PC is an extremely lethal disease, with a five year survival rate of less than 5% and a median survival period of 5-6 months. At the time of diagnosis, PC metastasizes to regional lymph nodes and distant organs and responds poorly to current chemo- and radiation therapies resulting in a high recurrence rate [1–3]. The poor prognosis and weak therapeutic responses are a consequence of late diagnosis of the majority of PC patients, primarily due to lack of early symptoms and reliable early diagnostic markers . Therefore, there is an urgent need to identify specific early biomarkers for early diagnosis and molecular targets for effective treatment of PC.
Previous studies done in human tissues have indicated an aberrant overexpression of various mucins in several epithelial malignancies including pancreatic, ovarian and lung cancers [7, 10]. Thus, not surprisingly, their potential in the diagnosis and targeted treatment of PC has been suggested and tested over the last decades [35, 36]. In cancer cells, mucins play an important role in cell growth, differentiation, transformation, adhesion, invasion and immune evasion [5, 8, 20]. In human PC tissues, MUC1, MUC4, and MUC5AC are aberrantly upregulated and their expression has been linked to the progression and poor prognosis of the disease. However, due to the late diagnosis of PC, the status of mucin expression in the earliest stages of the disease remains unknown.
Genetically engineered mouse models can facilitate the discovery of tumor biomarkers in order to design powerful techniques to diagnose, treat, and monitor therapeutic efficacy in cancer patients more effectively . Mouse Muc1 shares 34% homology with human MUC1 in the tandem repeat region mainly sharing threonine, serine and O-linked sugars but it is 87% homologous at transmembrane and cytoplasmic regions. Due to high degree of conservation in the promoter region (74%), the patterns of expression of mouse Muc1 is quite similar to human MUC1 . Similarly, the mouse and human MUC4 have identical exon/intron structure . Further, human MUC4 homology analysis with mouse, dog, rat, and chicken Muc4 revealed that NIDOgen-like (NIDO), Adhesion associated domain of MUC4 and Other Proteins (AMOP), von Willebrand factor D (vWD), Epidermal Growth Factor (EGF), transmembrane (TM), and cytoplasmic tail (CT) domains are highly conserved across the species suggesting that individual domains evolved from common ancestral domains and share common functions . In the case of mouse Muc5AC (located on chromosome 7), it shares 52% homology with human MUC5AC (located on human chromosome 11) and TATA box regions in both the species are fully conserved . Because mucin genes are conserved between humans and mice, such mouse models provide a unique opportunity to examine the expression profile and possibly functional role of mucin genes at the earliest stages of the disease.
We used a well characterized KrasG12D;Pdx1-Cre spontaneous PDAC mouse model, which recapitulates human PC genetically, histologically and pathologically , to investigate if the expression pattern of murine mucins (i.e. Muc1, Muc4 and Muc5AC) mirrors the altered mucin profile of the human disease. The KrasG12D;Pdx1-Cre genetically engineered mouse PDAC model was chosen over other spontaneous PDAC models because it recapitulates the full spectrum of human PanIN lesions, which are recognized as early events in PC. Moreover, mass spectrometry proteomics analysis in this mouse model identified a distinct serum proteome having preinvasive PanIN lesions compared to healthy controls , emphasizing its utility as a suitable platform to understand early stages of PC that may lead to the optimization of diagnostic and therapeutic techniques against this malignancy.
MUC1 is a transmembrane mucin with basal level expression in normal epithelial cells lining various organs including the pancreas. It has been shown to be overexpressed and aberrantly glycosylated in PC and play a role in the invasion and metastasis of PC [6, 8, 19]. Overexpression of MUC1 has been observed during the early stages of PC development, with a subsequent increase in expression in invasive carcinoma, both in humans and p48; KrasG12D; MUC1.Tg mouse model [18, 41]. Similarly, IPMNs like lesions from KrasG12D;TGFÎ±;Pdx-1-Cre transgenic mice showed elevated Muc1 and Muc5AC expression at 3 months of age  and recent reports also revealed that KrasG12D;P48-Cre; Muc1KO mice had slower tumor progression and metastasis compared to both KrasG12D;P48-Cre and KrasG12D;P48-Cre; MUC1 transgenic animals . On the other hand, Muc1 null mice are phenotypically normal and exhibit normal reproduction and survival rate . Previous studies in human pancreatic tissues also reported an increase in MUC1 expression which correlated with grade of PanIN lesions and PDAC . In our study, mRNA and protein levels of Muc1 progressively increased from 10 weeks to 50 weeks of age in the pancreas of KrasG12D;Pdx1-Cre mice compared to unfloxed LSLKrasG12D mice, and correlated with the development of PDAC from PanIN precursor lesions (Figure 2). Thus, the expression of Muc1 in the KrasG12D;Pdx-1-Cre spontaneous PDAC progression model corroborates its resemblance with the human disease.
