Aberrant activation of Rho/ROCK signaling in impaired polarity switching of colorectal micropapillary carcinoma

Abstract

Micropapillary carcinoma (MPC) is a morphologically distinctive form of carcinoma, composed of small nests of cancer cells surrounded by lacunar spaces. Invasive MPC is associated with poor prognosis. The nests of tumor cells in MPC reportedly exhibit reverse polarity, although the molecular mechanisms underlying MPC patterns are poorly understood. Using the cancer tissue‐originated spheroid (CTOS) method, we previously reported polarity switching in colorectal cancer (CRC). When cultured in suspension, the apical membrane promptly switches from the outside surface of the CTOSs to the surface of the lumen inside the CTOSs under extracellular matrix (ECM)‐embedded conditions, and vice versa. Here, we investigated two CTOS lines from CRC patient tumors with MPC lesions. Xenograft tumors from the CTOSs exhibited the MPC phenotype. The MPC‐CTOSs did not switch polarity in vitro. Time‐course analysis of polarity switching using real‐time imaging of the apical membrane revealed that local switching was continually propagated in non‐MPC‐CTOSs, while MPC‐CTOSs were unable to complete the process. Integrin β4 translocated to the outer membrane when embedded in ECM in both MPC and non‐MPC‐CTOSs. Protein levels, as well as the active form of RhoA, were higher in MPC‐CTOSs. The suppression of RhoA activity by GAP overexpression enabled MPC‐CTOSs to complete polarity switching both in vitro and in vivo, while overexpression of active RhoA did not affect polarity switching in non‐MPC‐CTOSs. Pretreatment with a ROCK inhibitor enabled MPC‐CTOSs to complete polarity switching both in vitro and in vivo, although delayed treatment after becoming embedded in ECM failed to do so. Thus, the inability to switch polarity might be a cause of MPC, in which the aberrant activation of RhoA plays a critical role. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Polarity switching of ovarian cancer cell clusters via SRC family kinase is involved in the peritoneal dissemination

“…Next, the role of SFK activation was investigated because our previous study revealed that SFK is the key molecule in the polarity switching of colorectal cancer 11,12 . In Ov114 and Ov129 OC organoids 48 hours after embedding in Matrigel, SFK phosphorylation upregulation was induced (Figure 3A).…”

“…Firstly, the polarity status in OC tissues and cell clusters in ascites was evaluated using immunofluorescent imaging. ZO‐1, a tight‐junction protein, and integrin beta 4 (ITGB4) were used as the apical and basal markers, respectively 11,12 . Unsurprisingly, in the OC tissue, ITGB4 was expressed on the membrane facing the stroma in the outermost cells of the cancer cell region (Figure 1A).…”

“…Next, the role of SFK activation was investigated because our previous study revealed that SFK is the key molecule in the polarity switching of colorectal cancer. 11,12 In Ov114 and Ov129 OC organoids 48 hours after embedding in Matrigel, SFK phosphorylation upregulation was induced (Figure 3A). This SFK activity upregulation was suppressed by SFK inhibitors, robustly by dasatinib and mildly with PP1 (Figure 3A) at the concentration below the toxic level (Figure S4).…”

“…ZO-1, a tightjunction protein, and integrin beta 4 (ITGB4) were used as the apical and basal markers, respectively. 11,12 Unsurprisingly, in the OC tissue, ITGB4 was expressed on the membrane facing the stroma in the outermost cells of the cancer cell region (Figure 1A). Meanwhile, ZO-1 is typically localized in the lining of the surface facing the lumen inside the cancer cell region (apical-in; Figure 1A).…”

Distinct and interchangeable growing patterns in colorectal cancer stem-like cells are regulated by Musashi-1

“…The PCR product was cloned into the pENTR4 no ccDB plasmid (Addgene #17424, a gift from Eric Campeau & Paul Kaufman) and transferred into the pLX304 plasmid by the Gateway cloning system. The lentiviral vectors were transfected (Roche, 6366236001; Addgene #12259 and #12260, both gifts from Didier Trono) as previously reported (Onuma et al, 2021). For the pLX304-GFP and -MSI1, blasticidin (2 µg/mL) (Millipore Sigma, 15205) selection was performed.…”