Clinical studies in cancer & Alzheimer's disease

Enhanced SphK1 and reduced S1P lyase expression and activity demonstrate dysregulated S1P metabolism in prostate cancer

In our initial study (Malavaud et al, Eur J Cancer, 2010), we reported the first-in-man study of quantification of SphK1 enzymatic activity in cancer. Using fresh tissues retrieved from 28 patients undergoing radical prostatectomy, we assessed SphK1 enzymatic activity in tumor and normal counterpart for each patient. Relationships with PSA, Gleason sum, pathological stage, resection margin status and treatment failure were researched. In addition, the SphK1 pattern of expression was then also on tissue microarray sections representative of prostate cancer Gleason scores (n=88, Gleason scores 6, 7 and 8).

A significant 2-fold increase in SphK1 enzymatic activity (P < 0.04) was observed in cancer when compared to the normal epithelium. Strong inter-individual variations were observed. SphK1 tumor activity, as expressed as a ratio of cancer/non-cancerous activities. The upper quartile of was associated with higher PSA (P = 0.04), higher tumor volumes (20.7% versus 9.8%, P = 0.002), higher rates of positive margins (85.7% versus 28.6%, P = 0.01) and surgical failure (71.4% versus 9.5%, P = 0.003) than the lower three quartiles. Odds ratios (OR) for treatment failure showed a strong relationship with SphK1 activity (OR: 23.7, P = 0.001), positive resection margins (OR: 15.0, P = 0.007) and Gleason sum (>4 + 3, OR: 8.0, P = 0.003). Tissue microarrays showed discrete epithelial expression that varied with Gleason sum with significant relationship between SphK1 expression and higher Gleason sum. In conclusion, SphK1 tumor activity was related to adverse clinical features (PSA, cancer volume) and predictive of surgical failure, suggesting a physiological function for SphK1 in the normal gland and supporting a role for SphK1 in prostate cancer progression.

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Stage 2, Gleason 4
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Stage 3, Gleason 8

Paraffin-embedded sections representative of prostate cancer natural history showed that the pattern of SphK1 expression was often heterogeneous with areas of higher aggressiveness stained more intensely.

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High power magnification of benign glands showing faint reactivity in the cytoplasm of basal and differentiated luminal cells.

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Poorly-differentiated Gleason score 8 cancer showing intense and diffuse staining in the cancer cells cytoplasm.

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Gleason score 6 cancer. Note the lack of staining in atrophic glands (*) and the homogeneous and intense cytoplasmic reactivity of cancer cells.

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Malavaud et al, Eur J Cancer, 2010

In a follow-up study, we analyzed the expression and enzymatic activity of sphingosine 1-phosphate lyase (SPL) in tissue microarray sections and fresh human prostate cancer specimens.

A remarkable decrease in SPL enzymatic activity was found in tumor samples, as compared to normal adjacent tissues. A significant relationship between loss of SPL expression and higher Gleason score was confirmed in tissue microarray analysis. Moreover, SPL protein expression and activity were inversely correlated with those of sphingosine kinase-1 (SphK1), the enzyme producing S1P, suggesting a deregulated S1P metabolism in prostate cancer.

SPL and SphK1 expressions were independently predictive of aggressive cancer on tissue microarray, supporting the relevance of S1P in prostate cancer.

SphK1 (left) and SPL (right) activities were quantified in 13 individual pairs of cancer (red) and noncancerous (blue) lesions. Median SphK1 activities are 88 (95% CI, 71-129) and 259 (95% CI, 223-289) pmol/min/mg of protein in noncancerous and tumor samples, respectively. Median SPL activities are 6 (95% CI, 5.2-6.2) and 4 (95% CI, 3.1-4.2) pmol/min/mg of protein in noncancerous and tumor samples, respectively.

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SPL expression is restricted to the epithelial layer in
non-cancer glands.
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Well-differentiated cancers showed conserved pattern of expression
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or abrogated cytoplasmic expression
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Poorly-differentiated cancers were characterized by equivocal

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Brizuela et al, Mol Cancer Ther, 2012

SphK1 is particularly overexpressed in neuroendocrine cells

Prostate cancer cell proliferation is regulated by androgens and androgen deprivation therapy (ADT) is the standard of care in the management of patients with advanced disease. ADT is initially effective but ultimately all patients become resistant to hormonal manipulation. ADT induces changes in prostate cancer biology promoting its progression to the androgen- refractory state or hormone-refractory prostate cancer (HRPC) phenotype, with an associated life expectancy of only 15 to 20 months. It is not clear how prostate cancer cells make the transition from androgen-dependent to androgen-independent status after ADT. Numerous studies have shown that, after long-term ADT, prostate cancer cells acquire a neuroendocrine (NE)-like phenotype leading to tumor populations enriched in NE cells. NE cells constitute a minor component of the normal prostate gland and secrete several neuropeptides that can induce mitogenic effects on adjacent cancer cells in androgen-depleted conditions

In vitro studies (
Dayon et al., Plos One, 2009) have uncovered a role for SphK1 during the transition to the HRPC state.

