Alternative splice variants of DCLK1 mark cancer stem cells, promote self‐renewal and drug‐resistance, and can be targeted to inhibit tumorigenesis in kidney cancer

Abstract

Renal cell carcinoma (RCC) is a common and devastating disease characterized by a hypoxic microenvironment, epithelial-mesenchymal transition and potent resistance to therapy evidencing the presence of cancer stem cells (CSCs). Various CSC markers have been studied in RCC, but overall there is limited data on their role and most markers studied have been relatively nonspecific. Doublecortin-like kinase 1 (DCLK1) is a validated CSC marker in the gastrointestinal tract and evidence for an equivalent role in other cancers is accumulating. We used bioinformatics, immunohistochemistry, flow cytometry, spheroid self-renewal and chemoresistance assays in combination with overexpression and siRNA-knockdown to study the stem cell-supportive role of DCLK1 alternative splice variants (DCLK1 ASVs) in RCC. To target tumor cells expressing DCLK1 ASVs directly, we developed a novel monoclonal antibody (CBT-15) and delivered it systemically to RCC tumor xenografts. DCLK1 ASVs were overexpressed, enriched together with CSC markers and predictive of overall and recurrence-free survival in RCC patients. In vitro, DCLK1 ASVs were able to directly stimulate essential molecular and functional characteristics of renal CSCs including expression of aldehyde dehydrogenase, self-renewal and resistance to FDA-approved receptor tyrosine kinase and mTOR inhibitors, while targeted downregulation of DCLK1 reversed these characteristics. Finally, targeting DCLK1 ASV-positive cells with the novel CBT-15 monoclonal antibody blocked RCC tumorigenesis in vivo. These findings establish DCLK1 as a CSC marker with implications for therapy, disease progression and survival in RCC and demonstrate the therapeutic value of DCLK1-targeted monoclonal antibodies against renal CSCs.

Highly‐expressed P2X7 receptor promotes growth and metastasis of human HOS/MNNG osteosarcoma cells via PI3K/Akt/GSK3β/β‐catenin and mTOR/HIF1α/VEGF signaling

“…EMT is associated with malignant stem cell function, and previous studies indicate that cancer stem cells (CSCs) are involved in tumor metastasis and relapse . To determine whether P2X7 affected stemness of osteosarcoma cells, we sorted CSCs from osteosarcoma cell lines using flow cytometry and treated them with BzATP or A740003.…”

“…EMT is associated with malignant stem cell function, 30 and previous studies indicate that cancer stem cells (CSCs) are involved in tumor metastasis and relapse. [31][32][33][34] To determine whether P2X7 affected stemness of osteosarcoma cells, we sorted CSCs from osteosarcoma cell lines using flow cytometry and treated them with BzATP or A740003. 15% to 50% more CD133 high CSCs were detected in 5 to 125 μM BzATP treated HOS/MNNG cells, but inhibition of P2X7 using an antagonist or shRNA decreased CD133 high CSCs by 50%.…”

Doublecotin-like kinase 1 increases chemoresistance of colorectal cancer cells through the anti-apoptosis pathway

“…miR-539, a tumor suppressor microRNA, directly inhibits DCLK1 expression to enhance chemosensitivity of non-small cell lung cancer to cisplatin [49]. Over-expression of DCLK1 significantly increased chemoresistance of kidney cancer cells to the receptor tyrosine kinase inhibitors and mTOR inhibitors [50]. miR-15a might overcome chemoresistance of CRC stem cells to 5-FU through suppression of DCLK1 [51].…”

Identification of prognosis‐related alternative splicing events in kidney renal clear cell carcinoma

“…The importance of the AS of enhancer of zeste 2 polycomb pre‐mRNA in the tumorigenic potential of kidney cancer has been reported . The AS variants of doublecortin‐like kinase 1 are overexpressed in kidney renal clear cell carcinoma (KIRC) and may be implicated in immune response . In addition, differentially splicing isoforms between KIRC and non‐tumour tissues have been studied, and a cassette exon differentially skipped in DAB adaptor protein 2 in KIRC has been identified .…”

“…16 The AS variants of doublecortin-like kinase 1 are overexpressed in kidney renal clear cell carcinoma (KIRC) and may be implicated in immune response. 17 In addition, differentially splicing isoforms between KIRC and nontumour tissues have been studied, and a cassette exon differentially skipped in DAB adaptor protein 2 in KIRC has been identified. 18 The importance of the systemic profiling of survival-associated AS has been emphasized in lung, ovarian and prostate cancers.…”