ERK-regulated αB-crystallin induction by matrix detachment inhibits anoikis and promotes lung metastasis in vivo

Abstract

Evasion of extracellular matrix detachment-induced apoptosis ('anoikis') is a defining characteristic of metastatic tumor cells. The ability of metastatic carcinoma cells to survive matrix detachment and escape anoikis enables them to disseminate as viable circulating tumor cells and seed distant organs. Here we report that αB-crystallin, an antiapoptotic molecular chaperone implicated in the pathogenesis of diverse poor-prognosis solid tumors, is induced by matrix detachment and confers anoikis resistance. Specifically, we demonstrate that matrix detachment downregulates extracellular signal-regulated kinase (ERK) activity and increases αB-crystallin protein and messenger RNA (mRNA) levels. Moreover, we show that ERK inhibition in adherent cancer cells mimics matrix detachment by increasing αB-crystallin protein and mRNA levels, whereas constitutive ERK activation suppresses αB-crystallin induction during matrix detachment. These findings indicate that ERK inhibition is both necessary and sufficient for αB-crystallin induction by matrix detachment. To examine the functional consequences of αB-crystallin induction in anoikis, we stably silenced αB-crystallin in two different metastatic carcinoma cell lines. Strikingly, silencing αB-crystallin increased matrix detachment-induced caspase activation and apoptosis but did not affect cell viability of adherent cancer cells. In addition, silencing αB-crystallin in metastatic carcinoma cells reduced the number of viable circulating tumor cells and inhibited lung metastasis in two orthotopic models, but had little or no effect on primary tumor growth. Taken together, our findings point to αB-crystallin as a novel regulator of anoikis resistance that is induced by matrix detachment-mediated suppression of ERK signaling and promotes lung metastasis. Our results also suggest that αB-crystallin represents a promising molecular target for antimetastatic therapies.

Figure 1. αB-crystallin is induced by matrix detachment-mediated inhibition of ERK signaling in cancer cells

(a) 435-LvBr1-mCherry and GILM2-mCherry cancer cells were grown for 72 h in adherent or suspension (susp.) culture. αB-crystallin and Hsp27 levels were analyzed by immunobloting (left), and αB-crystallin mRNA levels were determined by RT-PCR (right). mRNA levels were normalized to expression in adherent cells. (b) 435-LvBr1-mCherry and GILM2-mCherry cancer cells were grown for 72 h in adherent or suspension culture, and the expression of αB-crystallin, ERK and p-ERK was analyzed by immunobloting. (c) and (d) 435-LvBr1-mCherry (c) or GILM2-mCherry cancer cells (d) were treated with vehicle (cont.), 40 μM LY294002 (LY), 50 μM PP2, 10 μM CI-1040 (CI) or 10 μM Selumetinib (AZD) for 48 h. αB-crystallin, ERK, p-ERK, Akt, p-Akt, Src, and p-Src levels were determined by immunoblotting (left), and αB-crystallin expression was determined by RT-PCR (right). (e) Immunoblot of MCF10A-Vector or MCF-10A-RasV12 cells (left panel) or GILM2 cells expressing empty Vector or constitutively active MEK-DD (right panel) grown in adherent or suspension culture in the absence or presence of 10 μM CI-1040 or 10 μM Selumetinib for 72 h. In (a), (c) and (d), **P < 0.01 and ***P < 0.001.

Figure 2. αB-crystallin inhibits anoikis in cancer cells

(a) Immunoblot analysis of αB-crystallin expression in 435-LvBr1-mCherry and GILM2-mCherry cancer cells stably expressing a non-silencing (NS) construct, αB-crystallin shRNA (sh1-αB or sh2-αB), or sh1-αB and a mutant αB-crystallin that is resistant to gene silencing but retains its coding sequence (sh1-αBM). (b) and (c) MTS cell viability assay of 435-LvBr1-mCherry (b) and GILM2-mCherry cells (c) stably expressing NS, sh1-αB, sh2-αB or sh1-αBM grown in adherent or suspension culture. The number of viable cells is expressed as fold change normalized to day 1 (n = 3). **P < 0.01, ***P < 0.001.

Figure 3. αB-crystallin inhibits matrix detachment-induced caspase activation and apoptosis and promotes cell survival in response to combined MEK inhibition and matrix detachment

(a) 435-LvBr1-mCherry and GILM2-mCherry cancer cells stably expressing NS, sh1-αB, sh2-αB or sh1-αBM were grown in suspension for 48 h. Full-length PARP and cleaved caspase-3 were detected by immunoblotting. (b) 435-LvBr1-mCherry and GILM2-mCherry breast cancer cells stably expressing NS, sh1-αB, or sh1-αBM were grown in suspension for 24 h. Cancer cells stably expressing sh1-αB were untreated or treated with 50 μM zVAD-fmk. Apoptosis was measured by Annexin V labeling using flow cytometry. (c) MTS cell viability assay of 435-LvBr1-mCherry and GILM2-mCherry cells stably expressing NS or sh1-αB treated with vehicle or 10 μM AZD6244 in adherent or suspension culture for 48 h (n = 3). *P < 0.05, **P < 0.01.

Figure 4. αB-crystallin promotes circulating tumor cell survival and lung metastasis in an orthotopic 435-LvBr1-mCherry model

(a) 435-LvBr1-mCherry cells stably expressing NS or sh-αB were injected intraductally into the 4^th mammary glands of female athymic nude mice. Mammary tumor volume was measured weekly with calipers and expressed as the percentage original tumor volume at 2 weeks (n=10 mice per group). (b) 435-LvBr1-mCherry-NS and 435-LvBr1-mCherry-sh-αB mammary tumors were immunoblotted. αB-crystallin expression in mammary tumors and lung metastases was determined by immunohistochemistry in both groups (c) Representative whole lung images by fluorescence microscopy. The number of fluorescent metastases per lung and the percentage of the surface area occupied by lung metastases in NS and sh-αB groups (n = 10 mice per group) are indicated. (d) The number of colonies per mouse derived from the blood of mice bearing 435-LvBr1-mCherry-NS or 435-LvBr1-mCherry-sh-αB xenografts (n=5 mice per group). In (a), (c) and (d), *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5. αB-crystallin promotes circulating tumor cell survival and lung metastasis in an orthotopic TNBC model

(a) GILM2-mCherry cells stably expressing NS or sh-αB were injected intraductally into the 4^th mammary glands of female athymic nude mice. Mammary tumor volume was measured weekly and expressed as the percentage original tumor volume at 2 weeks (n=10 mice per group). (b) GILM2-mCherry-NS and GILM2-sh-αB mammary tumors were immunoblotted, and αB-crystallin expression was determined by immunohistochemistry of mammary tumors and lung metastases in both groups. (c) Representative whole lung images by fluorescence microscopy. The number of fluorescent metastases per lung and the percentage surface area of lung metastases in NS and sh-αB groups (n = 10 mice per group) are shown. (d) The number of colonies per mouse derived from the blood of mice bearing GILM2-mCherry-NS or GILM2-mCherry-sh-αB xenografts (n=5 mice per group). In (a), (c) and (d), **P < 0.01 and ***P < 0.001.