Immunohistochemical studies of prostate cancer human samples showed for the first time that NE cells strongly expressed SphK1.

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Sections from a representative patient who underwent palliative transurethral resection for local recurrence under complete androgen blockade were immunostained for Chromogranin A (CgA), a marker of NE cells, and SphK1 expression. CgA staining was observed in a minority of cancer cells, approximately 10%, in the form of secretory granuli. Co-expression of CgA in blue with brown SphK1 resulted in intense dark brown signal (see arrows)

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Dayon et al, Plos One, 2009

Reduced SphK1 and enhanced S1P lyase expression and activity demonstrate deregulated S1P metabolism in Alzheimer’s Disease

The accumulation of beta amyloid (Aβ) peptides, a hallmark of Alzheimer's disease (AD) is related to mechanisms leading to neurodegeneration. We have previously reported that Aβ peptides were able to decrease the activity of SphK1 in cell culture models, an effect that could be blocked by the prosurvival IGF-1/IGF-1R signaling (Gomez-Bouchet et al., Mol Pharmacol, 2007).

We showed for the first time the expression of both SphK1 and SPL by immunohistochemistry in frontal and entorhinal cortices from 56 human AD brains. Immunohistochemical analysis revealed a decreased expression of SphK1 and an increased expression of SPL both correlated to amyloid deposits in the entorhinal cortex. Analysis of brain tissue extracts showed a decrease of protein SphK1 expression in AD brains whereas SPL expression was increased. The content of IGF-1R, an activator of SphK1, was found decreased in AD brains as well as S1PR1, the major receptor for S1P.

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Immunohistochemical study of SphK1/SPL expression and Aβ deposits. (a) Double labeling of SphK1 and Aβ in AD brain section from entorhinal cortex. Diffuse and focal deposits are seen in different areas. X200. Inset: Strongly stained neurons (SphK1++, black arrow) and mild stained neurons (SphK1+, white arrow) are clearly distinguishable. X400 (b) Double labeling of SPL and Aβ in AD brain section from entorhinal cortex. A senile plaque is mainly surrounded by strong labeled neurons (SLP++). X400.

Collectively, these results highlight the importance of S1P in AD suggesting the existence of a global deregulation of S1P signaling in this disease from its synthesis by SphK1 and degradation by SPL to its signaling by the S1PR1 receptor.

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Ceccom et al, Acta Neuropath Comm, 2014

SphK2 expression and subcellular location are altered in Alzheimer’s Disease

The subcellular localization of SphK2, hence the compartmentalization of generated S1P, is recognized to play a crucial role in dictating either its pro-survival or pro-apoptotic function. We therefore aimed at studying the expression of SphK2 and notably its subcellular localization in brain tissues from patients with AD. Here we showed that a decrease in SphK2 protein cytosolic expression correlated with the density of amyloid deposits in a cohort of 25 post-mortem brains. In addition, we observed that the equilibrium between cytoplasmic and nuclear SphK2 is disrupted and showed that SphK2 is preferentially localized in the nucleus in AD brain extracts as compared to control extracts, with a marked increase of cleaved SphK2.

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Negative correlation between amyloid deposits and the percentage of SphK2 positive neurons in AD brain The percentage of SphK2 positive neurons (cytoplasm) was inversely correlated with the amount of amyloid deposits (Aβ surface percentage) in the frontal cortex (r=0.037, p=0.007; left), in the entorhinal cortex (r=0.062, p<0.001; middle) and in CA1 (r=0.29, p<0.001; right). Black arrows show SphK2 positive neurons, green arrows show SphK2 negative neurons and asterisks show SphK2 positive oligodendrocytes.

Our results suggest that a shift in the subcellular localization of the S1P generating SphK2 may compromise the well established pro-survival cytosolic S1P by favoring the production of nuclear S1P associated with adverse effects in AD pathogenesis.

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Dominguez et al, Acta Neuropath Comm, 2018