Doublecortin-Like Kinase Protein 1 in Cholangiocarcinoma: Is This the Biomarker and Target We Have Been Looking For?

Cholangiocarcinoma (CCA) is a rare but aggressive cancer of the biliary tree with high mortality. The asymptomatic nature of CCA development makes diagnosis difficult, causing a great need for diagnostic biomarkers. Surgical resection is the only curative “treatment”; however, this is only possible in a few patients. Late-stage CCA diagnosis has a high incidence of chemo- and radioresistance, with palliative care as the only therapeutic option.⁽¹⁾ CCA is classified as intrahepatic (iCCA), perihilar (pCCA), and distal (dCCA), with each CCA subtype displaying distinct tumor phenotype and tumor microenvironments (TMEs).⁽¹⁾ iCCA develops around the second-order bile ducts generally from cuboidal cholangiocytes, whereas pCCA and dCCA develop from the highly secretory columnar cholangiocytes along large bile ducts.⁽¹⁾ In lieu of these commonalities, all CCAs are heterogenous within each subtype; histological and molecular diagnosis has identified various cells of origin and gene mutations as probable cause of disease.⁽¹⁾

CCA tumors secrete inflammatory mediators to promote immune cell and tumor-associated macrophages (TAMs) infiltration, creating an ideal environment to sustain tumor growth.⁽²⁾ Cancer stem cells (CSCs) contribute to CCA aggressiveness and metastasis, making them prime targets for treatment strategies.⁽²⁾ Markers of CCA CSCs include: epithelial cell adhesion molecule, CD133, Laminin-332, CD274, aldehyde dehydrogenase, and CD117, among others.^(1,2) Despite many studies performed on CCA CSCs, and their downstream signaling cascades, little has been learned of CCA CSCs and their pathogenicity.

Doublecortin-like kinase protein 1 (DCLK1) is a microtubule-associated protein kinase that is a marker of tuft cells and gastrointestinal (GI) chemosensory cells and functions in the growth and plasticity of brain development.⁽³⁾ DCLK1 has been implicated as an important CSC marker in pancreatic cancer, colorectal cancers, and HCC.⁽³⁾ Discovery of DCLK1 as a CSC has increased investigation of its function in unrestrained growth, epithelial-mesenchymal transition (EMT), immunoregulation of TAMs, and inhibition of apoptosis.^(3,4) Because of its role in GI CSCs, Nevi et al. investigated the role of DCLK1 in CCA CSCs.

New Findings

Using extensive evaluation, the investigators found DCLK1 expression in various CCA CSCs, including iCCA and pCCA, but not in healthy interlobular bile ducts, hepatic ducts at the hilum, or tissues of origin of the small bile duct type found in iCCA and pCCA, implicating DCLK1 as a novel diagnostic marker for CCA.⁽⁵⁾ CCA samples from both iCCA and pCCA with accompanying noncancerous tissue were enzymatically digested, labeled, and separated through immune-magnetic sorting.⁽⁵⁾ Using multiple CS markers from two anatomically and clinically distinct CCA subtypes increases this impact of the study on personalized medicine to treat defined CCA subtypes with specific CSC marker expression. Notably, the investigators have previously characterized the CSC populations in iCCA subtypes, demonstrating the emerging importance in identification of CCA CSC markers.⁽⁶⁾ The iCCA case analyzed in this study recapitulates the previously published CSC profile of the small bile duct iCCA (LGR5^low), whereas the pCCA CSC profile matches the large bile duct type iCCA (LGR5^high).^(1,6) This investigation addresses the heterogeneity of CCA and its related tissues/cells of origin, increasing its impact on CCA progression.⁽¹⁾

