Studying Chemotactic Migration in Dunn Chamber: An Example Applied to Adherent Cancer Cells.

Cell migration is a vital process in the development of multicellular organisms. When deregulated, it is involved in many diseases such as inflammation and cancer metastisation. Some cancer cells could be stimulated using chemoattractant molecules, such as growth factor Heregulin ß1. They respond to the attractant or repellent gradients through a process known as chemotaxis. Indeed, chemotactic cell motility is crucial in tumour cell dissemination and invasion of distant organs. Due to the complexity of this phenomenon, the majority of available in vitro methods to study the chemotactic motility process have limitations and are mainly based on endpoint assays, such as the Boyden chamber assay. Nevertheless, in vitro time-lapse microscopy represents an interesting opportunity to study cell motility in a chemoattracting gradient, since it generates large volume image-based information, allowing the analysis of cancer cell behaviours. Here, we describe a detailed time-lapse imaging protocol, designed for tracking T47D human breast cancer cell line motility, toward a gradient of Heregulin ß1 in a Dunn chemotaxis chamber assay. The protocol described here is readily adapted to study the motility of any adherent cell line, under various conditions of chemoattractant gradients and of pharmacological drug treatments. Moreover, this protocol could be suitable to study changes in cell morphology, and in cell polarity.

TP53 Arg 72Pro and MDM2 SNP309 polymorphisms and colorectal cancer risk: a west Algerian population study.

The tumor suppressor gene TP53 and its regulator MDM2 are both key players involved in multiple pathways including apoptosis, cellular transcriptional control and cell cycle regulation. Common germline polymorphisms in these genes may affect colorectal cancer (CRC) susceptibility. An arginine-to-proline substitution at codon 72 in the TP53 gene is reported to decrease apoptotic potential, while a thymine-to-guanine polymorphism at nucleotide 309 (named SNP309) of murine double minute 2 MDM2 gene increases its transcription. These two polymorphisms therefore may be of importance in colorectal carcinogenesis. The relation of these polymorphisms to colorectal cancer in the Algerian population was addressed in this study. DNA samples from 121 controls and 116 cases were genotyped for these two polymorphisms by PCR/RFLP then confirmed by sequencing. Unexpectedly no significant association was found between this potential marker TP53 Arg72Pro and CRC (p > 0.05). However, our findings reveal that individuals with the MDM2 SNP309 GG genotype have a low risk of CRC as compared to the TT genotype (OR = 0.49; 95 % CI: 0.24-0.98, p = 0.04), with more significance for females (OR = 0.16; 95 % CI: 0.06-0.41, p < 0.05). Moreover, no significant association was observed between the combined TP53 and MDM2 genotypes and CRC. Contrary to initial expectations that the GG genotype with high MDM2 levels would increase cancer risk, our results demonstrate that the MDM2 SNP309 GG genotype is associated with decreased risk of colorectal cancer. This is suggesting that other mechanisms independent of increased MDM2 levels can influence cancer susceptibility.

ErbB2-dependent chemotaxis requires microtubule capture and stabilization coordinated by distinct signaling pathways.

Activation of the ErbB2 receptor tyrosine kinase stimulates breast cancer cell migration. Cell migration is a complex process that requires the synchronized reorganization of numerous subcellular structures including cell-to-matrix adhesions, the actin cytoskeleton and microtubules. How the multiple signaling pathways triggered by ErbB2 coordinate, in time and space, the various processes involved in cell motility, is poorly defined. We investigated the mechanism whereby ErbB2 controls microtubules and chemotaxis. We report that activation of ErbB2 increased both cell velocity and directed migration. Impairment of the Cdc42 and RhoA GTPases, but not of Rac1, prevented the chemotactic response. RhoA is a key component of the Memo/ACF7 pathway whereby ErbB2 controls microtubule capture at the leading edge. Upon Memo or ACF7 depletion, microtubules failed to reach the leading edge and cells lost their ability to follow the chemotactic gradient. Constitutive ACF7 targeting to the membrane in Memo-depleted cells reestablished directed migration. ErbB2-mediated activation of phospholipase C gamma (PLCγ) also contributed to cell guidance. We further showed that PLCγ signaling, via classical protein kinases C, and Memo signaling converged towards a single pathway controlling the microtubule capture complex. Finally, inhibiting the PI3K/Akt pathway did not affect microtubule capture, but disturbed microtubule stability, which also resulted in defective chemotaxis. PI3K/Akt-dependent stabilization of microtubules involved repression of GSK3 activity on the one hand and inhibition of the microtubule destabilizing protein, Stathmin, on the other hand. Thus, ErbB2 triggers distinct and complementary pathways that tightly coordinate microtubule capture and microtubule stability to control chemotaxis.

ErbB2 receptor controls microtubule capture by recruiting ACF7 to the plasma membrane of migrating cells.

Microtubules (MTs) contribute to key processes during cell motility, including the regulation of focal adhesion turnover and the establishment and maintenance of cell orientation. It was previously demonstrated that the ErbB2 receptor tyrosine kinase regulated MT outgrowth to the cell cortex via a complex including Memo, the GTPase RhoA, and the formin mDia1. But the mechanism that linked this signaling module to MTs remained undefined. We report that ErbB2-induced repression of glycogen synthase kinase-3 (GSK3) activity, mediated by Memo and mDia1, is required for MT capture and stabilization. Memo-dependent inhibition of GSK3 allows the relocalization of APC (adenomatous polyposis coli) and cytoplasmic linker-associated protein 2 (CLASP2), known MT-associated proteins, to the plasma membrane and ruffles. Peripheral microtubule extension also requires expression of the plus-end binding protein EB1 and its recently described interactor, the spectraplakin ACF7. In fact, in migrating cells, ACF7 localizes to the plasma membrane and ruffles, in a Memo-, GSK3-, and APC-dependent manner. Finally, we demonstrate that ACF7 targeting to the plasma membrane is both required and sufficient for MT capture downstream of ErbB2. This function of ACF7 does not require its recently described ATPase activity. By defining the signaling pathway by which ErbB2 allows MT capture and stabilization at the cell leading edge, we provide insights into the mechanism underlying cell motility and steering.