J Natl Cancer Inst. translocation of JAK2 resulting in phosphorylation of Histone-3 up-regulating stemness-specific genes and Jak2 dependence was demonstrated using Jak2+/+ and Jak2?/? cells. Using eGFP-Luciferase labeled cells, we demonstrate enhanced tumorigenic and metastatic potential of MUC16-Cter [28] and [29] implicated in inducing stem cell-like features during carcinogenesis [30-32]. In our previous study, we showed expression of MUC16 in the high-grade preneoplastic lesion, primary as well as metastatic PC with metastatic tumors having stronger MUC16 expression compared to the primary tumors from the same patient [33]. In the present study, we report (i) the generation of a 17-kDa cleaved MUC16 (MUC16-Cter) using dual-epitope tagged 114 amino Cyromazine acids of carboxyl-terminal MUC16 in PC cells, (ii) MUC16-Cter mediated enrichment of ALDH+ cancer stem-like cells imparts tumorigenic, metastatic and drug resistant properties to PC cells and (iii) MUC16-Cter mediated enrichment of stemness specific genes and is dependent on nuclear JAK2. RESULTS Expression of dual-tagged 114 amino acids of carboxyl-terminal MUC16 generates a ~17 kDa cleaved MUC16 and imparts proliferative advantage to PC cells Although Cyromazine previous studies addressed the functional significance of various lengths of carboxyl-terminal MUC16 fragments (283 and 413 amino acids) in ovarian, breast and colon cancer cells, none demonstrated whether a cleaved MUC16 is generated following ectopic expression of these fragments [19,24,34]. Since the cleavage of MUC16 in the last (56th) SEA domain is predicted to be at NFSPLARRVDR site that lies 50 residues upstream to the transmembrane domain in the last SEA domain [10], we reasoned that use of carboxyl-terminal 114 amino acids that includes the above mentioned cleavage site would be the smallest fragment that can generate the functional cell-associated MUC16. Due to lack of antibodies for the juxta-membrane region of MUC16, we generated a dual epitope-tagged mammalian expression construct using 114 carboxyl-terminal fragment of MUC16 with N-terminal FLAG-tag and a C-terminal HA-tag (Figure ?(Figure1A).1A). The resultant control (p3X-FLAG-CMV9 or CMV9) and MUC16-Cter (p3X-FLAG-114HA or Cyromazine F114HA) expression constructs were stably transfected into MUC16 non-expressing MiaPaCa-2 and expressing T3M4 PC cells. Expression of MUC16-Cter was verified by immunoblot and immunofluorescence analyses using anti-FLAG and anti-HA antibodies (Figures 1B and 1C). A unique ~17 kDa product representing the cleaved carboxyl-terminus of MUC16 was present in HA but not in FLAG-immunoblot (Figure ?(Figure1B).1B). Although we are not able to show cleavage of endogenous MUC16 owing to commercial unavailability of CTD specific antibody, Davies proliferation of PC cells(A) Schematic representation of full-length and 114 amino acids of carboxyl-terminal MUC16 with N-terminal FLAG and C-terminal HA-tag (F114HA) cloned into the p3X-FLAG-CMV9 vector (CMV9) with a preprotyrpsin leader peptide (LP). (B) MiaPaCa-2 (MUC16-non-expressing) and T3M4 (MUC16-expressing) PC cells were stably transfected with F114HA plasmids along with their vector only (CMV9) controls. Cell lysates were immunoblotted with indicated antibodies. (C) Immunofluorescence analysis of MiaPaCa-2 and T3M4 cells stably transfected with F114HA plasmids along with their vector only (CMV9) controls using anti-FLAG and anti-HA antibodies. DAPI was used to stain the nucleus. Scale bars, 10 m. (D and E) Proliferation of MiaPaCa-2 (D) and T3M4 (E) cells was measured by the WST1 assay: control cells (black line) and F114HA expressing cells (grey line). Cyromazine Data represent mean s.e.m of a representative experiment (n=4, Student’s is time interval between two stages of growth, and proliferation was measured using WST1 assay. Both MiaPaca-2 and T3M4-F114HA cells exhibited a significant increase in the proliferative potential with a ~ 6 C 7 h reduction in the doubling time (Figure 1D and 1E, *P 0.05, **P 0.001) compared to the control (CMV9) cells. MUC16-Cter promotes G2/M block with apoptotic resistance, a property associated with cancer stem-like cells, in PC cells Previously MUC16 was shown to induce rapid G2/M transition in MDA-MB-231 breast cancer cells [23]. However, cell cycle Cyromazine analysis to gaze at the role of MUC16 C-ter in PC cells, resulted in significant accumulation of cells in the G2/M phase (Figure ?(Figure2A,2A, P=0.03) as opposed to rapid G2/M transition [23]. Interestingly, this was unaccompanied by an increase in apoptosis (Figure ?(Figure2B),2B), a property expected of cells blocked in the G2/M phase. Extended G2/M phase with increased resistance to apoptosis is a property commonly ascribed to Rabbit Polyclonal to SKIL cancer stem cells (CSCs) [36-38]. To examine whether ectopic expression of MUC16-Cter confers CSC phenotype in PC cells, ALDH activity was measured that is shown to be more relevant [39] and efficient [40] in identification of PC stem cells..