Loss of the integrin-activating transmembrane protein Fam38A (Piezo1) promotes a switch to a reduced integrin-dependent mode of cell migration

Abstract

Lung cancer is one of the most common fatal diseases in the developed world. The disease is rarely cured by currently available therapies, with an overall survival rate of ∼10%. Characterizing novel proteins that offer crucial insights into the processes of lung tumour invasion and metastasis may therefore provide much-needed prognostic markers, and influence therapeutic strategies. Aberrant function of the integrin family of heterodimeric cell surface receptors is a common theme in cancer--investigation into novel integrin activity regulators may offer crucial insights into the processes of tumour invasion and metastasis and may reveal insights into potential therapeutic targets. We previously described that depletion of the novel multi-transmembrane domain protein Fam38A, located at the endoplasmic reticulum (ER), inactivates endogenous beta1 integrin affinity, reducing cell adhesion. We now show that depletion of Fam38A, also now known as Piezo1, causes anchorage independence and a switch to a reduced integrin-dependent mode of cell migration/invasion, a novel phenotype for this integrin-regulating protein. Normal lung epithelial cells show increased rates of migration by 2D time-lapse microscopy and increased capacity to invade into matrigel, despite having decreased integrin affinity. We confirm greatly depleted Fam38A expression in small cell lung cancer (SCLC) lines where a form of reduced integrin-dependent migration, i.e. amoeboid migration, is a known phenotype. We propose that loss of Fam38A expression may cause increased cell migration and metastasis in lung tumours.

Figure 1. Fam38A expression is reduced in SCLC cell lines.

Fam38A expression was measured by Real-time PCR in the indicated cell lines. Expression analysis showed a severe reduction (>90%) of Fam38A expression levels in SCLC cells, compared to 16HBE and Beas2B normal lung epithelial control cells.

Figure 2. Fam38A siRNA causes cell detachment and anchorage independence in normal bronchial epithelial 16HBE cells.

(a) Quantitation of Fam38A knock-down in 16HBE by Real-time PCR, compared to control siRNA treated cells. (b) Flow cytometry quantitation of control- and Fam38A-siRNA cells stained with the affinity-dependent integrin beta1 integrin antibody CD29 (HUTS-21). Fam38A-depleted cells show a reduction in HUTS-21 staining to 54% (+/−5%) that of control cell. N = 4. (c) Adhesion of control and Fam38A-siRNA cells - Fam38A-siRNA depleted cells showed a significant reduction in adhesion down to approximately 20% that of control cells. N = 3. (d) Colony formation in soft agar of 16HBE treated with control- or Fam38A-siRNA, showing a significant increase (approximately 7-fold) in colony number in Fam38A-depleted cells. N = 4.

Figure 3. Fam38A-depletion results in increased 2D cell migration and 3D cell invasion intro matrigel.

(a) Representative examples (4 per condition) of control- and Fam38A-siRNA treated 16HBE cell 2D migration using time-lapse microscopy. Fam38A-depleted cells migrate up to 250 µm from their starting point, in contrast to control cells which migrate up to 50 µm in the same time period. (b) Quantitation of invasion into matrigel of control vs. Fam38A-siRNA treated 16HBE cells, or GFP-vector vs. GFP-Fam38A transfected cells, towards a serum gradient. Representative confocal projections at a 90° angle to the stage are shown, demonstrating the extent of cell invasion.

Figure 4. TNS4 upregulation, actin rearrangement and calpain activity in Fam38A-depleted cells.

(a) Control- and Fam38A-siRNA treated 16HBE cells were stained for TNS4 (red channel), actin cytoskeleton (green channel), and nuclei (blue channel) showing upregulation of TNS4 expression and actin rearrangement in Fam38-depleted cells. Scale bar = 5 µm. (b) Quantitation of calpain activity (DABCYL cleavage) in 16HBE cells transfected with control- or Fam38A-siRNA. Mean N = 4, +/− SEM, p<0.05.