Differentiation and Elimination of Chemo-surviving Colon Tumors

描述（由申请人提供）： 尽管使用了积极的手术切除和化疗，但近50%的结直肠癌患者（美国第三大致命癌症）将出现复发性疾病，这突出了改善治疗的必要性。未能在CRC患者中产生长期临床缓解可能反映了无法有效靶向癌症干细胞或干细胞样细胞（CSC/CSLC;表达癌症干细胞标志物），因为已知这些细胞对常规疗法具有抗性。分化治疗可导致CSLCs自我更新能力丧失，诱导终末分化或凋亡，可能是治疗复发性结直肠癌的有效选择。 我们已经证明，miR-21是一种新出现的癌基因，它通过激活结肠癌细胞中的Wnt/β-catenin和EGFR信号传导来诱导干性，从而刺激肿瘤的促进、侵袭和转移。miR-21在5-FU +奥沙利铂（FUOX）耐药结肠癌细胞中也大幅升高; miR-21的下调导致这些细胞分化，如CK-20表达和碱性磷酸酶活性增加所证明。此外，这种下调使得FUOX抗性结肠癌细胞不仅对FUOX敏感，而且对3，4-二氟苯并姜黄素或二氟化姜黄素（CDF）以及CDF和FUOX的组合敏感。CDF是膳食成分姜黄素的新型类似物，具有比母体化合物更高的生物利用度和生长抑制特性。此外，我们已经观察到，响应于CDF或CDF和FUOX的组合，miR-21下调的FUOX抗性结肠癌细胞表现出EGFR和CD 44表达降低和细胞凋亡增加。CDF单用及CDF与FUOX联合应用的疗效均优于FUOX单用。我们建议在临床前环境中扩展这种分化治疗。因此，我们将使用SCID小鼠异种移植和原位结肠癌模型来检验以下假设：在化疗存活的结肠肿瘤中，miR-21（反义miR-21）下调miR-21将导致已知在CSC/CSLC中富集的化疗抗性肿瘤细胞分化。我们进一步假设，虽然随后用FUOX治疗在消除难治性结肠肿瘤方面仅部分有效，但用CDF或CDF和FUOX的组合治疗将通过抑制Wnt/β-连环蛋白和EGFR信号传导杀死分化的化学耐药结肠CSC/CSLC，在引起肿瘤完全或接近完全消退方面非常有效。最后，我们将证明miR-21在体外调节结肠癌化疗耐药细胞中的<$-Wnt/catenin和EGFR信号通路。

DESCRIPTION (provided by applicant): Despite the use of aggressive surgical resection and chemotherapy, nearly 50% of patients with colorectal carcinoma, the 3rd deadliest cancer in the US, will develop recurrent disease, highlighting the need for improved therapies. The failure to produce long-term clinical remission in CRC patients could reflect the inability to target cancer stem or stem-like cells (CSCs/CSLCs; that express cancer stem cell markers) efficiently since these cells are known to be resistant to conventional therapies. Differentiation therapy, which results in loss of self-renewal and induction of terminal differentiation or apoptosis in CSLCs, may be a valid option for the treatment of recurrent colorectal cancer. We have shown that miR-21, an emerging oncogene which induces stemness by activating Wnt/¿-catenin and EGFR signaling in colon cancer cells stimulates tumor promotion, invasion and metastasis. miR-21 is also greatly elevated in 5-FU + Oxaliplatin (FUOX) resistant colon cancer cells; downregulation of miR-21 leads to differentiation of these cells, as evidenced by increased CK-20 expression and alkaline phosphatase activity. Moreover, this downregulation renders the FUOX-resistant colon cancer cells susceptible not only to FUOX but also to 3, 4-difluorobenzo-curcumin or difluorinated curcumin (CDF) and the combination of CDF and FUOX. CDF is a novel analog of the dietary ingredient curcumin with much greater bioavailability and growth inhibitory properties than the parent compound. Additionally, we have observed that in response to CDF or the combination CDF and FUOX, miR-21-downregulated FUOX-resistant colon cancer cells exhibit decreased expression of EGFR and CD44 and increased apoptosis. CDF alone and the combination of CDF and FUOX were much more effective than FUOX alone. We propose to extend this differentiation therapy in a preclinical setting. Therefore, we will use SCID mice xenograft and orthotopic models of colon cancer to test the hypothesis that down regulation of miR-21 by antagomir-21 (antisense miR-21) in chemotherapy surviving colon tumors will lead to differentiation of chemotherapy-resistant tumor cells known to be enriched in CSCs/CSLCs. We further hypothesize that while subsequent treatment with FUOX will only be partially effective in obliterating the refractory colon tumors, treatment with either CDF or the combination of CDF and FUOX will be highly effective in causing a complete or near complete regression of the tumor by killing differentiating chemo-resistant colon CSCs/CSLCs through inhibition of Wnt/¿-catenin and EGFR signaling. Finally, we will demonstrate that miR-21 regulates ¿-Wnt/catenin and EGFR signaling pathways in colon cancer chemo-resistant cells in vitro.