Loss of the volume-regulated anion channel components LRRC8A and LRRC8D limits platinum drug efficacy.

In recent years platinum (Pt) drugs have been found to be especially efficient to treat patients with cancers that lack a proper DNA damage response, e.g. due to dysfunctional BRCA1. Despite this knowledge, we are still missing helpful markers to predict Pt response in the clinic. We have previously shown that volume-regulated anion channels, containing the subunits LRRC8A and LRRC8D, promote the uptake of cisplatin and carboplatin in BRCA1-proficient cell lines. Here, we show that the loss of LRRC8A or LRRC8D significantly reduces the uptake of cis- and carboplatin in BRCA1;p53-deficient mouse mammary tumor cells. This results in reduced DNA damage and in vivo drug resistance. In contrast to Lrrc8a, the deletion of the Lrrc8d gene does not affect the viability and fertility of mice. Interestingly, Lrrc8d-/- mice tolerate a two-fold cisplatin maximum-tolerable dose. This allowed us to establish a mouse model for intensified Pt-based chemotherapy, and we found that an increased cisplatin dose eradicates BRCA1;p53-deficient tumors, whereas eradication is not possible in WT mice. Moreover, we show that decreased expression of LRRC8A/D in head and neck squamous cell carcinoma patients, who are treated with a Pt-based chemoradiotherapy, leads to decreased overall survival of the patients. In particular, high cumulative cisplatin dose treatments lost their efficacy in patients with a low LRRC8A/D expression in their cancers. Our data therefore suggest that LRRC8A and LRRC8D should be included in a prospective trial to predict the success of intensified cis- or car-boplatin-based chemotherapy.

GLCE rs3865014 (Val597Ile) polymorphism is associated with breast cancer susceptibility and triple-negative breast cancer in Siberian population.

d-Glucuronyl C5-epimerase (GLCE) is one of key enzymes in heparan sulfate biosynthesis and possesses tumour-suppressor function in breast carcinogenesis. Here, we investigated a potential involvement of GLCE polymorphism(s) in breast cancer development in Siberian women population. Comprehensive analysis of SNP databases revealed GLCE rs3865014 (Val597Ile) missense polymorphism as the main significantly present in human populations. According the TaqMan-based SNP assay, allele distributions for the rs3865014 (A>G) were similar in healthy Siberian women (n=136) and cancer patients (n=129) (A0,73:G0,27) and intermediate between the European and Asian populations, while genotype distributions were different, with the increase of AG rate in breast cancer patients (OR=1.76; 95% CI=1.04-1.90; P(Y)=0.035 χ2=4.44). Heterozygous AG genotype was associated with tumour size (OR=3.67, P(Y)=0.004), ER-negative tumours (OR=3.25, P(Y)=0.0028), triple-negative tumours (OR=4.94, P(Y)=0.015) but not menopausal status, PR and HER-2 status, local or distant metastasis. Homozygous GLCE genotypes (AA/GG) were more common for ER+PR+ luminal A breast cancer (OR=0.25, P(Y)=0.031). Loss-of-heterozigosity was identified in 5 of 51 breast tumours and the loss of G allele was associated with the decreased GLCE expression. Epidemiologic data for the GLCE SNP in different racial/ethnic groups demonstrated high AG genotype rates as a risk factor not for breast cancer incidence but for poor prognosis of the disease. The obtained data suggest an involvement of GLCE rs3865014 in breast cancer development. Heterozygous AG genotype might be a risk factor for breast cancer susceptibility in Siberian women and is associated with aggressive ER-negative and triple-negative cancer subtypes.

Tissue-specificity of heparan sulfate biosynthetic machinery in cancer.

Heparan sulfate (HS) proteoglycans are key components of cell microenvironment and fine structure of their polysaccharide HS chains plays an important role in cell-cell interactions, adhesion, migration and signaling. It is formed on non-template basis, so, structure and functional activity of HS biosynthetic machinery is crucial for correct HS biosynthesis and post-synthetic modification. To reveal cancer-related changes in transcriptional pattern of HS biosynthetic system, the expression of HS metabolism-involved genes (EXT1/2, NDST1/2, GLCE, 3OST1/HS3ST1, SULF1/2, HPSE) in human normal (fibroblasts, PNT2) and cancer (MCF7, LNCaP, PC3, DU145, H157, H647, A549, U2020, U87, HT116, KRC/Y) cell lines and breast, prostate, colon tumors was studied. Real-time RT-PCR and Western-blot analyses revealed specific transcriptional patterns and expression levels of HS biosynthetic system both in different cell lines in vitro and cancers in vivo. Balance between transcriptional activities of elongation- and post-synthetic modification- involved genes was suggested as most informative parameter for HS biosynthetic machinery characterization. Normal human fibroblasts showed elongation-oriented HS biosynthesis, while PNT2 prostate epithelial cells had modification-oriented one. However, cancer epithelial cells demonstrated common tendency to acquire fibroblast-like elongation-oriented mode of HS biosynthetic system. Surprisingly, aggressive metastatic cancer cells (U2020, DU145, KRC/Y) retained modification-oriented HS biosynthesis similar to normal PNT2 cells, possibly enabling the cells to keep like-to-normal cell surface glycosylation pattern to escape antimetastatic control. The obtained results show the cell type-specific changes of HS-biosynthetic machinery in cancer cells in vitro and tissue-specific changes in different cancers in vivo, supporting a close involvement of HS biosynthetic system in carcinogenesis.

Modulation of the ATPase and transport activities of broad-acting multidrug resistance factor ABCC10 (MRP7).

The cell surface molecule ABCC10 is a broad-acting transporter of xenobiotics, including cancer drugs, such as taxanes, epothilone B, and modulators of the estrogen pathway. Abcc10(-/-) mice exhibit increased tissue sensitivity and lethality resulting from paclitaxel exposure compared with wild-type counterparts, arguing ABCC10 functions as a major determinant of taxane sensitivity in mice. To better understand the mechanistic basis of ABCC10 action, we characterized the biochemical and vectorial transport properties of this protein. Using crude membranes in an ABCC10 overexpression system, we found that the ABCC10 transport substrates estrogen estradiol-glucuronide (E(2)17ßG) and leukotriene C4 (LTC(4)) significantly stimulated ABCC10 beryllium fluoride (BeFx)-sensitive ATPase activity. We also defined the E(2)17ßG antagonist, tamoxifen, as a novel substrate and stimulator of ABCC10. In addition, a number of cytotoxic substrates, including docetaxel, paclitaxel, and Ara-C, increased the ABCC10 basal ATPase activity. We determined that ABCC10 localizes to the basolateral cell surface, using transepithelial well assays to establish that ABCC10-overexpressing LLC-PK1 cells exported [(3)H]-docetaxel from the apical to the basolateral side. Importantly, we found that the clinically valuable multikinase inhibitor sorafenib, and a natural alkaloid, cepharanthine, inhibited ABCC10 docetaxel transport activity. Thus, concomitant use of these agents might restore the intracellular accumulation and potency of ABCC10-exported cytotoxic drugs, such as paclitaxel. Overall, our work could seed future efforts to identify inhibitors and other physiologic substrates of ABCC10.