FNIP1 and FNIP2 inhibit Hsp90 chaperone cycle and enhance drug binding

Heat shock protein-90 (Hsp90) is a molecular chaperone required for folding, stability and activity of many proteins, known as clients, including drivers of tumour initiation, progression and metastasis (Rohl et al. 2013). ATPase binding and hydrolysis is essential for the chaperone function of Hsp90. ATPase function is regulated by other proteins known as co-chaperones. In an interesting new study, Woodford et al. (2016) show that the stability of the tumour suppressor folliculin (FLCN), whose mutations cause Birt-Hogg-Dubé syndrome, is dependent on the chaperone function of Hsp90. Authors report that folliculin-interacting protein (FNIP)1 and FNIP2 act as co-chaperones of Hsp90 by regulating its ATPase activity and chaperoning. They also show that the Aha1 co-chaperone competes with FNIPs and can stimulate Hsp90 ATPase activity and that FNIPs enhance the binding of Hsp90 to its inhibitors.

Using human embryonic kidney 293 (HEK293) cells, authors used mass spectrometry to show that that FLCN interacts with the chaperones Hsp70 and Hsp90, their regulators and members of the chaperonin system TRiC. Hsp90 helps to protect its clients from degradation (Rohl et al. 2015), so the authors treated HEK293 cells with the Hsp90 inhibitor ganetespib (GB) and found that FLCN is then ubiquitinated and degraded in the proteasome.

FNIPs were shown to interact with Hsp90 by immunoprecipitation. Treating HEK293 cells with GB did not affect FNIP stability, suggesting that the FNIPs act as co-chaperones of Hsp90 rather than clients. Direct interaction between FNIP1 and Hsp90 was shown and although Hsp90 and FLCN did not directly interact, pre-incubation of Hsp90 with FNIP1 facilitated the Hsp90–FNIP1–FLCN complex formation suggesting that FNIPs are involved in loading of FLCN to Hsp90. Silencing either FNIP1 or FNIP2 with small interfering RNA (siRNA) caused a small decrease in Hsp90 ‘client’ protein levels, while silencing both significantly decreased the stability of ‘clients’ suggesting that the FNIP1 and FNIP2 are functionally redundant here. Overexpression of FNIPs caused an increase in the activity of Hsp90 ‘clients’, including FLCN. Because Hsp90 chaperone function is coupled to its ATPase activity (Panaretou et al. 1998), the impact of FNIPs on Hsp90 ATPase activity was assessed. The presence of FNIPs significantly inhibited the ATPase activity of Hsp90 suggesting that they are potent inhibitors of the chaperone cycle. The authors depleted FNIP protein levels in HEK293 cells to study FNIP effects on Hsp90 interactions with other co-chaperones. Silencing FNIPs increased Hsp90 interaction with the co-chaperones Aha1 and PP5, while overexpressing FNIPs led to significantly reduced Hsp90 interaction with Aha1 and PP5. While FNIPs inhibited the ATPase activity of Hsp90, subsequently adding Aha1 stimulated Hsp90 ATPase activity. These data suggest that Aha1 and the FNIPs compete to bind Hsp90. The impact of FNIPs on Hsp90 binding to ATP or GB was also examined. Overexpression of FNIPs in HEK293 cells led to increase in Hsp90 binding to ATP and to the inhibitor GB. Silencing of FNIPs did not have an impact on Hsp90 binding to ATP but significantly decreased binding to GB.

Tumour cells were already known to be sensitive to Hsp90 inhibitors (Dunn et al. 2015; Woodford et al. 2016), so the authors tested the effect of FNIPs on this sensitivity. FNIPs were highly expressed in a variety of different cancer cell lines and Hsp90 interaction with FNIPs was detected via immunoprecipitation. Two apoptotic markers were abundant in cancer cells treated with GB inhibitor but this effect was not seen in samples lacking FNIPs suggesting that overexpression of FNIPs contributes towards cancer cell sensitivity to Hsp90 inhibitors. Interestingly for BHD, tumours and adjacent normal tissues from patients with renal cell carcinoma (RCC) were analysed and the data showed that FNIPs were overexpressed in RCC tumours compared with adjacent tissues and the Hsp90 from tumours had a higher affinity for binding to GB. As expected, there was a greater association between Hsp90 and FNIPs in tumours compared with normal tissues. However, Hsp90 bound more strongly to Aha1 in normal tissues than in tumours even though both tissues expressed equal levels of Aha1. Addition of FNIP to the protein lysates from normal tissues displaced Aha1 interaction with Hsp90 and increased Hsp90 binding to GB suggesting that FNIPs make renal tumours sensitive to Hsp90 inhibitors.

In summary, the authors show that FNIPs expression correlates with the cellular response to Hsp90 inhibitors, that they are co-chaperones of Hsp90, and that they inhibit Hsp90 ATPase activity.  The results suggest that FNIPs expression level could potentially serve as a predictive indicator of tumour response to Hsp90 inhibitors used in cancer therapy.

  • Woodford MR, Dunn DM, Blanden AR, Capriotti D, Loiselle D, Prodromou C, Panaretou B, Hughes PF, Smith A, Ackerman W, Haystead TA, Loh SN, Bourboulia D, Schmidt LS, Marston Linehan W, Bratslavsky G, & Mollapour M (2016). The FNIP co-chaperones decelerate the Hsp90 chaperone cycle and enhance drug binding. Nature communications, 7 PMID: 27353360
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