Identification of an Intrinsic Determinant Critical for Maspin Subcellular Localization and Function

<div><p>Maspin, a multifaceted tumor suppressor, belongs to the serine protease inhibitor superfamily, but only inhibits serine protease-like enzymes such as histone deacetylase 1 (HDAC1). Maspin is specifically expressed in epithelial cells and it is differentially regulated during tumor progression. A new emerging consensus suggests that a shift in maspin subcellular localization from the nucleus to the cytoplasm stratifies with poor cancer prognosis. In the current study, we employed a rational mutagenesis approach and showed that maspin reactive center loop (RCL) and its neighboring sequence are critical for maspin stability. Further, when expressed in multiple tumor cell lines, single point mutation of Aspartate³⁴⁶ (D³⁴⁶) to Glutamate (E³⁴⁶), maspin^D346E, was predominantly nuclear, whereas wild type maspin (maspin^WT) was both cytoplasmic and nuclear. Evidence from cellular fractionation followed by immunological and proteomic protein identification, combined with the evidence from fluorescent imaging of endogenous proteins, fluorescent protein fusion constructs, as well as bimolecular fluorescence complementation (BiFC) showed that the increased nuclear enrichment of maspin^D346E was, at least in part, due to its increased affinity to HDAC1. Maspin^D346E was also more potent than maspin^WT as an HDAC inhibitor. Taken together, our evidence demonstrates that D³⁴⁶ is a critical <i>cis</i>-element in maspin sequence that determines the molecular context and subcellular localization of maspin. A mechanistic model derived from our evidence suggests a new window of opportunity for the development of maspin-based biologically competent HDAC inhibitors for cancer treatment.</p></div

Maspin nuclear localization correlates with increased histone acetylation and release of HDAC-repressed gene expression.

<p>(<b>A</b>) Western blot of recombinant maspin, HDAC1, and HDAC1 target protein (acetylated Histone 3 at Lysine 9 (H3 Acetyl K⁹)) in DU145 cells. GAPDH was used as a loading control. (<b>B</b>) Q-RT-PCR of HDAC1 targeted genes differentially regulated by maspin. The threshold cycle (ct) numbers obtained from qRT-PCR were normalized by the internal GAPDH controls and presented as the fold change. Maspin^WT: black bar; maspin^D346E: white bar.</p

Increased nuclear localization of maspin^D346E correlates with increased HDAC1 interaction.

<p>(<b>A</b>) Confocal imaging of endogenous maspin (green) and HDAC1 (red) immunofluorescence staining in MCF10A cells. Scale bar = 20 µm (<b>B</b>). Bimolecular fluorescence complementation (BIFC) of live PC3 and DU145 cells transiently transected with pBiFC maspin^WT YC or pBiFC maspin^D346E YC in combination with pBiFC HDAC1 YN. The BiFC of bJunYN and BiFC bFosYC and BiFC of bJunYN and BiFC bFosYC Δ179–193 were used as positive and negative controls, respectfully, in PC3 cells. Scale bar = 10 µm (<b>C</b>) Immunofluorescence imaging (x40) of live PC3 cells after co-transfection with either pEGFP N1-maspin^WT or pEGFP N1 maspin^D346E in combination with pcDNA3.1 HDAC1-RFP. → indicates cytosolic maspin/HDAC1 interaction and ▸ indicates nuclear maspin/HDAC1 interaction. Scale bar = 20 µm. (<b>D</b>) Western blot of recombinant maspin and HDAC1 after immunoprecipitation (IP) with maspin antibody in DU145 cells. The mouse IgG was used as a negative control. Total levels of maspin, HDAC1, and loading control GAPDH are shown in the input panel. Numbers below represent normalized HDAC1/maspin ratio.</p

A hypothetical model explaining disregulation of maspin in tumor progression.

<p>(<b>A</b>) Benign tumor or better differentiated cancer with better prognosis is associated with nuclear maspin. Nuclear maspin has a stronger affinity towards HDAC1 as compared to hypothetical HDAC1-associated nuclear factor X. (<b>B</b>) High grade carcinoma exhibiting a less differentiated phenotype and worse prognosis is associated with cytosolic maspin or loss of maspin. In cancer, hypothetical HDAC1- associated nuclear factor X is X′ and it has a stronger affinity towards HDAC1 as compared to maspin. (<b>C</b>) Therapeutic potential of bioengineered maspin mutant or its derivative in treating advanced disease. Maspin mutant restores maspin nuclear localization and HDAC1 inhibition in advanced disease.</p