Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 134 Sinchon-dong, Seodaemun-gu, Seoul, 120-752, South Korea.
Breast Cancer Res. 2012 Jun 6;14(3):R88. doi: 10.1186/bcr3203.
Although development of anoikis-resistant myofibroblasts during tissue remodeling is known to be associated with tumor invasion, the mechanism by which myofibroblasts become resistant to anoikis is unknown. We previously demonstrated laminin-332 upregulation in the fibrosis around invasive ductal carcinoma (IDC). Because laminin-332 promotes cell survival through binding to integrins, we hypothesized that invasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts by upregulating laminin-332 expression during tissue remodeling. Here, we demonstrate that invasive breast cancer cells induce laminin-332 upregulation and integrin β4 neoexpression in myofibroblasts to confer an anoikis-resistant phenotype.
Three types of fibroblasts were isolated from the tumor burden, the fibrosis, and normal tissue of patients with early stage IDC (less than 10 mm diameter), designated cancer-associated fibroblasts (CAFs), interface fibroblasts (InFs), and normal breast fibroblasts (NBFs), respectively. To investigate direct and indirect crosstalk with tumor cells, fibroblasts were co-cultured with invasive MDA-MB-231 or noninvasive MCF7 cells or in conditioned medium. Anoikis resistance of fibroblasts was measured by cell viability and caspase-3 activity after incubation on poly-HEMA coated plates for 72 hours. Involvement of laminin-332/integrin α3β1 or α6β4 signaling in anoikis resistance was confirmed by treatment with purified laminin-332 or blocking antibodies against laminin-332, integrin β1, or integrin β4.
MDA-MB-231 cells induced laminin-332 upregulation and integrin β4 neoexpression in fibroblasts, leading to anoikis resistance. InFs showed a higher endogenous level of laminin-332 than did CAFs and NBFs. After stimulation with MDA-MB-231-conditioned medium, laminin-332 expression of InFs was dramatically increased and maintained under anoikis conditions. Laminin-332 upregulation was also observed in CAFs and NBFs, but at a lower level than in InFs. Laminin-332 induced Akt (Ser473) phosphorylation by binding to integrin α3β1. Integrin β4 neoexpression induced laminin-332-independent Rac1 activation and promoted anoikis resistance in fibroblasts approximately twofold more effectively than did laminin-332, regardless of the type of fibroblast. In addition, integrin β4 expression suppressed fibroblast aggregation in conditions of anoikis.
Invasive breast cancer cells confer an anoikis-resistant phenotype on myofibroblasts during tissue remodeling by inducing laminin-332 upregulation and integrin β4 neoexpression. Interface fibroblasts appear to be the primary myofibroblasts that interact with invasive tumor cells during tissue remodeling.
尽管已知组织重塑过程中抗失巢凋亡的肌成纤维细胞的发展与肿瘤侵袭有关，但肌成纤维细胞如何获得抗失巢凋亡的能力尚不清楚。我们之前证明了层粘连蛋白-332 在浸润性导管癌（IDC）周围纤维化中的上调。因为层粘连蛋白-332 通过与整合素结合促进细胞存活，所以我们假设在组织重塑过程中，浸润性乳腺癌细胞通过上调层粘连蛋白-332 表达使肌成纤维细胞获得抗失巢凋亡表型。在这里，我们证明浸润性乳腺癌细胞通过诱导肌成纤维细胞中层粘连蛋白-332 的上调和整合素β4 的新表达，赋予肌成纤维细胞抗失巢凋亡表型。
从早期 IDC（直径小于 10 毫米）患者的肿瘤负担、纤维化和正常组织中分离出三种类型的成纤维细胞，分别命名为癌相关成纤维细胞（CAFs）、界面成纤维细胞（InFs）和正常乳腺成纤维细胞（NBFs）。为了研究与肿瘤细胞的直接和间接串扰，将成纤维细胞与浸润性 MDA-MB-231 或非浸润性 MCF7 细胞或在条件培养基中共同培养。在聚-HEMA 包被的平板上孵育 72 小时后，通过细胞活力和 caspase-3 活性测量成纤维细胞的抗失巢凋亡能力。通过用纯化的层粘连蛋白-332 或针对层粘连蛋白-332、整合素β1 或整合素β4 的阻断抗体处理，确认层粘连蛋白-332/整合素α3β1 或 α6β4 信号通路在抗失巢凋亡中的作用。
MDA-MB-231 细胞诱导成纤维细胞中层粘连蛋白-332 的上调和整合素β4 的新表达，导致抗失巢凋亡。InFs 比 CAFs 和 NBFs 具有更高的内源性层粘连蛋白-332 水平。在 MDA-MB-231 条件培养基刺激后，InFs 中的层粘连蛋白-332 表达显著增加，并在失巢凋亡条件下维持。CAFs 和 NBFs 中也观察到层粘连蛋白-332 的上调，但水平低于 InFs。层粘连蛋白-332 通过与整合素α3β1 结合诱导 Akt（Ser473）磷酸化。整合素β4 的新表达诱导层粘连蛋白-332 非依赖性 Rac1 激活，并在失巢凋亡条件下比层粘连蛋白-332 更有效地促进成纤维细胞的抗失巢凋亡，无论成纤维细胞类型如何。此外，整合素β4 的表达抑制了失巢凋亡条件下成纤维细胞的聚集。
在组织重塑过程中，浸润性乳腺癌细胞通过诱导层粘连蛋白-332 的上调和整合素β4 的新表达，赋予肌成纤维细胞抗失巢凋亡表型。界面成纤维细胞似乎是在组织重塑过程中与浸润性肿瘤细胞相互作用的主要肌成纤维细胞。
