevalent high-risk HPV type 16. Constitutive activation of AP-1 leading to its high binding activity was observed in most of the esophageal tumors, irrespective of their clinical stage and histo - pathological grade; whereas normal adjacent tissues showed low or no AP-1 activation. Aberrant AP-1 activity is one of the most frequent mechanisms of tumor promotion in epithelial tissues irrespective of the tumor site and is mediated through activation of its upstream kinases such as ERK, JNK and p38 . Similar to our observation, constitutively active AP-1 has also been observed in other epithelial cell malignancies [21, 23–25]. But the mechanism(s) by which AP-1 or its upstream regulatory kinases get activated in ESCC is not known. Apart form physiological regulators like growth factors, cytokines or hormones, AP-1 activity is also induced by bacterial and viral infections as well as many carcinogens . Recent study showed that HPV which is found in almost all cervical cancer, can also promote AP-1 activation to a significant level [13, 21]. Apart from HPV, other infections such as H. pylori, prevalence of which is notably high in Kashmir region, are also known to induce AP-1 activation . Another factor that could significantly contribute to constitutive activation of AP-1 is consumption of sodium-bicarbonate-brewed hot salted tea which is very widely consumed by majority of people in snow-capped Kashmir Valley . This salted tea it is rich in carcinogenic N-nitroso compounds  that primarily act through AP-1 . Interestingly, majority of ESCC cases investigated in the present study showed AP-1 activation had a high intake of salted tea.
Functional AP-1 complex is constituted either by homo- or hetero-dimerization between different members of Jun and Fos family of proteins . Our results demonstrate a preferential participation of JunB and c-Fos in active AP-1 complex formation in almost all tumors irrespective of HPV infection. Interestingly, HPV positive tumors demonstrated an additional participation of JunD in AP-1 DNA binding complex. Also, Fra-1 which showed minor participation in HPV negative tumors was completely absent in HPV positive cases. Apart from formation of heterodimer between Jun and Fos proteins, homodimerization of Jun proteins is also known to form active AP-1 complex but complete absence of cJun in supershift assays indicate that cJun do not participate in functional AP-1 complex formation and hence no jun homodimer formation takes place in ESCC. It is interesting to note here that though c-Jun showed elevated expression in western blotting (see Figure 5), it does not participate in DNA binding activity. It is quite possible that even if a protein is overexpressed, it may not always participate in DNA binding activity and trans-activation possibly due to mutation within the binding sites or otherwise. It can also be speculated that in addition to posttranslational modifications of c-Jun, a simple competition with JunB  and JunD to bind with cFos, may also account for the exclusion of cJun from AP-1 complex in esophageal cancer. It is interesting to note that presence of active JunB/cFos dimers may provide a favorable niche for establishment of HPV infection and viral propagation as two AP-1 binding sites are present in URR of HPV18 that essentially require JunB containing hetrodimers for HPV transcription . These observations, therefore, indicate presence of suitable co-operativity between the virus and the host for expression of viral oncogenes.
Most of the ESCC tissues irrespective of their clinical and histopathological grade showed a high expression of AP-1 family of proteins in tumor tissues as compared to their normal counterparts. Upregulated AP-1 activity is frequently associated with overexpression of its family members [30, 31]. Since majority of AP-1 members are part of immediate early response genes and express differentially in non-neoplastic and neoplastic tissues as well as contribute both in early events of tumorigenesis and tumor progression [30, 31], elevated AP-1 expression and activity appears to be generic carcinogenesis-associated event. Though it is well established that AP-1 regulates the expression of HPV oncogenes , recent observations indicate that HPV reciprocally modulate AP-1 expression and its activity . We stratified expression levels of AP-1 which revealed a differentially higher expression of JunD and cFos proteins in HPV-positive tumors, whereas Fra-1 levels were characteristically low or nil in them. Fra-1 was, however, highly expressed in HPV- negative tumors. Over-expression of Fra-1 and cFos have been demonstrated in esophageal cancers in some sporadic studies [32, 33]. On the contrary, an in vitro study demonstrated increase of cFos in HPV16-transformed cell lines is associated with decreased expression of its negative regulator Fra-1 as well as their reduced contribution in active DNA-binding activity . In an ingenious experiment, overexpression of cFos in HPV 18 positive non-tumorigenic HeLa - Fibroblast hybrid 444 cells having low or no cFos but a very high amount of Fra-1 expression resulted in tumorigenic cells which showed complete loss of Fra-1 but a high expression of cFos . These observations perfectly match with in vivo situations in HPV-infected esophageal tumors, where Fra-1 expression was diminished or completely absent. These observations, therefore, indicate a potential antagonistic effect of HPV infection on Fra-1. Fra-1 have been proposed to have tumor suppressor function as it is located in chromosome 11q13 region which is known to harbor tumor suppressor genes  and its over-expression have recently been shown to inhibit cell proliferation, induce apoptosis and reduce tumorigenesity . Moreover, change in AP-1 composition resulting in elimination of Fra-1 has been found to be associated with enhanced tumorigenesity [13, 34]. Thus, lack of Fra-1 appears to contributing to more aggressiveness of the disease, as majority of these patients were infected with HPV and in poorly-differentiated state. This is in sharp contrast to our recent observation in oral cancer, where presence of HPV but lack of Fra-1 expression was observed mainly in well-differentiated oral squamous cell carcinomas that showed better prognosis . Thus, it appears that the role of Fra-1 as a tumor suppressor or oncogene may vary from tumor to tumor and on host-virus interactions.
Considering oncogenic role of HPVs  and their co-operative interaction with AP-1 signaling [10, 13], infection with HPV has been implicated as a possible etiological factor in the development of squamous cell carcinoma of the esophagus. Our results demonstrated unlike cervical cancers where HR-HPV infection is an essential etiological factor, the percentage of cases with HPV infection in ESCC was small and accounted for about one fifth of total cases. Occurrence of HPV infection in esophageal cancer is variable and conflicting, ranging from complete absence to detection of up to 60 to 70% mainly of high - risk HPV types 16 and 18 [8, 39]. Our earlier study on different subset of samples from the same region showed similar frequency of HPV16 infection . Though considerable proportions of esophageal cancers have been shown to have HPV infection and that too of high risk types, the etiological role of HPV in these cancers is not established. It is suggested that HPV infection when present may act as a co-factor or have synergistic effects with environmental carcinogens in the genesis of ESCC and/or their progression. We also demonstrate a significant correlation of HPV infection with smoking habit and intake of nitrosated compounds in salted tea [Table 2]. The entry of HPV in esophageal cancers appears to be in later stages as the infection was more prevalent in clinically advanced tumors. Therefore, HPV may significantly influence the disease progression rather than initiation. In contrast to genital cancers, it has been shown that HPV infection may be associated with better prognosis  as it is found more in well-differentiated squamous cell carcinomas of the head and neck  and may positively influence the treatment outcome . Therefore, determination of HPV status in esophageal cancer may also be helpful in predicting the treatment outcome.