Complex polymorphisms in endocytosis genes suggest alpha-cyclodextrin as a treatment for breast cancer

Abstract

Most breast cancer deaths are caused by metastasis and treatment options beyond radiation and cytotoxic drugs, which have severe side effects, and hormonal treatments, which are or become ineffective for many patients, are urgently needed. This study reanalyzed existing data from three genome-wide association studies (GWAS) using a novel computational biostatistics approach (muGWAS), which had been validated in studies of 600-2000 subjects in epilepsy and autism. MuGWAS jointly analyzes several neighboring single nucleotide polymorphisms while incorporating knowledge about genetics of heritable diseases into the statistical method and about GWAS into the rules for determining adaptive genome-wide significance. Results from three independent GWAS of 1000-2000 subjects each, which were made available under the National Institute of Health's "Up For A Challenge" (U4C) project, not only confirmed cell-cycle control and receptor/AKT signaling, but, for the first time in breast cancer GWAS, also consistently identified many genes involved in endo-/exocytosis (EEC), most of which had already been observed in functional and expression studies of breast cancer. In particular, the findings include genes that translocate (ATP8A1, ATP8B1, ANO4, ABCA1) and metabolize (AGPAT3, AGPAT4, DGKQ, LPPR1) phospholipids entering the phosphatidylinositol cycle, which controls EEC. These novel findings suggest scavenging phospholipids as a novel intervention to control local spread of cancer, packaging of exosomes (which prepare distant microenvironment for organ-specific metastases), and endocytosis of β1 integrins (which are required for spread of metastatic phenotype and mesenchymal migration of tumor cells). Beta-cyclodextrins (βCD) have already been shown to be effective in in vitro and animal studies of breast cancer, but exhibits cholesterol-related ototoxicity. The smaller alpha-cyclodextrins (αCD) also scavenges phospholipids, but cannot fit cholesterol. An in-vitro study presented here confirms hydroxypropyl (HP)-αCD to be twice as effective as HPβCD against migration of human cells of both receptor negative and estrogen-receptor positive breast cancer. If the previous successful animal studies with βCDs are replicated with the safer and more effective αCDs, clinical trials of adjuvant treatment with αCDs are warranted. Ultimately, all breast cancer are expected to benefit from treatment with HPαCD, but women with triple-negative breast cancer (TNBC) will benefit most, because they have fewer treatment options and their cancer advances more aggressively.

Fig 1. GWAS quantile-rank (QR) plots.

Left: ssGWAS, right: muGWAS (each point represents the most significant result among all diplotypes centered at the same SNP) Results are ranked by significance (bottom). For the most significant results and other results of interest, the location of SNPs to genes is shown in. Upper curve (red): convex fit against points; dashed extension: projection; lower curve (blue): population-specific expectation. Vertical lines between curves connect the highest s-values (−log₁₀ p) of a gene (dot) with its expected value for genes with known function. Light gray vertical lines indicate genes omitted from the list because of low reliability (either low µIC or reliance on a single SNP), respectively. Genes to the right of the vertical dark line are above the aGWS cut-off. See Fig A in S1 File for Manhattan plots. The horizontal solid line at highest point at the end of the expected curve indicates the estimate for adjusted GWS (aGWS). All points above the horizontal line (and genes to the right of the vertical blue line) are “significant” at the aGWS level.

Fig 2. EEC of β1 Integrin underlying mesenchymal tumor cell migration and invasion.

Cell migration necessitates trafficking of β1 integrin, whose internalization is controlled by dynamin. Both clathrin- and caveolin 1 (CAV1)-coated domains of the plasma membrane are involved. Once in early endosomes (EE), integrins may be sorted for degradation in lysosomes, recycled to the plasma membrane through a RAB4-dependent route, or transported to the recycling endosome (RE). Recycling from the RE requires Rab11 family members, such as RAB25 which is often aberrantly expressed in human tumors, including luminal B breast cancer.[49] (adopted from [–48]).

Fig 3. Endo-/exocytosis pathway.

