Association of the Combination of Stemness Gene Amplifications and Copy Number Aberrations of WNT-Signaling Genes in Breast Tumors With Metastasis

Abstract

<jats:p>We studied the association between the presence of 2 or more stemness gene amplifications as well as copy number aberrations (CNAs) of WNT signaling genes in residual breast tumor and metastasis. WNT pathway genes associated with metastasis were identified.</jats:p><jats:p><jats:bold>Material and Methods.</jats:bold> The study included 30 patients with breast cancer, who had 2 or more stemness gene amplifications in the residual tumor after neoadjuvant chemotherapy. Fifteen of the thirty patients developed hematogenous metastases; they constituted a group with metastases, the remaining 15 patients entered the second group without metastases. The tumor DNA was examined using a CytoScanHD Array microarray (Affymetrix, USA).</jats:p><jats:p><jats:bold>Results</jats:bold>. By subtracting amplification and deletion frequencies in 852 cytobands between groups with metastases and without metastases, 21 cytobands were identified with the largest difference in deletion and amplification frequencies. They contain 19/150 of WNT genes (12 activators: SKP1, WNT8A, MAPK9, CCND3, FZD9, WNT8B, CCND1, PLCB2, PRKCB, FZD2, WNT3, WNT9B and 7 negative regulators: GSK3B, APC, CSNK2B, SFRP5, BTRC, TCF7L2, CSNK2A2). A point system was developed: when amplifying WNT-signaling activators or deletion of negative regulators, one point was added to the total score, and vice versa when deleting WNT-signaling activators or amplification of negative regulators, one point was taken from the total amount. It was shown that 93% (14/15) of patients with metastases had a total score higher than 0, while 93% (14/15) of patients without metastases had a total score of zero or less than zero. The differences between the groups were statistically significant according to the two-sided Fisher test with a high level of confidence probability (p=0.000003) and the log-rank test (p=0.00004) when assessing non-metastatic survival by the Kaplan-Mayer method.</jats:p><jats:p><jats:bold>Conclusion.</jats:bold> Nineteen WNT signaling genes were identified. Copy number aberrations of these genes in combination with stemness gene amplifications in residual tumors were associated with metastasis. A new highly effective prognostic factor for breast cancer was identified. </jats:p>

WNT-Conditioned Mechanism of Exit from Postchemotherapy Shock of Differentiated Tumour Cells

“…The most important WNT signalling pathway genes responsible for its activation have previously been identified: 15 WNT signalling activator genes (WNT2B, SKP1, TCF7, PPP2CA, WNT8A, MAPK9, CCND3, PPP2R5D, WNT8B, CCND1, FZD2, WNT3, FZD9, WNT3, WNT9B) and 7 negative regulator genes (GSK3B, APC, CSNK2B, SFRP5, BTRC, TCF7L2, CSNK2A2), whose amplifications and deletions, respectively, should stimulate the WNT signalling pathway [32]. CNA-genetic landscape analysis of breast cancer tumour lines (BT-474, BT-549, MDA-MB-231, MDA-MD-468, MCF7, SK-BR-3 and T47D) revealed cultures with the ability/inability to dedifferentiate (due to amplifications of stemness genes [24]) and to the exit from postchemotherapy shock after chemotherapy exposure (due to CNA of WNT signalling pathway genes presented above).…”

“…Cell lines with a presumed ability to dedifferentiate and to emerge from postchemotherapy shock after exposure to chemotherapy drugs were established for mechanistic culture studies. The ability to emerge from postchemotherapy shock was established using a scoring system that we developed earlier: one point was added to the total sum of points if WNT signalling activators were amplified or negative regulators were deleted, and vice versa; one point was subtracted from the total sum if WNT signalling activators were deleted or negative regulators were amplified [32].…”

“…We analysed WNT signalling status in these patients and found that only those patients display metastases whose WNT signalling is in a potentially activated state due to activator gene amplifications and/or deletions of negative regulator genes of WNT signalling pathway. If, on the contrary, activator deletions and negative regulator amplifications are observed in tumour patients, these patients do not metastasise despite the presence of stemness gene amplifications [32]. Stem genes are the key transcription factors that support self-renewal and phenotype of stem cells.…”

“…In total, we identified 48 such genes, more details about them are written in our article [24]. In the absence of stemness gene amplifications, no metastasis is observed regardless of WNT signalling pathway genes status [32]. Based on our findings and those of other researchers, we hypothesized that the spontaneous emergence of tumour cells from postchemotherapy shock (replication arrest, senescence) is associated with ectopic expression of WNT signalling pathway genes, which condition cell activation through amplification of activator genes and deletion of negative regulator genes.…”

“…Based on our findings and those of other researchers, we hypothesized that the spontaneous emergence of tumour cells from postchemotherapy shock (replication arrest, senescence) is associated with ectopic expression of WNT signalling pathway genes, which condition cell activation through amplification of activator genes and deletion of negative regulator genes. In total, we have identified 15 genes of WNT signalling activators (WNT2B, SKP1, TCF7, PPP2CA, WNT8A, MAPK9, CCND3, PPP2R5D, WNT8B, CCND1, FZD2, WNT3, FZD9, WNT3, WNT9B) and 7 negative regulator genes (GSK3B, APC, CSNK2B, SFRP5, BTRC, TCF7L2, CSNK2A2) whose amplifications and deletions (respectively) should stimulate the WNT signalling pathway [32]. Thus, the present study aims to prove or disprove the hypothesis that the state of CNA activation of WNT signalling pathway genes (activator amplification and deletion of the negative regulators we have identified previously) accounts for the ability of differentiated tumour cells to emerge from postchemotherapy shock after chemotherapy exposure.…”