MUC4 is a high molecular weight, type I transmembrane glycoprotein that is overexpressed in PC but absent in normal pancreas and chronic pancreatitis . Although previous studies in human specimens have shown an increased expression of MUC4 in PC progression and metastasis [10, 11], it remains unknown if MUC4 overexpression is an early event in PC. MUC4 expression has been observed in precursor PanIN lesions in clinical samples , which is suggestive of, but not a definitive proof of MUC4 overexpression as an early event in PC. In the present study, we observed that Muc4 mRNA and protein levels increased progressively from 10 weeks of age, which is when we observed the appearance of PanIN I lesions and continued to increase up to 40 weeks of age where we observed advanced PanIN III lesions (Figure 3). Our findings establish that Muc4 expression is indeed an early event in PC progression, which recapitulates the MUC4 expression profile in human PC. Future studies using Muc4 knock out and MUC4 transgenic animals on the KrasG12D murine background will help delineate the molecular mechanisms and contribution of Muc4 in PC progression and metastasis. Nonetheless, the present study establishes the suitability of KrasG12D model for evaluating the potential of Muc4 as an early diagnostic marker and therapeutic target.
The expression of the gel-forming secretory mucin MUC5AC in human PC increases progressively with the increase in grade of PanIN lesions and PDAC, whereas it is undetected in normal pancreas [19, 29]. Similar to the expression of the transmembrane mucins MUC1 and MUC4, MUC5AC expression has also been related to PC progression  and it is associated with a shorter survival period of PC patients . In the present study, Muc5AC expression in the pancreas of KrasG12D;Pdx1-Cre spontaneous PDAC mice increased progressively from 10 to 50 weeks of age (Figure 4) as compared to unfloxed LSLKrasG12D mice, corroborating studies of the human disease. It is important to emphasize the particular usefulness of the detection of Muc5AC in early lesions of PC, as its secretory nature is advantageous for non-invasive serum based diagnostics.
Previous studies with human tissues have implicated Kras activation in rigorous inflammatory responses in PC, mainly by activating the NF-κB pathway . In agreement with these studies, recent studies reported the observation of proinflammatory responses in the KrasG12D;PdxCre spontaneous PDAC mouse model, which suggested that chronic inflammation is indeed a precursor and potentially a key factor in promoting PC . These studies suggested that constitutive NF-κB activation and inflammatory responses induced by oncogenic Kras are one of the earliest events in PC development. Mucins are known to be transcriptionally regulated by inflammatory cytokines like IFN-γ (MUC4)  and neutrophil elastase (MUC1 and MUC5AC), which is a serine proteinase secreted by neutrophils during inflammation [33, 48]. Moreover, a recent study demonstrated that glycosylation of mucins can be altered in response to proinflammatory conditions in PC cells . Given the functional and pathological significance of MUC1, MUC4 and MUC5AC in PC progression and their regulation by inflammatory environment in the human disease, we analyzed the inflammation in the pancreas of KrasG12D;Pdx1-Cre mice. Increased inflammation in the pancreas of KrasG12D;Pdx1-Cre spontaneous PDAC mice correlated with an increase in inflammatory cytokines/chemokines such as INFγ (p < 0.0062), CXCL1 (p < 0.00014), CXCL2 (p < 0.08) and lymphocyte (Figure 5C) and macrophage infiltration (Figure 5D, G-H). These results correlate with an increase in the expression of Muc1, Muc4 and Muc5AC in the pancreas of KrasG12D;Pdx1-Cre spontaneous PDAC mouse model, suggesting a possible link between inflammation and mucin expression, which further recapitulates the studies done in the human disease.