Frequent PTEN loss and differential HER2/PI3K signaling pathway alterations in salivary duct carcinoma: Implications for targeted therapy

Abstract

Background: Patients with advanced primary and recurrent salivary duct carcinoma (SDC), a rare and lethal malignancy, have limited therapeutic options. Novel small-molecule agents aimed at targeting critical signaling associated with SDC tumorigenesis may lead to new therapeutic options for patients with these tumors. The human epidermal growth factor receptor 2 (HER2)/phosphoinositide 3-kinase (PI3K) axis, an important oncogenic pathway, has been targeted for therapy in several solid tumors. Currently, little is known about the role and clinical implications of alterations of the HER2/PI3K pathway in patients with SDC.

Methods: The authors investigated the clinicopathologic features, genetic alterations, and expression of key members of the HER2/PI3K pathway in 43 primary tumors and conducted in vitro functional and targeted drug-response analyses on cell lines derived from salivary epithelial carcinomas.

Results: In primary tumors, loss of phosphatase and tensin homolog (PTEN) expression was identified in 22 of 43 tumors (51%), overexpression of HER2 was observed in 12 of 43 tumors (28%), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutations were identified in 12 of 43 tumors (28%). Phosphorylated protein kinase B (p-AKT) was highly expressed in most tumors. Most tumors (70%) displayed mutually exclusive alterations of PI3K members, whereas 8 tumors (19%) had 2 or more concurrent abnormalities. In vitro studies demonstrated a direct association between PTEN loss and PI3K pathway activation and evidence of response to combined PI3Kα and PI3Kβ and/or pan-PI3K inhibitors.

Conclusions: The current analyses reveal frequent PTEN loss and mutually exclusive alterations of key PI3K pathway members in SDC and demonstrate in vitro evidence of a response to pan-PI3K inhibitors. These results provide a framework for a biomarker-based substratification of patients with SDC in future targeted therapy. Cancer 2018;124:3523-32. © 2018 American Cancer Society.

Keywords: phosphatase and tensin homolog (PTEN) loss; phosphoinositide 3-kinase (PI3K) pathway; salivary duct carcinoma; salivary gland carcinoma; targeted therapy.

Figure 1. HER2/PI3K/pathway alterations in salivary duct carcinomas

(A) Selected examples of IHC results for PTEN, HER2, and p-AKT in SDC cases. SDC-02 showed PTEN negative (positive in stroma) and high HER2 (+3) with high p-AKT. SDC-06 showed PTEN positive and negative HER2 expression without p-AKT. SDC-24 showed PTEN loss and negative of HER2 with high p-AKT. (B) PIK3CA mutation (p.S130_R131del) in SDC-10. A distribution of PIK3CA mutation in our study showed that p.E542K was most frequent mutation type. (C) A grid plot of alterations profile in PTEN, HER3, and p-AKT expressions and PIK3CA mutation. The numbers above a grit plot indicate the classification of SDC group and applied to our salivary tumor cell lines; Group I (A253): PTEN loss (or PIK3CA mutation)/HER2 negative, Group II (RET981 and MAC): intact PTEN/HER2 positive, Group III (RET981/MAC-PTEN-kd): concomitant alterations with PTEN loss/HER2 positive and/or PIK3CA mutation, and Group IV: no alteration.

Figure 2. PTEN status in A253 and RET981 salivary gland tumor cell lines

(A) PTEN is expressed in RET981 salivary gland tumor cell line. PTEN loss and its mutation were found in A253 cell line. (B) Left panel, PTEN knockdown (kd) led to increases of p-AKT expression in RET981 cell lines. Right panel, PTEN knockdown (kd) increases cell proliferation as measured by MTT assay and value was normalized to the point of day 0. Error bards represent SD. Asterisks (*) represents significant difference of proliferation activities between PTEN-kd and control lines (p <0.01).

Figure 3. Effect of PI3K inhibitors on the proliferation in PTEN deficient (Group I) A253 salivary gland cells

(A) Treatment of LNCaP cells with AZD (0.25μM) showed significant decrease of cell growth. P-AKT was inhibited after 3 hours but rebounded after 12 hours treatment. (B) and (C) High dose (5μM) of PI3Kβ inhibitors (AZD8186 and GSK2636771) had no impact on cell growth and p-AKT expression level of A253 salivary tumor cell. (D) Effects of BYL719 (BYL, 5μM), AZD8186+BYL719 (A+B, 5μM), and GDC941 (941, 5μM) on A253 cell. Left upper panel, A253 cell growth inhibition was repressed by combination treatment of PI3Kα and PI3Kβ inhibitors (A+B, 5μM each) and pan-PI3K inhibitor (941, 5 μM). Left lower panel, comparison of dosage differences (2.5 μM and 5 μM at 4 days treatment) against AZD, BYL, A+B, and 941 on A253 cells. Asterisk (*) indicates that A253 was impacted on cell proliferation by 5 μM of A+B and 941 treatment. (E) P-AKT level after 3h and 24h of treatments. Both combination and pan-PI3K inhibitors maintained inhibition of p-AKT level after 24 h. Error bars in all graph related to cell proliferation indicate SD.

Figure 4. Effect of PI3K inhibitors on the proliferation in HER2 positive (Group II) salivary gland cells

Effects of BYL719 (BYL, 5μM), AZD8186+BYL719 (A+B, 5μM), and GDC941 (941, 5μM) on RET981 (A) and MAC (B) cells. Differential responses to both lines were observed, where RET981 cells was suppressed completely by 5 μM of both PI3Kα and PI3Kβ alone. In contrast, MAC cells demonstrated inhibition of cell proliferation at 5 μM doses of pan-inhibitor or combined PI3Kα and PI3Kβ but not by either PI3Kα or PI3Kβ alone.

Figure 5. Growth suppression by combined PI3Kα and PI3Kβ inhibition in PTEN-knockout/HER2 overexpression (Group III) SDC cell lines

(A) Upper panel, anti-proliferative activity in both PTEN-kd lines was increased by combined treatment (5 μM each) and pan-PI3K (5 μM) inhibitor in PTEN-kd RET981 cells. Lower panel, comparison of dosage differences (2.5 μM and 5 μM at 4 days treatment) against single or combined PI3K inhibitors on PTEN-kd and control RET981 cells. Asterisk (*) indicates that PTEN-kd lines show no changes in cell proliferation at 5 μM of AZD 8186 only compared with other inhibitors. Error bars in all graph related to cell proliferation indicate SD. (B) P-AKT level after 24h of AZD (5μM), BYL (5μM), A+B (5μM), and 941 (5μM) treatments on PTEN-kd of RET981 cells. (C) Both PTEN-kd MAC cell lines indicate more resistance to AZD (5μM), BYL (5μM), A+B (5μM), and 941 (5μM) treatments than control line. (D) P-AKT level after 24h of AZD (5μM), BYL (5μM), A+B (5μM), and 941 (5μM) treatments on PTEN-kd of MAC cells. Of note, p-AKT level retained in control line after single treatment with AZD.