Pink: genes identified in this analysis, most of which have been implicated in breast cancer previously (Table A of S1 File), by stage of EEC: Formation of clathrin-coated vesicles, E3 ubiquitination, separation of inactive integrin (fast recycling) from active integrins (slow recycling), sorting between secretory, lysosomal, and (slow) recycling pathway, and lysosomal degradation. Red and green genes are known breast cancer promoters and suppressors, respectively (Table C of S1 File). Clathrin-mediated endocytosis (CME) begins with co-assembly of the heterotetrameric adaptor complex AP-2 with clathrin at PI(4,5)P₂-rich plasma membrane sites. AP-2 in its open conformation recruits clathrin and additional endocytic proteins, many of which also bind to PI(4,5)P₂. Maturation of the clathrin-coated pit (CCP) may be accompanied by SHIP-2-mediated dephosphorylation of PI(4,5)P₂ to PI(4)P. Synthesis of PI(3,4)P₂ is required for assembly of the PX-BAR domain protein SNX9 at constricting CCPs and may occur in parallel with PI(4,5)P₂ hydrolysis to PI(4)P via synaptojanin, thereby facilitating auxilin-dependent vesicle uncoating by the clathrin-dependent recruitment and activation of PI3KC2α, a class II PI3-kinase. PI(3,4)P₂ may finally be converted to PI(3)P en route to endosomes by the 4-phosphatases INPP4A/B, effectors of the endosomal GTPase Rab5. Adapted from [51]. In the early endosome, β1 integrins are sorted. Inactive integrins undergo fast “short loop” recycling; active integrins go to the late endosome / multivesicular body for slow “long group” recycling (RAB11), lysosomal degeneration (unless rescued by RAB25/CLIC3), or secretion via the trans-Golgi-network mediated by RAB9. Fast recycling of epidermal GF receptor drives proliferation,[52] so one would expect gain-of-function mutations in the upper part of the Figure. Lysosomal and synaptic vesicle exocytosis share many similarities. Endolysosome-localized PIs may regulate lysosomal trafficking in early onset lysosomal storage diseases.[53] and, particularly in ageing, insufficient lysosomal degradation contributes to Alzheimer’s disease (PSEN1, GLB1), Parkinson’s disease (ATP13A2),[54] atherosclerosis (vATPase),[55, 56] and type 2 diabetes (GLB1, HEXA).[57], (derived, in part, from KEGG pathways hsa04144, hsa04721, hsa00531, and hsa04142).

Fig 4. Functional relation of the PI/EC genes.

PI is synthesized from myo-inositol (imported by HMIT) and PA (via CDP-DAG) which can be synthesized from lysophosphatic acid (LPA), PC, or PS, or salvaged from IP₃ and DAG. It can also be synthesized from 1-acyl GPI. Arrows: PIs are phosphorylated at a 3-, 4-, or 5- position by PI-kinases (left to right) and hydrolyzed by phosphatases (right-to-left). Genes associated with breast cancer in this GWAS are highlighted in pink (bold: aGWS). See Table 1 for other box colors. Colored arrows in the center indicate the sequence of PIs involved in EEC (Fig 3). Percent values indicate approximate proportion of phospholipids.[58].

Fig 5. Structure of cyclodextrins.

Cyclodextrins are toroids formed of six (n = 4, αCD), seven (n = 5, βCD), or eight (n = 6, γCD) starch molecules. The cavity is lipophilic, while the surface is hydrophilic.

Fig 6. Wound healing by cyclodextrins in breast cancer cell lines.

Cells were grown in triplicates for 12h and incubated with either of the CDs for 2h at the concentration indicated (0–4mM), before a 0.9mm wide gap was opened and cells were allowed to migrate into the “wound” for 12h. HPβCD is more than 10× as toxic as HPαCD, which at <100mM does not affect epithelial cell viability.[75, 76] Dashed horizontal line indicates inhibition of wound healing in HPαCD at 1 and 4 mM respectively. ANOVA results: indep: HPαCD vs HPβCD (fixed) block: MCF-7/MDA-MB-231 (fixed) dep: %change in wound healing 1mM α vs 1mM β, p = .0001 ***1mM α vs 2mM β, p = .0252 *4mM α vs 4mM β, p = .0442 *.