Notably, the investigators demonstrate that inhibition of DCLK1 with LRRK2-IN-1 results in reduced cell viability, proliferation/population doubling, and colony formation, prominently in CSC populations with high DCLK1 expression, namely the CD133⁺ iCCA CSCs, LGR5⁺ pCCA CSCs, and LGR5⁺ HT29 cells, a colon adenocarcinoma cell line.⁽⁵⁾ Coexpression of DCLK1 and LGR5 is of interest, given that LGR5 is a stem/progenitor cell marker in normal liver, primary liver cancers, and iCCA,^(3,6,7) and, in large bile duct iCCA, LGR5 marks cell populations with the highest tumorigenic potential.⁽⁶⁾ In this study, following DCLK1 inhibition, all CCA CSCs had increased cell apoptosis, whereas EMT markers, like SNAIL1/-2 and TWIST1/-2, were transcriptionally reduced.⁽⁵⁾ To this regard, Nakanishi et al. previously showed that elimination of DCLK1 in intestinal CSCs led to tumor collapse.⁽⁸⁾ It is profound that cholangiocytes lining the hepatic ducts and interlobular bile ducts do not express DCLK1 and do not have a reduction in cell viability following DCLK1 inhibition, adding credibility to DCLK1 as a specific marker of CCA CSCs.⁽⁵⁾

CLINICAL IMPACT

To add to the clinical impact of their study, Nevi et al. demonstrated that serum DCLK1 levels were elevated in patients with liver diseases, including primary sclerosing cholangitis and cirrhosis, relevant risk factors for CCA and HCC, respectively.⁽⁵⁾ Remarkably, DCLK1 serum levels were highest in patients with diagnosed iCCA and pCCA. A similar trend was found with iCCA and pCCA having the highest serum levels of IL-6, TNFα, and carbohydrate antigen 19-9, which were undetectable or lowly expressed in healthy human controls.⁽⁵⁾ These findings provide a rationale for a multifactor approach in the early diagnosis of CCA, whereas the investigators demonstrate that DCLK1 serum levels could represent an effective diagnostic biomarker for CCA, perhaps helpful for early diagnosis; further experimentation needs to be performed.⁽⁵⁾

The role of CSCs in CCA is a developing field, attributable to CCA’s aggressiveness and high patient mortality. The investigators thoroughly investigated DCLK1 as a CSC marker in iCCA and pCCA, adding to the growing body of evidence of DCLK1 as an important CSC marker.^(3,4) This study confirmed that DCLK1 (1) is up-regulated in CCA and CSCs, but not healthy cholangiocytes; (2) is coexpressed with other CSC markers; (3) promotes proliferation, inflammation, and EMT; and (4) inhibition of DCLK1 reverses these phenotypes (Fig. 1). The minimal expression in DCLK1 healthy intrahepatic bile ducts and liver parenchyma, accompanied with no expression in healthy isolated cholangiocytes, further demonstrate the potential for specific targeting of cancer populations. These findings also indicate how peribiliary glands could be the source of CCA CSCs.⁽⁹⁾ In support of the current findings, DCLK1 has been found to contribute to EMT in various GI cancers, and reduction of its expression in tumor cells reduced tumor growth and increased apoptosis and miRNA let-7a, a negative regulator of cell-cycle progression.^(3,8)

FIG. 1.

DCLK1 (1) is up-regulated in CCA and CSCs, but not healthy cholangiocytes; (2) is coexpressed with CSC markers; (3) promotes proliferation, inflammation, and EMT; and (4) may interact with T cells and induce a macrophage phenotype switch. Inhibition of DCLK1 blocks these phenotypes.

The investigators did not find any significant correlation between CD68⁺ TAMs and DCLK1 expression, likely attributable to single staining for CD68⁺ macrophages instead of costaining with M2 phenotype markers like CD-206, YM-1, and TGM-2, which may allow the determination of M1 and M2 macrophages near tumor CSCs.⁽¹⁰⁾ Further investigation is required to fully assess the role of DCLK1 in CCA immunoregulation of the TME, given that increased DCLK1 expression positively correlates with regulatory T-cell infiltration and T-cell exhaustion in colon and gastric adenocarcinoma TMEs.⁽⁴⁾ Nevi et al. have made great strides in characterizing DCLK1 in CCA CSCs and create a rationale for further investigation into the role of DCLK1 on CCA initiation, development, and progression. Expansion of DCLK1 studies with a larger number of CCA patients is crucial to confirm DCLK1 as a biomarker/possible target of treatment for CCA-CSCs; however, this study is a giant step in the right direction.

Abbreviations:

CCA

cholangiocarcinoma

CSC

cancer stem cell

dCCA

distal CCA

DCLK1

doublecortin-like kinase protein 1

EMT

epithelial-mesenchymal transition

GI

gastrointestinal

iCCA

intrahepatic CCA

pCCA

perhilar CCA

TAMs

tumor-associated macrophages

TME

tumor microenvironment